No difference between the groups was seen in postoperative pain intensity measured on the visual analog scale, except for the evaluation at hour 2, which was in favor of the QLB group (p = 0.03). Patients who received QLB were more satisfied with postoperative pain management than the CON group. Persistent postoperative pain was assessed with the Neuropathic Pain Symptom Inventory (NPSI) at months 1, 3, and 6, and was found to be significantly lower in the QLB group at each evaluation (p < 0.001). We also analyzed the impact of the surgery type on persistent pain severity, which was significantly lower after laparoscopic procedures than open procedures at months 1, 3, and 6. Conclusions: QLB reduces oxycodone consumption in patients undergoing open and laparoscopic nephrectomy and decreases persistent pain severity months after hospital discharge. Background: New regional techniques can improve pain management after nephrectomy. Methods: This study was a randomized controlled trial conducted at two teaching hospitals. Patients undergoing elective open and laparoscopic nephrectomy were eligible to participate in the trial. A total of 100 patients were divided into a quadratus lumborum block (QLB) group (50 patients) and a control (CON) group (50 patients). At the end of surgery, but while still under general anesthesia, unilateral QLB with ropivacaine was performed on the side of nephrectomy for patients in the QLB group. The main measured outcome of this study was oxycodone consumption via a patient-controlled anesthesia (PCA) pump during the first 24 h following surgery; other measured outcomes included postoperative pain intensity assessment, patient satisfaction with pain management, and persistent pain evaluation. Results: Patients undergoing QLB needed less oxycodone than those in the CON group (34.5 mg (interquartile range 23 to 40 mg) vs. 47.5 mg (35–50 mg);  Although a multimodal analgesia approach is recommended following nephrectomy, some individuals still suffer from severe pain after the procedure . RegionaQuadratus lumborum block (QLB) is a relatively new regional anesthesia technique; it was developed by Blanco  and has Our study aimed to evaluate the impact of QLB on postoperative oxycodone consumption in patients undergoing laparoscopic and open nephrectomies. The secondary measures included postoperative pain intensity assessment, patient satisfaction with pain management, and persistent pain evaluation. Our primary hypothesis was that a 25% reduction in oxycodone use would be observed in the QLB group.The RCT was conducted in the urology departments of two teaching hospitals. The Institutional Review Board of the Medical University of Lublin, Poland approved the study protocol (permit number KE-0254/328/2017), which was registered with ClinicalTrials.gov (NCT03529201) before patient recruitment. Written informed consent was obtained from each patient, and the study was conducted in accordance with the principles for medical research involving human subjects of the Declaration of Helsinki.Adult patients younger than 80 years who were scheduled for nephrectomy procedures (due to cancer) could volunteer to participate in the study. Patients with known coagulopathy, allergies to the studied drugs, psychiatric disorders requiring antidepressant usage, or addiction to alcohol or other recreational drugs were excluded. Before recruitment, the investigators assessed the patients’ ability to handle a patient-controlled analgesia (PCA) pump and to understand the visual analog scale (VAS) and the Neuropathic Pain Symptom Inventory (NPSI).2), a three-lead electrocardiogram (ECG), non-invasive blood pressure (NIBP), capnography, oxygen, and volatile anesthetic concentrations. The type of surgery—open or laparoscopic nephrectomy—was chosen at the surgeon’s discretion.General anesthesia was induced and maintained by an anesthesiologist who was unaware of the patient’s group allocation. To induce general anesthesia, each patient received fentanyl , propofol , and rocuronium . Anesthesia was maintained with 1.5–2.0% sevoflurane  in a mixture of oxygen and air, and additional doses of fentanyl and rocuronium were administered. Standard monitoring during the procedure included blood oxygen saturation  group and half to the QLB group. Computer-generated randomization was prepared by an investigator who was not involved in the procedure or further assessment of the participants. Both the primary investigator and the statistician were blinded to data collection throughout the study.Patients in the CON group emerged from anesthesia and were transferred to the post-anesthesia care unit (PACU). In the other group, QLB was performed before the end of anesthesia, under ultrasound guidance, and with the patient in the lateral position. Only one person in each center performed the QLBs. The anesthesiologist used a 10 cm Ultraplex 360 needle  to perform a QLB type II . The needle was introduced in the medial to posterolateral direction. Infiltration with 0.9% saline and the needle’s position were confirmed, and 2 mL/kg 0.375% ropivacaine  was then deposited (up to 40 mL) only on the side of the surgery. Patients were not aware of the group to which they had been allocated.In the PACU, oxygen was supplied and the patient’s vital signs, including respiratory rate, plethysmography, oxygen saturation, heart rate, and blood pressure, were monitored. The standard postoperative pain management in both groups included oxycodone and non-opioid drugs. The investigator began pain treatment with oxycodone administered intravenously (i.v.) via a PCA pump, 1 mg/mL with a 5-minute lockout. The attending nurse could add 5 mg of oxycodone in the case of severe pain exceeding 40 mm on the VAS. The other painkillers comprised i.v. paracetamol, 1 g every 6 h, and i.v. ketoprofen, 100 mg twice daily. The patient also received 4 mg of ondansetron, twice daily, as prophylaxis for nausea and vomiting. The nurse, who was unaware of the participants’ group allocations, reminded the patients about the pain severity assessment.The total patient consumption of oxycodone via PCA during the first 24 postoperative hours was the primary measure of the study. The other outcome measures included pain severity on the VAS at hours 2, 4, 8, 12, and 24; rescue doses of oxycodone administered by nurses in the PACU; patient satisfaction with pain management; and persistent pain evaluation. Satisfaction with pain treatment was assessed on a five-point Likert scale on which the patient and the investigator could describe the pain management as very poor (1), poor (2), moderate (3), good (4), or excellent (5). The presence and intensity of persistent postoperative pain were evaluated during a phone interview at months 1, 3, and 6 using the NPSI , which wContinuous data were compared using the Mann–Whitney U test or the Kruskal–Wallis test by ranks (for more than two comparisons). Medians (interquartile ranges) are used to present these data. Categorical variables were compared using Fisher’s exact test. Logistic regression was used to reveal the parameters that affect chronic pain presentation. The odds ratio (OR) was used to describe the predictors that were included in the model. The receiver operating characteristic (ROC) curve was calculated for the best model. All analyses were performed using Statistica 13.1 software , which was also used to generate the random group assignments.A sample size analysis was performed for the primary outcome of the study. In a pilot study, we recruited 20 patients (10 per group) and found a mean oxycodone consumption of 42 mg in the CON group and 30 mg in the QLB group, with an SD of 18. The calculated sample size for a significance level of 0.05 and power of 0.9 that would detect a hypothesized 25% reduction in oxycodone use was 49 participants per group; we therefore recruited 105 patients with a 1:1 randomization ratio.The study was conducted from May 2018 to February 2019. p < 0.001).p = 0.028), and patients in the QLB group were more satisfied with the postoperative pain management  and 6 (red arrows). Pairwise comparisons were made with the Mann–Whitney U test using a significance level of 0.008 after Bonferroni correction. CON: control group; QLB: quadratus lumborum block group.Three factors were associated with the incidence of persistent pain after nephrectomy at month 6. The severity of persistent pain at month 3 increased the risk of chronic pain prevalence at month 6 ). Conversely, a lower amount of oxycodone administered via PCA had a reduced risk of persistent pain after 6 months ). Moreover, patients after laparoscopic nephrectomy who received QLB had a lower chance of chronic pain ). The area under the ROC curve for this model was 0.91 .The primary hypothesis of our study—A 25% reduction in PCA-administered oxycodone in the QLB group—was confirmed by the results . FurtherFurther analysis showed that patients undergoing open nephrectomies had more severe chronic pain at months 1, 3, and 6 than those undergoing laparoscopic surgery . In our Our study had some methodological differences from previous RCTs examining QLB use in patients after nephrectomy. First, both open and laparoscopic procedures were studied; in previous RCTs, only laparoscopic nephrectomies were investigated ,5,12,13.Similar to our results, in all RCTs in which QLB was compared with a control, the total consumption of opioids administered via PCA was significantly lower at hour 24 following the surgery ,13; notaFew researchers have investigated the problem of persistent pain in patients following nephrectomy. Alper and Yüksel compared 27 laparoscopic nephrectomy patients with 25 open surgery patients  and founFurthermore, more severe pain was found after open nephrectomy than laparoscopic nephrectomy in our RCT 6 months after surgery, but only in the CON group. The difference between our results and those of Alper and Yüksel could be a result of the NPSI in our trial allowing more detailed identification of persistent pain. However, Owen et al. also found chronic pain in 33% of kidney donors following open surgery , and theOur study presented a significant reduction in persistent pain severity in patients following the plain block, however, the QLB was performed postoperatively. It is possible that chronic pain severity would have been even lower in the QLB group if the block had been done pre-emptively. However, the block was performed under general anesthesia. Thus, patients had reduced nociception due to opioids and anesthetics. Our study has some limitations. Patients undergoing both laparoscopic and open nephrectomies were included, and our population was thus more heterogeneous than in previous RCTs. Patients in the CON group also did not receive a sham block with saline. Although a significant difference in persistent pain severity between the QLB and the CON groups was found in our study, its impact on patient recovery and quality of life was not assessed. To conclude, QLB reduces opioid consumption and alleviates persistent pain severity in patients undergoing laparoscopic and open nephrectomies.


Sirt1 and its downstream targets in the brain and liver, mitochondrial enzymatic activity in skeletal muscle, and exploratory behavior in offspring. We also observed differential responses of both lactation and gestational food restriction on gene expression, thermogenesis, organ masses, and behavior, in response to adult caloric restriction. We conclude that the early nutritional state could determine the magnitude of responses to food scarcity later in adulthood, mediated by the pivotal metabolic sensor Sirt1. Our results suggest that maternal gestational weight gain could be an important life history trait and could be used to predict features that improve the invasive capacity or adjustment to seasonal food scarcity of the offspring.Variations in the availability of nutritional resources in animals can trigger reversible adjustments, which in the short term are manifested as behavioral and physiological changes. Several of these responses are mediated by Sirt1, which acts as an energy status sensor governing a global genetic program to cope with changes in nutritional status. Growing evidence suggests a key role of the response of the perinatal environment to caloric restriction in the setup of physiological responses in adulthood. The existence of adaptive predictive responses has been proposed, which suggests that early nutrition could establish metabolic capacities suitable for future food-scarce environments. We evaluated how perinatal food deprivation and maternal gestational weight gain impact the transcriptional, physiological, and behavioral responses in mice, when acclimated to caloric restriction in adulthood. Our results show a strong predictive capacity of maternal weight and gestational weight gain, in the expression of  Food access for animals may be limited by spatial or temporal resource availability . These variations in the availability of nutritional resources in animals can trigger reversible adjustments, which in the short term are expressed behaviorally and physiologically . Among m+-dependent deacetylase activity. Indeed, Sirt1 controls a global genetic program to cope with changes in the nutritional status. Absence of Sirt1 results in a metabolically inefficient animal that fails to adapt to CR conditions , has been proposed as a key factor in the response to CR, acting as an energy status sensor through its NADnditions . Sirt1 anditions . In the nditions , whereasnditions . Hence, nditions .The role of Sirt1 as a master regulator of metabolism occurs mainly through two mechanisms. Its deacetylation activity has been reported acting both on non-histone proteins and histones. Sirt1 actively deacetylates proteins involved in signal transduction or gene transcription. For example, Sirt1 deacetylates PGC-1α, a potent coactivator of a plethora of transcription factors (such as peroxisome-proliferator-activated receptors or PPARs) impacting whole-body energy expenditure see . Sirt1 iMus musculus and Rattus sp.) indicate that the reduction in energy intake during pregnancy and lactation produces persistent effects in the progeny during adulthood under ad libitum feeding conditions, such as altered proteomic profile, reductions in insulin and leptin sensitivity, modifications in hypothalamic function, hyperphagia, reduction in thermogenesis, and abnormalities in body mass, among others  acclimated to both ad libitum feeding and CR in adulthood. Our results suggest that Sirt1 is involved directly in the CR-response strategy playing a central role in metabolic changes setting the ground for further studies on its potential long-lasting epigenetic effects on offspring.To gather evidence suggesting that Sirt1 is a mediator of predictive adaptive responses, here, we evaluated the effect of perinatal food deprivation  and maternal body condition  on physiological  and behavioral features (exploratory activity in an open field test) in adult rodents  were obtained from the central animal housing facilities at the Faculty of Sciences, Universidad de Chile. These females were bred in pairs with nine males (3 months old) in the same conditions. Pregnancy was identified through a vaginal plug, and the females were assigned to three conditions: ad libitum feeding , CR in pregnancy  with 70% of normal feed and CR in lactation  with 70% of normal feed. For the latter two treatments, we estimated the daily food consumption previously, in an exploratory study on other females from the same origin, strain, and age. Specifically, we observed that during pregnancy (embryonic days E10-E19) and lactation , females eat 7.86±3.0 and 13.77±3.28 g of dry food per day , respectively. Then, despite the progressive increment in food intake during pregnancy and lactation, we determine to provide 70% of the mean value , implying a greater energetic challenge, in females with larger litters, late pregnancy, and after the first week of weaning. To compensate for the energetic effort of lactation between mothers, we regrouped pups in litters of eight individuals per mother in all treatments. We measured the body mass of all females, before pregnancy and one day before birth, on day 18 of pregnancy. Additionally, we measured the body mass of the offspring weekly from birth to the end of the experiment.Eighteen adults BALB/c strain of ad libitum  for three months, until adulthood. Then, they were separated into six groups of 9 to 11 animals, with no siblings in the same group , offspring were housed individually in standard cages with paper flakes and kept in a temperature-controlled room, at 25° ± 1°C, in a LD=12:12cycle, with water and food me group . In thisad lib food intake, i.e., we provided 0.6* 3.61 g of dried food  for 20days. This commercial food was employed during their growth, in lactation and pregnancy. Besides, the colony of mice was bred for at least three generations with this food. All animal procedures were in accordance with the Chilean legislation and were approved by the Institutional Animal Care and Use Committees at the Universidad de Chile (Certificate no: 17068-FCS-UCH) and Comisión Nacional de Investigación Científica y Tecnológica .For each maternal condition, in the half of the adult individuals, we restricted the food intake by 40% of the Body temperature during the experiments was measured by means of a subcutaneous transponder , with an accuracy of ±0.2°C in the calibrated range of 32–43°C, as well as a handheld reader . At the age of two months, the transponders were implanted subcutaneously in the lower back of animals using a sterilized needle assembly provided by the manufacturer , under anesthesia (isoflurane + oxygen). Temperature was measured during acclimation in the light phase.2 and a height of 30 cm. These measurements were recorded within the activity phase of the animals (9 pm to 2 am) as briefly described: at the beginning of the measurement, the rodents were placed in the center of the field and for 10 min, they were filmed in darkness using an infrared camera (Wanscam HW0026) located 2 meters above the exploration field, in articulated support. The camera was connected to a computer, in an adjoining room where filming was recorded. Between each session, the soil surface was completely cleaned with 70% ethanol. Running or walking is considered as an exploration index, therefore, as a proxy of exploration or trajectory, we measured the number of squares crossed in the open field . Video analysis was performed with Phobos software.The exploratory behavior was assessed through an open field test both at the beginning and end of the acclimation period in 58 individuals (we lost two recordings). Tests were performed in a square acrylic box (thickness of 2 mm) of white color, with an internal surface of 1 mn=60  was dissected on ice. The tissues were stored at −80°C, pending the completion of enzymatic assays. Brain, liver fraction, and brown adipose tissue (BAT) were conserved in RNAlater (Thermo Fisher) at −80°C, for further extraction of RNA.After all measurements, animals were sacrificed by cervical dislocation, and inner organs were weighed  for all individuals, n=60 see . The int−1 cm−1 at 550 nm for cytochrome c. The citrate synthase (CS) activity was measured as follows: The enzyme assay medium contained 10 mm Tris–HCl (pH 8.0), 10 mm 5,5’dithiobis- (2 nitrobenzoic acid), 30 mm acetyl Coenzyme A (acetyl CoA), and 10 mm oxaloacetic acid (OAA) in a final volume of 0.2 ml; these reagents were omitted in controls. Citrate synthase catalyzes the reaction between acetyl CoA and OAA to form citric acid. The increase in extinction at 412 nm was measured in a Thermo Scientific Multiskan GO at 25°C. Enzyme activity was calculated using an extinction coefficient of 13.6 mm−1 cm−1 at 412 nm. All enzyme activities are reported as specific activity per gram of protein (μmol min −1 mg protein−1).The enzymatic activity determinations were carried out using hind legs muscles homogenates of six individuals per treatment. Samples were homogenized on ice (1:10 w/v) in 0.1 M phosphate buffer supplemented with 2 mm EDTA (pH 7.3) using an Ultra Turrax homogenizer . The samples were then sonicated at 130 W using an Ultrasonic Processor VCX 130 on ice 14 times in 20-s cycles with 10-s intervals between cycles. Homogenates were then centrifuged at 15,000 rpm for 15 min at 4°C to obtain a post-mitochondrial fraction. The supernatant was transferred into a new tube, to avoid transferring the upper lipid layer present in the homogenates. Protein concentration was determined using the Bradford method, with bovine serum albumin as the standard. We measured the activity of two mitochondrial enzymes: cytochrome c oxidase (E.C. 1.9.3.1) and citrate synthase (E.C. 4.1.3.7). An increase in the activity of these enzymes likely reflects changes in both the functional properties and the density of mitochondria. The cytochrome c oxidase (COX) activity was quantified using a microplate spectrophotometric method. Enzyme activity was determined in a reaction mixture containing 10 mm Tris–HCl (pH 7), 120 mm KCl, 250 mm sucrose, and cytochrome c reduced with dithiothreitol in a final volume of 0.2 ml. The decrease in extinction at 550 nm was monitored in a Thermo Scientific Multiskan GO UV/VIS spectrophotometer at 25°C. Enzyme activity was calculated using an extinction coefficient of 21.84 mmWe analyzed the deacetylase activity in nuclear protein extracts from liver tissues of three individuals per treatment. For the isolation and extraction of nuclear proteins from liver tissues, we used an EpiQuik Nuclear Extraction Kit  and measured enzymatic activity by mean of Epigenase Universal SIRT Activity/Inhibition Assay Kit , following the manufacturer’s protocol.Sirt1 was assessed by qPCR  using specifically designed primers as indicated in via 2-ΔΔCt, and gene expression was normalized to that of tissue specific reference/housekeeping genes for CR, as suggest by Gapdh, B2m, Hmbs, and Hprt, see Total RNA was obtained from brain, liver, and BAT from five individuals per treatment, by phenol-chloroform extraction using Trizol. cDNA was synthesized using 1 μg of RNA and a M-MLV reverse transcription kit, employing a DNAse treatment. Relative expression of several genes related to The averages of the morphological, biochemical, and behavioral variables between treatments were compared through nested and factorial ANOVA, using perinatal and adult feeding as factors. In the cases in which the variables correlated with body mass, the residuals of each variable against body mass were used from the regression analyses.Sirt1 related gene expression and phenotypic changes to the effect of maternal feeding, we performed Pearson correlations between physiological variables and relative gene expression. To identify differential relationships between inner fat deposits, organ mass, and body mass changes during CR, we evaluated the relationship of these variables by mean Pearson correlation, for each treatment. We evaluated the association between physiological traits of offspring and variables related to maternal weight  or maternal gestational weight gain , using linear regression analysis.To associate levels in To evaluate the effect of maternal mass in gene expression and offspring body mass, a path analysis using AMOS (SPSS) was performed. For that, we computed path coefficients among measured variables to test the overall path diagram of the observed data. We used maximum likelihood to estimate parameters. The significance of each parameter is presented only in significant cases, and the relative magnitude of regression weights was reported in color key (edited in Microsoft Publisher).Sirt1 expression?Sirt1 mediates effects of CR in mammals, as widely demonstrated in the literature see . SeveralF=0.709, p=0.496] indicating that the ranges of variation were similarly , with a range of 2–9 litter size.Maternal gestational weight gain was not affected by the experimental treatment . Nevertheless, maternal feeding regime did not modify the mass reduction of offspring during CR in adulthood =3.592, p=0.028] but only showed statistical difference in the RP treatment group between adult control and the CR condition (Fisher LSD test). In a similar fashion, rodents under CR had higher Pepck expression, using B2m and Hmbs as housekeeping genes . Interestingly, Ppar gamma expression was affected by the perinatal condition, with a lower expression found in RL animals compared to AL and RP groups . We observed a similar trend in Pepck expression .We found a significant effect by the maternal food regime on he brain , ~2.6-foUcp1 expression under CR in BAT . We also observed that the RP-AL group had a significantly higher expression of Ucp1 compared to RP-CR . In consequence, variation in Sirt1 mRNA levels is affected by maternal body weight and perinatal treatments.To analyze the independent effect of perinatal treatment on Sirt1 expression, we accounted for the maternal weight effect by performing an ANCOVA using maternal gestational weight gain and maternal weight at birth as covariates. This analysis revealed a significant effect of treatments . Of note, we found that the number of squares crossed before acclimation is correlated with maternal gestational weight gain in the RP group , but not in either the RL or AL . The RP group had half of the exploratory activity as AL and RL  at the end of acclimation; averaging 35.5°C in these animals (A positive relationship between Δ mass and Δ temperature (before and after acclimation) could be established when all data were pooled =5.864, p=0.004]. We observed a positive association between Ucp1 and body mass and temperature . These results are consistent with using both Gapdh and Hprt as the housekeeping gene for both body mass and temperature parameters .Ppar gamma in the liver. Specifically, RL and RP had a negative correlation  and AL had a positive correlation . When all data were pooled Ppar alpha had a positive relationship with body mass when using either Hprt and Gapdh as housekeeping genes . An intriguing result was observed in the RP-CR group since there was an inverse relationship between Ppar gamma and Sirt1 in both the liver and body temperature parameters . In the RP-CR group, we observed a negative association between the intestine length and BAT mass . A negative correlation between n-aminopeptidase activity and temperature  was identified between AL-CR and RL-CR groups.We found a difference in correlation between BAT mass and Sirt1 expression in the liver in the RP treatment group, suggesting that a lower increase in body mass during the pregnancy implies a higher expression of Sirt1 in offspring. This finding, is in line with the negative association found between both variables, observed when data were pooled  or when the treatments AL-CR and RL-CR were considered separately . The model showed a positive and significant relationship between P53 and Sirt1 expression levels in the liver, but only in perinatal restricted females (RP and RL conditions). Sirt1 expression in the liver had a significant effect on offspring body mass in all treatment groups; however, the direction and magnitude differed between experimental groups depending on the type of perinatal feeding. In control animals acclimated to adult CR , we found that Sirt1 mRNA levels were positively related with changes in body mass in adult offspring (before and after acclimation). Contrary, in perinatal restricted animals, this relationship was negative , the relationship was positive.The aforementioned differences in organ mass and fat deposits suggest the development of trade-offs between lipid droplet formation in the liver and BAT mass under perinatal food restriction. There was a different association between BAT mass and Pepck was in individuals under prenatal CR. These findings suggest an important role of perinatal food restriction in the modification of nutritional resource management. Thus, a differential metabolic predisposition to use energetic fuels or sources (from organs and adipose tissues) could be potentially determined by Sirt1 promoting acetylation of downstream targets, such as PPARs. The latter would agree with the glucose intolerance and lipid metabolic adaptations in response to intrauterine and postnatal CR documented in male adult rats  and the expression of ult rats . It was ult rats . FurtherUcp1 expression in BAT in the RP-CR group. This finding is in line with previous studies in rats showing that moderate CR during gestation programs offspring for lower BAT thermogenic capacity, probably by thyroid and sympathetic signaling  and mitochondrial activity  should consider early nutritional status, particularly during pregnancy. Based on the results of our study, metabolic programming is greatly influenced by the maternal energetic condition. These observations shed light on the importance of the predictability of physiological features from gene expression to phenotype. The predictability found in our study agrees with the employment of maternal anthropometry as a predictor of birth weight in humans  was identified. Paradoxically, in control condition, Sirt1 expression was positively associated with an increase in body mass in CR rodents. The negative association between maternal weight gain and Sirt1 expression was detected in the RP-CR group, with an increase of 1.95 (± 0.26) in Sirt1 relative expression, for each gram of body weight not gained at pregnancy (with respect to the ad libitum condition). This finding supports the predictive adaptive responses since the presence of Sirt1 has been considered a condition for metabolic efficiency during CR  and promote physiological and behavioral adjustments to seasonal food scarcity of offspring.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committees at the Universidad de Chile (Certificate no: 17068-FCS-UCH) and Comisión Nacional de Investigación Científica y Tecnológica .IP-V, VP, and PS: conceptualization, writing-original draft, and writing-review and editing. IP-V, FO, and BC: investigation. IP-V: formal analysis. All authors contributed to the article and approved the submitted version.This work was supported by the Fondo Nacional de Desarrollo Científico y Tecnológico Postdoctoral ANID FONDECYT Postdoctoral: 3180108 to IP-V; ANID PIA/BASAL FB0002 and CONICYT/ANID Fellowships for PhD # 21150781 (BC).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Good bedding materials can increase the comfort potential of the lying surface and enhance the welfare of cows in intensive dairy farms. The preference, behaviour, hygiene, and health of cows are affected by different bedding surfaces. In the current study, we evaluated the preference, behaviour, cleanliness, and health of cows on three bedding materials, peanut shells (PS), rice husks (RH), and a combination of two-thirds peanut shells, one-third rice husk (PRC). The daily behaviour, serum metabolites, and productivity of dairy cows were all within normal values, and no statistical differences were found between all three bedding materials, although cows showed a preference for rice husk when given access to all three bedding materials at the same time. Finally, the results suggest that bedding comprised of peanut shells and peanut–rice combinations are all suitable for maintaining the health and comfort of dairy cows.p < 0.05), and lay down most often on rice husk (4.35 bouts/d) than on peanut shell (2.55 bouts/d) (p < 0.05) but did not differ between peanut shells and peanut–rice combinations in terms of lying time and lying bouts. In Experiment 2, 12 nonlactating cows were used to assess the effects of the 3 bedding materials on dairy cow behaviour, cleanliness, serum indicators, and productivity. The total duration of lying down , the frequency of bouts of lying down , and the mean duration of lying bouts  did not differ between treatments. Similarly, no differences in eating or drinking behaviour of dairy cows were observed. In terms of hygiene, cleanliness scores did not differ between the three bedding materials, but udder and flank cleanliness decreased and improved, respectively. In addition, treatments did not affect serum metabolites or productivity of the cows. In summary, daily behaviour, serum metabolites, and productivity of dairy cows were all within the normal range, and no statistical differences occurred between the three bedding materials, although cows showed a preference for rice husk when given access to all three bedding materials at the same time. Finally, the results suggest that bedding comprised of peanut shells and peanut–rice combinations are all suitable for maintaining the health and comfort of dairy cows.The provision and quality of bedding materials affect the behaviour, welfare, and health of dairy cows. The objective of this study was to evaluate the preference, behaviour, cleanliness, and physiological status of cows on three bedding materials, peanut shells, rice husks, and a combination of two-thirds peanut shells, one-third rice husk. In an initial experiment, 15 nonlactating, pregnant Holstein cows had free access to all 3 bedding treatments for 39 d. Cows spent more time lying down on rice husk (337 min/d) than on peanut–rice combination (212 min/d) and peanut shell (196 min/d) ( Lying down is essential in the daily activities of dairy cows. The duration of time that cows normally spend lying down is between 8 and 16 h each day . When cohttp://www.hnagri.org.cn/, accessed on 5 April 2021). The broken peanut shells are dry and soft, characteristics usually favoured by cows for increased comfort [Rice husk has been used as a bedding material in commercial farms , althoug comfort ,23. HencScientific studies of dairy cow bedding usually record cow behaviour , body hygiene (particularly of the udder and legs), and health (which may be determined from serum metabolites) ,15,24. WThis study was conducted at the research farm of Henan Agricultural University, in Zhengzhou, China, between November 2019 and January 2020. All procedures involving animals were approved by the animal ethics committee of Henan Agricultural University under the Guidelines for Ethical Review of Laboratory Animal Welfare (GB/T35892). The 55 × 20 m barn had open sides and was naturally lit for 11.5 h each day. The experimental barn orientation was in the east/west direction, and the mean daily temperature at the experiment barn ranged from 0.3 to 18.4 °C. In total, 15 nonlactating and pregnant (mean 37 ± 8 d before parturition (dbp)) Holstein dairy cows between 2 and 6 years old (3 primiparous heifers and 12 multiparous cows) from the research dairies were used. Before the experiment, these cows were kept on a hard bedding surface (dry manure) with other dry cows over 15 days. The barn was divided into three adjacent pens containing the three resting areas by a steel pipe fence, and each resting area  was freshly filled with a sand base and the following three bedding materials to a depth of 10 cm: (1) peanut shell (PS) (dry matter content (DM): 91.67%); (2) peanut–rice combination (PRC) ); (3) rice husk (RH) (DM: 90.4%). Then, the cows were kept in 3 pens of 5 cows, balanced by parity and calving date, and they had free access to all three bedding types in each pen.Cows were fed with a total mixed ration at 12 kg/cow (DM basis) containing corn silage, peanut vine, and a commercial concentrate offered daily at 06:00 h. Ad libitum access to water was provided in a trough in each pen. The bedding was levelled daily and faeces were removed while the cows were eating. Fresh bedding material (30 kg) was added to each resting area once weekly. The water trough was emptied, cleaned, and refilled with fresh water daily. Two portable weather stations   Co., Ltd., Shanghai, China) were installed in the centre of the experimental barn (at a height of 197 cm) to automatically record temperature and relative humidity at 10 min intervals. Data from the portable weather station were downloaded at the end of the test by Testo Comfort Software Basic 5.0 .Four high-definition digital video cameras with built-in infrared lights  Co., Ltd., Shenzhen, China) attached to a digital video recorder  Co., Ltd., Shenzhen, China) were installed in the experimental barn to continuously capture the behaviour of the cows for 43 d. The cows in each pen were tested simultaneously and monitored continuously in individual pens until calving. The first seven days of the experiment were an adaptation period for the cows, and the behaviour was not recorded. In each pen, there were no physical obstacles to prevent the cows from lying down on any of the three bedding surfaces. Behaviour was coded from the video recordings by a trained observer using focal animal sampling with continuous recording, who distinguished individuals by their unique coat colour patches. The following behaviours were recorded: the treatment area in which each cow was lying, the total duration of lying down, the frequency of bouts of lying down, and mean duration of lying bouts .A handheld infrared thermometer  was used to assess the surface temperature of bedding materials in 5 locations  daily at2. Mean daily temperature ranged from −1.2 to 18.1 °C for inside and −1.1 to 19.6 °C for outside of the experiment barn. This research was conducted from January to April 2020 for a total of 60 days.In total, 12 nonlactating, pregnant (mean 38 ± 11 dbp) Holstein cows between 2 and 5 years old (7 primiparous heifers and 5 multiparous cows) with no history of mastitis were used. Cows were divided into three bedding treatment groups with the same three bedding materials as used in Experiment 1: peanut shell, peanut–rice combination, and rice husk, balanced for parity (but with two primiparous heifers in the last pen) and calving date. Each treatment group of cows was kept in two pens and was given only one choice of bedding surface to lie down in each pen. The dimensions of the bedding area in each pen were 5.00 m long and 5.15 m wide, with a total area of 25.75 mThe same equipment in Experiment 1 was used to monitor the behaviour of dairy cows and environmental conditions. Individual cows in each pen were distinguished by their unique coat colour patches at the time of recording. Behavioural observations and procedures were carried out as per Experiment 1, with an ethogram of behaviour presented in https://www.dairyresearch.ca/cow-comfort.php#self, accessed on 2 January 2021 [Before cows entered the experimental pen, the cleanliness of their right abdomen, right flank, and udders were separately scored on a scale designed by the Dutch Udder Health Centre  . High amary 2021 ). The log for 10 min, and the serum collected in a sterile 1.5 mL centrifuge tube and stored at −20 °C until analysis. Serum samples were analysed for serum nonesterified fatty acid (NEFA), serum total calcium (Ca), and B-hydroxybutyrate (BHB) using a fully automated bio-analysis machine . The calves were weighed immediately on a scale  at birth. The majority of births were assisted by a veterinarian; therefore, dystocia in the cows was not recorded. Cows were milked in an automatic milking system, between 06:00 and 07:00 h, and between 15:00 and 16:00 h. Milk yield for the first seven days after calving was recorded from each cow twice daily using a 28 L glass bottle collecting milk.Blood samples were collected from the coccygeal vein into 10 mL tubes without anticoagulants within 24 h of calving and centrifuged at 4000× Individual cows were considered the observational unit (15 cows for Experiment 1 and 12 cows for Experiment 2). Behaviour results from the single observer were checked for intrarater reliability by that person coding a subset  of the same videos twice as a consistency check, using Cohen’s kappa in SPSS . The average reliability was 0.96. The average interobserver reliability (Cohen’s kappa) for cleanliness scores was 0.773. Assumptions of normality and homoscedasticity were checked using the Shapiro–Wilk test and Levene’s test, respectively, in SPSS. Behaviour, serum metabolites, milk yield, and bedding properties data were analysed using a one-way ANOVA with SPSS. The variables were included as follows: the bedding treatment as an independent variable; all variables as a dependent variable. When the omnibus test was significant, Tukey post hoc tests were conducted to determine which treatments were significantly different from each other. The differences in cleanliness scores within and between treatments over the study were analysed by Kruskal–Wallis and Wilcoxon test. The results are expressed as means and standard errors. Figures were generated with GraphPad Prism 8 software . A statistical difference was assumed if Alpha was <0.05.Due to insufficient device memory, 14 days of weather station data were not recorded. The average daily temperature and relative humidity obtained from the remainder are shown in Cows chose to spend more time lying on rice husk (337 min/d) compared with the peanut-rice combination (212 min/d) and peanut shell (196 min/d) and had more frequent lying bouts on rice husk (4.35 bouts/d) than on peanut shell (2.55 bouts/d), but these variables did not differ between peanut shell and peanut–rice combination bedding . No diffAmbient temperature and relative humidity varied throughout the experiment. There were some differences in temperature and humidity between the inside and the outside of the barn. The lowest temperature readings were similar for the two locations (inside: −2.6 °C and outside: −2.8 °C). The maximum temperature was lower inside (25.8 °C) than outside the barn (36.5 °C). Similar minimum (26.4% vs. 24.3%) and maximum (96.4% vs. 96.5%) humidity values were observed in and out the barn.The total lying time was higher for rice husk than other bedding but no significant differences were found between the three bedding treatments . SimilarCleanliness scores of cows were not affected by bedding treatment, but the udder cleanliness score increased and the flank cleanliness score descended over the study .Serum metabolite concentrations were all within a normal range, with no statistical differences between bedding materials . In addiThe goal of this study was to compare the preference of dairy cows for three bedding materials and to determine the effects of different bedding on animal behaviour, welfare, and health. During the period when cows had a free choice of three bedding types, cows lied down most often and spent more time lying down on rice husk than on peanut shells and peanut–rice combinations , while tOn the other hand, the heat distribution of the bedding was elucidated by thermal images when the cows were lying down , indirecIn Experiment 2, cows were only given one of three potential bedding types. Cow behaviour was unaffected by the type of bedding material presented . There wCows tended to spend the most resting time on rice husk and the least time in the peanut–rice combination, but these differences were not significant. This aligns with the number of lying bouts, which was most frequent for the rice husk bedding, followed by peanut shell and the peanut–rice combination . In the Feed intake is related to animal energy balance . BeddingCow cleanliness was measured using a Likert scale from 1 (free of dirt) to 4 (very dirty). The cleanliness of cows was not affected by different bedding treatments. Greater improvement in the cleanliness of flanks was found for those cows in post-test than in pre-test, while the udder cleanliness score increased over the study . It is wAdequate energetic balance is important for maintaining both cow welfare and adequate production levels. Serum BHB and NEFA concentrations are used as indicators of energy balance and hyperketonemia in cows ,43. KetoAccording to the results of this experimental study, peanut shells and peanut–rice combinations appear to be suitable bedding materials for dairy cows. Daily behaviour, serum metabolites, and productivity measures of the cows were all within the normal range, and no statistical differences were found between bedding types, although cows showed a preference for rice husk when given access to all three bedding materials. Further research is needed to confirm the long-term effect of these deep bedding materials on dairy cow health, welfare, and production.


Hydrogelsand organogels are widely used as cleaning materials,especially when a controlled solvent release is necessary to preventsubstrate damage. This situation is often encountered in the personalcare and electronic components fields and represents a challenge inrestoration, where the removal of a thin layer of aged varnish froma painting may compromise the integrity of the painting itself. Thereis an urgent need for new and effective cleaning materials capableof controlling and limiting the use of solvents, achieving at thesame time high cleaning efficacy. In this paper, new sandwich-likecomposites that fully address these requirements are developed byusing an organogel (poly(3-hydroxybutyrate) + γ-valerolactone)in the core and two external layers of electrospun nonwovens madeof continuous submicrometric fibers produced by electrospinning  or polyamide 6,6). The new composite materialsexhibit an extremely efficient cleaning action that results in thecomplete elimination of the varnish layer with a minimal amount ofsolvent adsorbed by the painting layer after the treatment. This demonstratesthat the combined materials exert a superficial action that is ofutmost importance to safeguard the painting. Moreover, we found thatthe electrospun nonwoven layers act as mechanically reinforcementcomponents, greatly improving the bending resistance of organogelsand their handling. The characterization of these innovative cleaningmaterials allowed us to propose a mechanism to explain their action:electrospun fibers play the leading role by slowing down the diffusionof the solvent and by conferring to the entire composite a microstructuredrough superficial morphology, enabling to achieve outstanding cleaningperformance. However,this approach is extremely risky for the painting because the solventaction is highly uncontrollable and leads to saponification, swelling,and leaching of the painting layer components, with decrease of thesurface mechanical strength.10 A valid alternative is the useof gels able to exert a controlled solvent action. Consequently, chemicaland physical gels have become popular for the removal of aged varnishesor coatings from artwork surfaces through a “press and peel”approach.13 Gels used in restoration have different physical aspects: viscousdispersions, sticky masses, flexible, and peelable films.4 Gel–solvent interaction and chemical/physical cross-linksaffect both solvent retention and mechanical properties, thus determiningthe final cleaning efficacy, the presence of gel residues, the safetyfor the underneath substrate, and the ease of use.15 Generally, low elastic moduli are desired to maximize the contactwith the surface to achieve better cleaning at the expense, however,of retention capability and gel cohesion, with a consequent uncontrolledrelease of solvent, undesired painting damages, and residues on theobject. Therefore, when designing gels for such a kind of applications,the challenge is to find a good compromise between gel softness andsolvent retention.Soft hydrogels andorganogels combined with suitable solvents arecommonly employed for the cleaning of dirty surfaces, especially whenthe substrate is susceptible to swelling and its functionality iscompromised by the solvent itself. This is a frequent issue encounteredin art restoration as well as in personal care and electronics. Inthese frameworks, the benefits of gels rely on their capability toentrap the solvent to minimize substrate damage and to adapt theirshape to maximize the contact with the surface, necessary to achievegood cleaning efficiency. A typical application of hydrogels and organogelsis the selective removal of varnish layers from paintings.18 addressing the demand of green bio-based cleaningapproaches,25 to the benefit not only of the artworks, but also of restorers andthe environment. A step forward for optimizing the use of these gelsis the improvement of their cleaning efficiency, solvent retention,handling, and mechanical properties in order to avoid their fragmentationswhen they are applied or removed. To this aim, the combination ofgels with mechanical resistant materials in a composite structureis expected to provide relevant benefits.We have recently proposed poly-3-hydroxybutyrate(PHB)-based gelswith green solvents as new cleaning systems for paintings and indoorbronzes27 which make them idealmaterials to act as reinforcing components for organogels. Electrospinningis a polymer processing technique that uses electrostatic forces tostretch a viscoelastic jet derived from a polymer solution. Electrospunfibers are currently used for a wide range of applications, includingfiltration, sensors, catalytic systems, energy storage, structuralcomposites, and biomedical applications.31 Electrospinning is a consolidatetechnology on a lab-scale dimension and the scaling up toward levelsfeasible for mass production has been realized in the past decadeby several companies.32Nonwoven fabrics ofcontinuous micrometric fibers made by electrospinningtechnology have unique properties, such as interconnected and openporosity, high surface area, liquid permeability, excellent flexibility,and mechanical resistance,33 have been exploitedfor enhancing gel mechanical properties36 and various combinationstrategies have been adopted to obtain different kind of fibers–hydrogelcomposites.41 Moreover, the use of fibers with submicrometric diameters confersto the gel a surface microstructuration that is considered highlyadvantageous for tissue engineering applications.42Electrospunmaterials have been already used in combination withsoft gels for applications in the biomedical field. Here, their uniquestructures and properties43 was also tested.Finally, a possible mechanism was suggested to explain why the compositematerials produce a limited and superficial release of solvent andat the same time are able to exhibit exceptional cleaning performances.With these premises, in this work, we propose forthe first timethe use of new sandwich-like composites based on the combination ofelectrospun polymers, that is, poly (PVA) and polyamide6,6 , with the PHB-based organogel. In these composites, electrospunmats act as external mechanically resistant layers and provide microstructurationon the surface of the composite. The gel constitutes the core andcontains the active solvent, which in turn exerts its function bydiffusing through the pores of the electrospun layer toward the paint.PA6,6/PHB–GVL and PVA/PHB–GVL composites have been producedand their mechanical properties, morphology, and composition havebeen characterized. Their capability to remove a varnish layer madewith a terpenic material applied on a paint mock-up and prepared byfollowing ancient recipesMw = 85,000–124,000g/mol, 87–89%hydrolyzed), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), absolute ethanol(EtOH), and γ-butyrolactone (BL) were purchased from Sigma-Aldrich.PA6,6 (Zytel E53 NC010) was kindly provided by DuPont. All of thechemical reagents were commercially available and directly used withouttreatment.γ-Valerolactone(GVL), PHB, PVA (2O (1/1 v/v) at a concentration of 10%w/v and stirred until all the polymer dissolved in the solvent atroom temperature (RT). PVA nonwoven was fabricated using the followingconditions: applied voltage 16 kV, needle-to-collector distance 18cm, solution flow rate 1.5 mL/h, at RT, and relative humidity 40–50%.PA6,6 was dissolved in HFIP at a concentration of 20% w/v and stirreduntil all of the polymer dissolved at RT. PA6,6 nonwoven was fabricatedusing the following conditions: applied voltage 20 kV, needle-to-collectordistance 15 cm, solution flow rate 0.5 mL/h, at RT, and relative humidity40–50%. Electrospun samples have a final dimension of about15 × 8 cm2 and a thickness in the range 70–110μm.The electrospinning apparatus, made in-house,was composed by ahigh voltage power supply (Spellman SL 50 P 10/CE/230), a syringecontaining the polymeric solution connected to a stainless steel blunt-endedneedle (inner diameter: 0.51 mm), and a grounded aluminum rotatingmandrel as a collector . The polymericsolution was dispensed through a polytetrafluoroethylene (PTFE) tubeto the needle that was vertically placed on the collector. PVA wasdissolved in EtOH/H16 Briefly, 300 mg of PHB were solubilized in 3mL of GVL, in a closed vial, by stirring at 110 °C for 5 minin an oil bath, until all the PHB powder dissolved in the solutionand became transparent. The solution was then poured in a Petri dishand cooled down to RT to obtain an opalescent and stiff gel about3 mm thick.PHB–GVLgels were produced according to a previously reported procedure.2). During this step, the PHB–GVL solutionwell-impregnated the pores of the electrospun tissue by homogeneouslyspreading over the entire surface of the nonwoven. A second layerof electrospun nonwoven was quickly laid on the top of the PHB solutionby manually applying a slight pressure. The duration of the droppingand of the application of the second layer was controlled to be notlonger than 30 s. The solution instantaneously wetted and impregnatedthe second fabric. The system was finally let to cool down to RT,obtaining a final sandwich-like composite with thickness in the range1.7–1.9 mm (see Table S1 for detailson the geometry of the composites). No shrinkage is observed immediatelyafter the preparation . Thecomposites were employed for mock-ups cleaning within 30 min fromthe preparation. In this time interval, no relevant modification ofthe sample shape was detected .T = 40 °C for 15 days to remove theGVL solvent, and sputtered with gold. The distribution of fiber diameterswas determined through the measurement of about 100 fibers and theresults were given as the average diameter ± standard deviation.The student unpaired t-test was used to test thestatistical significance of the difference between the mean values(p < 0.05). Thermogravimetric analyses (TGA) werecarried out using a TGA Q500 thermogravimetric analyzer (TA Instruments).The thermograms were recorded from RT to 700 °C at a heatingrate of 10 °C/min under the N2 atmosphere. TGA analysiswas performed twice on the composites to verify the reproducibilityof the result. The mechanical characterization of the materials wascarried out using a Dynamic Mechanical Analyzer DMA Q800 (TA Instruments)in single cantilever configuration at RT. The analyses were performedon rectangular strips  whosethickness was measured by a digital caliper. Two replicas for eachsample were carried out by applying a ramp force of 1 N/min untilsample break. Differential scanning calorimetry (DSC) was carriedout under the N2 atmosphere by means of a differentialscanning calorimeter  equipped with a refrigeratedcooling system. Electrospun samples were kept at 50 °C for 1h under N2 flow (50 mL/min) to eliminate most of the absorbedwater and were subsequently subjected to two heating scans at 20 °C/minand one fast cooling applied between the heating scans.Sample morphology was observed with a scanningelectron microscope  at an acceleratingvoltage of 20 kV. Prior to SEM analysis, the samples were dried inan oven at Rheologicalexperiments were carried out on an Anton Paar rheometer MCR 102 usinga cone-plate configuration (50 mm diameter). The temperature was controlledby an integrated Peltier system and a Julabo AWC100 cooling system.To keep the sample wet during the measure, a solvent trap was used(H-PTD200). The temperature sweep analyses during cooling rate scans were performed with a fixed gap value of0.102 mm at a constant strain of 0.5% and frequency of 10 rad/s. ThePHB–GVL solutions were prepared at 120 °C and transferredon the hot plate of the rheometer kept at 140 °C. The solutionwas maintained for 3 min in the hot cone-plate before starting thetest. The viscosity of the solution was also monitored during a coolingscan at 10 °C/min by applying a strain rate of 10 1/s.To evaluate the cleaning efficiency,the sandwich-like composites were tested on mock-ups and comparedwith the PHB–GVL gel. The mockups were prepared according totraditional painting techniques, and naturally aged over 2.5 years.The preparation layer was made of gypsum and rabbit glue, dissolving1.0 g of animal glue in 5.0 mL of hot distilled water and mixing itwith 6.0 g of grinded gypsum. The pigment layer was obtained by dissolving2.0 g of red burnt ochre in 2.0 mL of 10% w/w of rabbit glue solution.The varnish layer was prepared by dissolving 1.0 g of dammar in 2.5mL of turpentine. PVA/PHB–GVL, PA6,6/PHB–GVL, and PHB–GVLgel were directly applied on the surface of painting mock-ups for5 min, then the samples were removed and the surface was further cleanedwith dry cotton swabs to eliminate the residues of dammar and theexcess solvent.–1, at a spectral resolution of4 cm–1 with 128 scans and an optical aperture of150 × 150 μm. Spectroscopic analysis was performed on 3different areas treated with the same cleaning procedure and 4 spectrawere recorded before and after treatment. Headspace solid phase microextraction(HS-SPME) was employed for the evaluation of the solvent retentioninto the substrate after the cleaning treatments.17 Analyses were performed exposing directly a carboxen–polydimethylsiloxanefiber into the headspace of a sealed vial containing the sample. Thesample consisted in a fragment of around 1.0 mg collected from thesurface of the painting mock-up. Because of the low volatility ofGVL, sampling was performed after 2 h and after 24 h from the applicationto better describe the release behavior of the cleaning systems. Thesample was placed into a 20 mL HS vial, spiked with 1 μg ofthe internal standard (BL) and sealed with a silicone/PTFE septa andaluminum cap . TheSPME fiber was inserted into the headspace vial and the sample wasthermally heated to 150 °C for 30 min. After reaching the extractiontime, the fiber was inserted into the injector of a 5977 Agilent gaschromatograph connected to a 7820A Agilent quadrupole mass spectrometer. Analytes werethermally desorbed at 250 °C for 15 min and separated with aDB-FFAP polar column , using helium as carrier gas. The thermal program was:100–250 °C at 10 °C min–1. Theabundances of the individual compounds were quantified from the m/z 86 for BL, 100 for GVL mass chromatograms.Calibration curves were performed for BL in the consistent concentrationof 1000 ppm in methanol and GVL in the concentration range of 500–8000ppm by the regression method. The areas corresponding to this rangeof concentration of standard solutions are in accordance with thoseobtained by sample measurements, at least two sample replicas treatedwith the same procedure were performed to quantify the amount of GVLin the substrate after the cleaning procedure.Cross sectionsof mock-ups varnished with dammar before and after the cleaning treatmentwere observed by optical microscopy in visible and ultraviolet light. Attenuated totalreflectance (ATR) analyses were performed with a Thermo Nicolet , iN10MX imaging microscope, fittedwith a mercury–cadmium–telluride detector cooled byliquid nitrogen. Measurements were performed using a slide-on ATRobjective, equipped with a conical germanium crystal, in the range4000–675 cmp > 0.05). Both electrospun samples are highly flexibleand mechanically resistant to handling. PVA is a water-soluble polymerlargely employed for pharmaceutical uses, protective water-solublefilms, textile processing, and cosmetics. PA6,6 is used for textiles,in automotive, and for electronic goods. Both polymers are semicrystalline, strong but ductile,and both are neither solubilized nor swollen by the GVL solvent.Nonwoven fabricsmade of randomly oriented fibers of PA6,6 andPVA were fabricated by applying optimized electrospinning conditionsto gain continuous, smooth, and bead-free fibers 1b,c,e,f Figure S3 shows that the temperature of gelationof PHB–GVL solution is in the range 75–113 °C,depending on the cooling rate. The data demonstrate that the PHB–GVLsystem massively changes its property in a narrow temperature range,thus making the preparation of the composite critical if not performedin the proper conditions. In particular, it was experimentally verifiedthat, to achieve the formation of composites with cohesive layers,the dropping step and the laying of the second electrospun mat mustbe performed in a time range of about 30 s, before the occurrenceof PHB–GVL gelation. It was also verified that, as soon asthe PHB–GVL is in the liquid state, the viscosity is not affectedby the temperature , meaningthat its percolation in the pores of the electrospun layers is notexpected to be influenced by the solution temperature. This informationallowed to set the proper dropping conditions in terms of temperatureand duration of the procedure, as reported in the “To optimize the procedure for the preparation of the sandwich-likecomposites, the rheological properties of the PHB–GVL solutionwere investigated with the aim to identify the temperature and thedropping conditions for obtaining reproducible results. Supporting Information for details).The stabilityof the fibers at the PHB–GVL solution wasevaluated by means of SEM and DSC. Thesandwich-like composites appear highly flexible and resistto handling and bending 1h,i. A rFigure S4 for additional images). This observation will be crucialfor elucidating the cleaning mechanism of the composites.The dried PHB–GVL gel and thesandwich-like composites wereobserved by SEM both at the cross section and at the external surface2. The PHTable S1.To gain information on the composition of the PHB–GVL geland of the composites, TGA analysis was carried out. Inorder to investigate the cleaning performance of the different materials,mock-ups varnished with a 40 μm thick layer of dammar were used.Cross-sections of paint samples collected from the mock-ups beforeand after the cleaning treatment were observed with optical microscopy4. It can–1 (C=Ostretching) and at 1455 and 1380 cm–1 (C–H2 scissoring) were just reduced. Moreover, residues of GVLwere also observed as the band at 1767 cm–1  suggests. After the cleaning with thesandwich-like composites, the dammar C=O stretching band at1704 cm–1 completely disappeared and no residueof GVL was observed. In addition, the glue binder characteristic bands, observed in the unvarnishedpaint layer, were also detected after cleaning with the composites,thus demonstrating the high efficiency of these new materials in removingthe dammar coating, without leaving solvent residues. It is pointedout that no significant differences in the cleaning performance ofPVA- and PA6,6-based composites were detected.The high cleaning performanceof the composites was confirmed byFTIR microscopy in the ATR mode 5 perform46FTIR analyses in the ATR mode underline also thatGVL residueswere not detected after the application of the sandwich-like composites.To better quantify the solvent residues left on the painting aftercleaning with the different materials HS-SPME was applied. This aspectis of key importance on a practical point of view because residualsolvent on the paint surface could lead to a series of adverse effects.The evaluation of the amount of solvent released to the paint layerwas aimed at comparing the different cleaning systems, thus acquiringinformation about their capability to retain the solvent. To makea realistic comparison, because 5 min treatment with PHB–GVLgel was not effective in completely removing the varnish, a secondapplication of 5 min was also carried out for this material, afterwhich the removal of the dammar was complete. In the case of sandwich-likecomposites, instead, only one 5 min-treatment was enough to achievethe same result in terms of cleaning efficiency. The amount of residualGVL detected after 2 and 24 h from the treatments are reported in Figure S5.To understand why the sandwich-like composites showhigher cleaningefficiency, SEM observation of the samples after cleaning was performed6 and 7. Figure S5. Differently, thedammar layer collected by the PHB–GVL gel appears not wellattached to the material , it turnsout that the dammar and the fibers do not directly come in contactduring cleaning and the same chemical interactions are establishedbetween dammar and composites and dammar and pure gel. Therefore,the role of the fibers is only to provide structural support for thegel and to increase the surface roughness, with the consequent increaseof mechanical adhesion between PHB and dammar.Thisstudy reports the first attempt to combine electrospun nonwovenfabrics with organogels for cleaning purposes. New materials witha sandwich-like structure, consisting of two external layers of electrospunfibers and a core of organogel, have been developed and tested forpainting cleaning. The presence of the external electrospun layersconfers to the composite new properties and advanced functionalities.First, the electrospun nonwoven exerts the function of mechanicallysupporting component, thus massively improving the mechanical resistanceof the system, as demonstrated by mechanical tests. This aspect isof key importance because facilitates the use and the handling ofthe cleaning material, especially in the laying and peeling-off procedures,reducing the presence of residual debris on the paints. Cleaning testsand SEM observations highlighted that the presence of a microstructuredsurface with an interconnected porosity provided by the electrospunlayer ensures, on one side, the possibility for the active solventto diffuse from the inner gel toward the paint surface with a moresuperficial action than the normal gel and, on the other side, actas anchoring sites for the swollen dammar. Overall, the new developedcleaning systems may be better handled, have better cleaning performances,and are safer toward the cultural object than the neat gel, thanksto their controlled and superficial action. Efforts are currentlydevoted to further investigate the role played by electrospun materialsin cleaning procedures. Thus, new tests, based on the applicationof PVA and PA with the use of different neat green solvents, are beingcarried out. Given these exceptional results, such a kind of materialsmay find application for the cleaning and disinfection of other delicatesurfaces, in personal care, and hygiene fields.


Wheat grain protein content and composition are important for its end-use quality. Protein synthesis during the grain filling phase is supported by the amino acids remobilized from the vegetative tissue, the process in which both amino acid importers and exporters are expected to be involved. Previous studies identified amino acid importers that might function in the amino acid remobilization in wheat. However, the amino acid exporters involved in this process have been unexplored so far. In this study, we have curated the Usually Multiple Amino acids Move In and out Transporter (UMAMIT) family of transporters in wheat. As expected, the majority of UMAMITs were found as triads in the A, B, and D genomes of wheat. Expression analysis using publicly available data sets identified groups of TaUMAMITs expressed in root, leaf, spike, stem and grain tissues, many of which were temporarily regulated. Strong expression of TaUMAMITs was detected in the late senescing leaves and transfer cells in grains, both of which are the expected site of apoplastic amino acid transport during grain filling. Biochemical characterization of selected TaUMAMITs revealed that TaUMAMIT17 shows a strong amino acid export activity and might play a role in amino acid transfer to the grains. Amino acids serve as the major carrier of nitrogen within the long-distance transport systems (i.e. xylem and phloem) of plants. Amino acids, typically synthesized in the photosynthetic leaf tissues from inorganic N, are carried to heterotrophic tissues such as seeds and roots. Such exchanges of amino acids between organs are indispensable for the plant growth.1, amino acid loading to the seeds4, xylem-to-phloem amino acid transfer5, amino acid transport from the nodules6, and root uptake of amino acids10. These transporters belong to amino acid -polyamine-choline (APC) family and the amino acid/auxin permease (AAAP) family, and most of them function as amino acid-proton symporters12. Another and more recently identified family, UMAMITs, encode bidirectional amino acid transporters and vacuolar auxin transporters. Although this family of transporters has not been studied as extensively as the members of APC and AAAP superfamilies, the physiological roles of some of the UMAMIT transporters have been identified. In Arabidopsis, UMAMIT11, 14, 18, 24, 25, 28 and 29 have been implicated in seed loading14, UMAMIT14 and 18 in phloem export and root amino acid secretion16, and WAT1/UMAMIT5 is involved in vacuolar transport of auxin17. The major functional difference between APC/AAAP family transporters and UMAMIT is that UMAMIT is capable of functioning as cellular amino acid exporters15.Due to their zwitterionic nature, the passing of amino acids through cellular membranes is mediated by dedicated carrier proteins. In the past three decades, many of the amino acid transporters involved in such processes have been identified. Cellular amino acid importers belonging to the families of Amino Acid Permeases (AAPs), Lysine and Histidine Transporters (LHTs), Cationic Amino acid Transporters (CATs), Proline and GABA transporters (ProT/GABAT), Auxin Transporters (AUXs), and Aromatic and Neutral amino acid transporters (ANTs) have been identified, and for some transporters, their physiological roles have been identified, including amino acid export from source leaves18.Using bioinformatics and the ever-increasing number of available plant genomes, it has been revealed that the APC, AAAP, and UMAMIT families of transporters are conserved among all higher plants investigated so far, the vast majority of which are uncharacterized. An inventory of these amino acid transporters in the crop of interest can be used as the starting point to modify and optimize the amino acid transport within the plant, a strategy with a record of success in multiple model and crop species3. Accumulation of glutenins and gliadins relies on amino acids supplied during the seed filling phase, and increased N supply during the seed filling stage is associated with higher grain protein content. Therefore, amino acid transporters expressed during this stage are of particular interest. Previous publications have already identified members of the APC and AAAP superfamilies in wheat, and identified the transporters that could be involved in seed loading20. Here we report the identification of potential amino acid transporters belonging to the UMAMIT family in wheat. We have identified 213 putative members, and ~ 70% of the members were found as triads on all three  genomes. Further, spatio-temporal analysis of UMAMIT expression revealed that many are developmentally controlled, and are expressed during the seed filling stage in either developing kernels or source leaves, suggesting that the function of UMAMITs in seed filling might be conserved in wheat.Among cereals, amino acid transport in wheat is of specific interest due to the wheat flour’s end-use. The dough quality is tightly linked to the quantity and quality of its storage proteins in the endosperm, glutenins and gliadins, which determines the viscosity and elasticity22. Interpro search of all 44 Arabidopsis UMAMIT genes classified them as WAT1-related family (IPR30183), and most carried EamA domain (IPR00620). Searching wheat proteins (Ensembl v47) for IPR30183 and IPR00620 yielded 194 and 192 translated sequences respectively. Among those, 182 sequences belonged to WAT1-related family (IPR30183) and contained EamA domain (IPR00620). Pfam search of Arabidopsis UMAMIT genes identified two repeats of PF00892 motifs in full-length proteins (EamA domain). Pfam search of wheat proteins with PF00892 yielded an identical list of 194 proteins with the Interpro search using IPR00620. Blastp search using all AtUMAMIT genes, at the e-value cutoff of 1e−5, yielded 186 entries, all of which were included in the list found in the Interpro search. In total, 204 genes have been identified. The domain structure of full-length UMAMITs is shown in Fig. Wheat UMAMIT homologs were searched using Interpro and Pfam programsIn addition, low confidence genes in the Refseq v1.1 cDNA annotation have also been mined. A tblastn search using all AtUMAMIT protein sequences at e-value cutoff of 1e−5, yielded an additional 28 genes, of which 12 were partial genes. Corresponding intervals for all low confidence proteins were identified in IWGSCv1.0 genome, and gene models were examined using the reads mapped to the corresponding intervals in CRAM files for 308 publicly available RNAseq datasets Table . Of the 23. To identify the clades to which the TaUMAMIT proteins belong, a phylogenetic tree including the sequences from six representative species 25. Genes belonging to clades I to IV, VII, IX, and X were found in both Arabidopsis and wheat. Clades I and III includes previously characterized amino acid transporters16. Two other clades include previously documented genes; clade V includes Arabidopsis WAT123, and clade IX includes RTP1/UMMAIT36, which has been identified as a negative regulator of SA-mediated pathogen response26. Clade VI, which contains many wheat and rice UMAMIT members, did not contain any Arabidopsis members. Clade IX, on the other hand, did not contain any UMAMIT from wheat, confirming the previous report showing that this clade is dicot-specific. In summary, all but one clade of UMAMITs found in Arabidopsis were conserved in wheat. TaUMAMIT genes were distributed on all 21 chromosomes and the unassigned scaffold. Some of the TaUMAMITs were found in multiple tandem duplications, such as the lower arm of chromosome 6, where 6 or 7 tandem repeats of TaUMAMITs belonging to clade III are found , as well as to the manually annotated UMAMITs identified in this study were counted. In total, the expression of 91 out of 98 homeologous groups was detected in at least one tissue. The largest cluster of genes .In order to understand the tissue specificity of TaUMAMITs, an RNAseq dataset prepared from grain, leaf, root, spike and stem tissues at three different developmental stages each8. In wheat, amino acids are released from the nucellar projection on the maternal side to be picked up by the filial tissues such as the embryo, transfer cells and the aleurone layer surrounding the endosperm9. To identify the UMAMITs involved in the amino acid transfer process described above, a publicly available RNAseq data set derived from whole endosperm at 10 days post-anthesis (DPA), starchy endosperm at 20 and 30 DPA, aleurone layer at 20 and 30 DPA, and transfer cells at 20DPA10 were queried as described above. The cell type that expressed the largest number of UMAMIT genes was transfer cells  throughout early and mid grain filling stages despite some changes in composition34. Therefore, it is likely that amino acid export out of the flag leaf is also a continuous process, and is mediated not only by those expressed in the late senescence stage but also by those expressed in the earlier stages. Understanding the main players in leaf N export will require additional biochemical analysis to identify the active amino acid exporters, as well as detailed temporal analysis of leaf N loss and the expression profile of TaUMAMITs.Earlier studies examining the leaf N content after anthesis showed a gradual decline in N throughout the filling stage and not a sharp decline at the late senescing stageTo validate the expression of some of the TaUMAMITs in grains, four strongly expressed TaUMAMIT genes were selected Fig. a. The ex12. The cells were grown in a minimal medium containing ammonium as the sole nitrogen source, and the excretion of L-amino acids to the medium was measured using a colorimetric assay. The results indicated that TaUMAMIT17 has a strong activity for amino acid export, resulting in > 100 fold concentration of amino acids in the growth media compared to the control expressing the empty vector, and higher than cells expressing previously characterized AtUMAMIT14 and 18 genes  and 51 (clade VII) did not result in a statistically significant increase in amino acid excretion compared to the WT, despite them belonging to the clades that include Arabidopsis members that show some activity as amino acid exporters25.The amino acid secretion from yeast expressing clade I, V, and VII transporters was lower than those expressing clade III genes from Arabidopsis in the yeast expression system. TaUMAMIT92 belongs to clade VI, which largely consists of monocot members. TaUMAMIT92 did not show a significant amino acid transport activity in our assay. Therefore, whether transporters in these clades indeed function as amino acid exporters remain to be seen.To test their functions as amino acid exporters, one homeolog each of TaUMAMIT17, 30, 51, 92 were selected Table  and exprPhyscomitrella patens, Selaginella moellendorffii, Arabidopsis thaliana, Oryza sativa) previously published were included31. The sequences were aligned using MUSCLE35, with gap open penalty of − 2.9, gap extend 0 and hydrophobicity multiplier 1.2, UPGMA clustering option. The aligned sequence was used to build a phylogenetic tree with RAxML version 8.236, using rapid bootstrap analysis (1000 iterations) and GAMMA model of rate heterogeneity using empirical base frequencies and the LG substitution model.The longest isoforms of wheat UMAMIT genes  and Arabidopsis UMAMIT genes  were used for the analysis. Additionally, UMAMIT genes in four representative species (https://www.ncbi.nlm.nih.gov/sra)36 were surveyed and datasets with the following SRA study IDs were selected for further processing: ERP004714 , ERP004505 (grain development), and SRP166963 (leaf senescence). For each SRA study, the read counts for high confidence UMAMI genes were obtained from iRAP pipeline (v1.0.1)37—mapped data available at European Nucleotide Archive (ENA)38 (https://www.ebi.ac.uk/ena/browser/home), obtained using RNASeq-er REST API39. The pipeline uses FASTX (v0.0.13) for filtering and adapter trimming, FASTQC (v0.11.7) for quality check, HISAT2 (v2.1.0)40 for alignment and featureCounts (v1.6.2)41 for quantification. The mapping quality for every dataset was confirmed to be above 75%. For determining counts of low confidence UMAMIT genes, the HISAT240 mapped CRAM alignment and corresponding CRAI index files from the ENA database were used to count the reads within the corresponding intervals identified in this study. The wheat genome assembly and GTF version 41 were downloaded from the Ensembl Plants website (ftp://ftp.ensemblgenomes.org/pub/plants/release-41). SAMTools (v1.9)42 was used to extract the reads within gene boundaries for the low confidence UMAMIT genes. FeatureCounts41 from Subread package (version 1.6.2) was then used with the GTF file corresponding to the low confidence UMAMIT genes to count the mapped reads. The read counts for the high- and low-confidence genes were processed in featureCounts, then normalized using GeTMM43, where gene length was obtained by summing lengths of unique exons for each gene from the GTF files. Mean was taken across replicates and the resulting expression was summed for each gene in the triad. For triads missing one or more genes in the triad, only the mean expression of available genes was added. This was followed by log2 transformation in GeTMM with an offset of 1 to reduce the dynamic range for creating the heatmap. The R-package pheatmap (v1.0.12)44 was used to generate the heatmaps by scaling the mean sample expression by row to show peak tissue expression. The row clustering was performed using the ward.D method and Euclidean distance11. The genes with zero variance across all samples were removed to resolve errors resulting from clustering in pheatmap.Wheat RNAseq projects publicly available on NCBI Sequence Read Archive (SRA) database .TaUMAMIT sequences were synthesized  and cloned into the pTwist_ENTR vector. TaUMAMIT17 and 92 were codon-optimized to avoid GC-rich sequences. The sequences of synthesized fragments are found in Table 12. Briefly, 22Δ10α  expressing individual UMAMIT proteins were grown in a minimum medium46 overnight at 28C. On the next day, the cultures were diluted to OD600 = 0.1 in 5 mL of minimal medium, then incubated at 28C for 5.5 h. The cultures were spun down and the supernatant was flitered using a 10 kDa exclusion membrane. Amino acid concentration of the supernatants was determined using L-Amino Acid Quantitation Kit  following the manufacturer’s instruction.TaUMAMITs amino acid export activity was determined via yeast cell secretion in liquid media according to the procedure previously describedTriticum aestivum cv. TAM11447) plants were grown in 2018 at Texas A&M Research and Extension Center in Bushland, TX . Standard agronomic practices were performed according to Texas A&M AgriLife extension recommendations. Spikes at five and 14 days after flowering were harvested and stored in liquid N2. The grains were mechanically isolated from the glumes while soaking in liquid N2, and stored at − 80 °C until use. Flag leaves and stems were harvested from plants grown at 28 °C with a 12 h light cycle in a greenhouse, at five and 14 days after flowering. Root tissues were collected from 10 day-old seedlings germinated on filter paper moistened with distilled water.Wheat  following the manufacturer’s instruction. Complementary DNA synthesis was performed using iScript™ Advanced cDNA Synthesis Kit , using 2 μg of total RNA as a template. Quantitative PCR was performed using SsoAdvanced™ Univseral SYBR® Green Supermix  on the CFX96™ Real-Time System platform, using the primers listed in Table t−Cqt/Er−Cqr, where Et and Er are the efficiencies of the primer set for the target and the reference genes, respectively, and Cqt and Cqr are the Cq values for the target and the reference, respectively. Et and Er values were calculated using LinRegPCR software49.Total RNA was extracted using a modified method described previouslySupplementary Figure S1.Supplementary Tables.


In spite of their biodegradability, the stability of both PGS and APS membranes was not deteriorated on ethanol/water pervaporation for one month. Furthermore, these two biodegradable membranes were applied in the pervaporation of simulated ABE (acetone-butanol-ethanol) fermentation solution, and the results were comparable with those reported in the literature.Biodegradable polymers are a green alternative to apply as the base membrane materials in versatile processes. In this study, two dense membranes were made from biodegradable PGS (poly(glycerol sebacate)) and APS ), respectively. The prepared membranes were characterized by FE-SEM, AFM, ATR-FTIR, TGA, DSC, water contact angle, and degree of swelling, in comparison with the PDMS (polydimethylpolysiloxane) membrane. In the pervaporation process for five organic solvent/water systems at 37 °C, both biodegradable membranes exhibited higher separation factors for ethanol/water and acetic acid/water separations, while the PDMS membrane attained better effectiveness in the other three systems. In particular, a positive relationship between the separation factor and the swelling ratio of organic solvent to water (DS Membrane separation has been a persuasive technology utilized broadly in industrial separation processes. Amid various membrane applications, pervaporation is a popular membrane approach commonly employed as an alternative to conventional separation processes, such as distillation, extraction, adsorption, etc. The principle of pervaporation, combining permeation and evaporation, is the separation of liquid solvents by partial vaporization through a nonporous or porous membrane, whereafter the vapor permeating through the membrane is removed by vacuum or sweeping inert gas in the permeate side ,2,3. ItsNonporous membranes or asymmetric membranes with dense active layers are preferentially utilized in pervaporation process. Molecular transport across the dense pervaporation membrane is usually governed by solution-diffusion mechanism ,7,17,19.Although good polymeric pervaporation membranes have a long-term stability and could be reused many times, the eventually exhausted membranes, if not degradable, will become the secondary pollutants because the combustion of these solid membrane wastes will emit plenty of gas contaminants or their disposal will produce environmentally polluted landfills. Since lots of polymers are resistant to degradation, causing a very critical issue in universal waste management, biodegradable polymers, thus, become the preferred candidate for the base material of membrane. Biodegradable substances can undergo deterioration and completely degrade when exposed to microorganisms in aerobic/anaerobic processes; such a degradation process will ultimately leave environmentally friendly byproducts. They are expected to become a strong competitor to conventional plastics. In this study, two kinds of dense membranes made from biodegradable poly(glycerol sebacate) (PGS) and poly (APS)  ,45 were ® 184 silicone elastomer kit) was purchased from Dow Corning . All the solvents used in this study, including ethanol (99.5%), isopropanol (99.5%), n-butanol (99.5%), acetone (95%), and acetic acid (99.8%), were supplied by ECHO Chemical  and Aencore . They were used as received.Commercially available PDMS , while the tapping-mode Atomic Force Microscope  with ScanAsyst was employed to evaluate the surface topography over a membrane area of 5 μm × 5 μm. The RMS (root mean square roughness) and Ra (arithmetic average roughness) parameters were analyzed with the software . Moreover, the functional groups of the prepared membranes were detected using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy .The thermal property of membrane was studied via a Thermogravimetric Analyzer  under an inert atmosphere from 110 °C to 800 °C at a heating rate of 10 °C/min. A sample of approximately 13–15 mg was used for the analysis. Moreover, to figure out the glass transition temperature (Tg) of the polymeric membrane, the measurement with a sample of 3–5 mg was carried out through a Differential Scanning Calorimeter  under the inert atmosphere from –80 °C to 270 °C at a scanning rate of 20 °C/min.The water contact angle measurement was performed by using the instrument FTA-125  to determine the surface hydrophilicity and water wettability of membrane.s and Wd are the weight of the solvent-swollen membrane and that of dry membrane, respectively.A dry membrane sample was weighed first, and later immersed in a specific solvent at 37 °C for 8 h. The membrane sample was taken out and weighed immediately after carefully wiping the liquid remained on the surface. The degree of swelling (DS) of the membrane in the solvent was defined by the following equation:2). The permeate was vacuumed (below 2 mmHg) and collected in cold traps (immersed in liquid nitrogen). After reaching steady state, the compositions of the aqueous solutions in the feed tank and the permeate were analyzed by GC equipped with a flame ionization detector (FID). The oven temperature was programmed from 100 °C to 150 °C at a rate of 20 °C/min. Both injector and detector temperatures were set at 225 °C.The schematic diagram of the pervaporation process in this study is illustrated in 2), and t is the duration time of the experiment (h). Considering that the permeation flux is usually affected by membrane thickness, a normalized flux JN was expressed in terms of a certain membrane thickness LN for normalization. The equation is as follows:N = 100 μm adopted in this study. The separation factor α was determined as:The pervaporation performance is commonly examined in connection with the total permeation flux and separation factor. In this study, the total flux J was calculated by the following equation:ex (PSI) :(5)PSI=JAll the prepared membranes had a thickness of ca. 200 μm (degree of error <3%). The surface morphology and topography of each polymeric membrane were analyzed using FE-SEM and AFM, and the images of PGS and APS membranes are presented in −1 and 1740 cm−1 [−1. Based on their general chemical structures [The ATR-FTIR spectra of PGS and APS membranes are plotted in 740 cm−1 , while t740 cm−1 ) displayructures ,51, the ructures ,47,52,53The DSC results of PGS and APS membranes are displayed in The water contact angle was measured for the as-prepared polymeric membranes. The results show that APS (85°) was more hydrophilic than PGS (94°), which should be attributed to the extra NH groups on the APS backbone. Moreover, the surface patterning and a little bit higher roughness of PGS membrane may have raised its hydrophobicity. On the other hand, these two biodegradable membranes had smaller water contact angle values than the PDMS membrane (104°).The solvent swelling effects at 37 °C for the two biodegradable membranes, along with the PDMS membrane, were investigated in this study. The results are shown in The DS values of APS for the three alcohols and water were slightly higher than those of PGS; on the contrary, the APS data for acetone and acetic acid were evidently smaller than the PGS results. These phenomena may be clarified in consideration of the hydrophobicity of organic solvent. Based on the Hansen solubility parameter (δ) ,56, the Furthermore, all the DS data of PDMS membrane were greatly lower than the two biodegradable membranes. The low-degree swelling behaviors for water (<2%) and n-butanol (<30%) at 40 °C had also been displayed in the literature  using PDorganic solvent/DSwater was evaluated, and the data are listed in o/DSw ratios than PGS in all the five organic solvent/water pairs. The APS data on acetone/water and acetic acid/water systems were noticeably smaller owing to its less hydrophobicity and poorer affinities with acetone and acetic acid, as mentioned previously. In addition, these two biodegradable membranes exhibited larger DSo/DSw values than PDMS for ethanol/water and acetic acid/water systems but lower results in the other three pairs. It may be worthy to indicate that ethanol (4.3 Å) and acetic acid (4.4 Å) have smaller kinetic diameters than the other three organic solvents  [Consider that, when the feed mixture is loaded and touches the frontal membrane surface, both solvents in the mixture will compete with each other to sorb into the membrane. The ratio of their swelling degrees should be a more important index accounting for pervaporation performance than the individual values of swelling degree. Hence, the ratio of DS6–4.7 Å) ,34. The 6–4.7 Å) ,34) but N in terms of 100 μm was also presented, for the later comparison with the literature data.The pervaporation process of organic solvent/water mixture was conducted at 37 °C using the membrane module with one piece of membrane disc. The results of the total permeation flux J and separation factor α are listed in From the data in o/DSw ratio in o/DSw, the higher the separation factor. The DSo/DSw value may be interpreted as the ratio of organic solvent affinity with the polymeric membrane to water affinity with the polymeric membrane. An increase in DSo/DSw corresponds to a higher membrane solubility for organic solvent in comparison with water, and henceforth, helps raising the concentration of organic solvent in the permeate. The separation factor is consequently improved. For all the five organic solvent/water systems, the PGS membrane presented better separation factor results than APS since it had higher DSo/DSw ratios, which resulted from its larger hydrophobicity and good affinities with organic solvents. In all the cases, the PGS membrane exhibited the superior performance on both permeation flux and separation factor than APS. On the other hand, in comparison with the PDMS membrane, these two biodegradable membranes showed higher effectiveness for the separation of ethanol or acetic acid from water, both of which were the organic solvents with smaller kinetic diameters.In Furthermore, in the three alcohol/water systems, with the increasing feed alcohol wt%, the total permeation flux was enhanced, but the separation factor was decreased for all the membranes. The increase in total permeation flux could be explained by the much larger quantity of sorbed alcohol in membrane at a higher feed concentration and the promoted diffusion, as well as the less significant change in water flux ,48. ThisAs an overall index of separation efficiency, the PSI value of each membrane was calculated by combining the normalized total flux and separation factor together (Equation (5)). The PSI data are also displayed in From the above analyses, the biodegradable PGS and APS membranes are very promising materials for achieving beneficial pervaporation performance, especially in the separation of ethanol/water and acetic acid/water mixtures. Although biodegradability is an attractive feature of these two membrane materials, it might cause a short lifetime and worsen the membrane reusability, eventually limiting their feasibility for practical applications. The PGS and APS membranes, synthesized mainly from glycerol and sebacic acid, have been known to degrade via hydrolysis and enzymatic degradation ,75. TheiN than both PDMS/PAN/silicatite-1 and HTPB-based PUU membranes. The selectivities towards ethanol and acetic acid for these two biodegradable membranes were better than those of the PDMS/PAN/silicatite-1 membrane, while their separation factors for acetone and n-butanol were superior to the HTPB-based PUU membrane. These phenomena were in good agreement with the previous results in To further explore the practical usage of PGS and APS membranes, a popular process of solvent recovery by pervaporation under the ABE fermentation  was simuo/DSw ratio and separation factor was observed, which may be used as an indicator for the assessment of pervaporation performance. Although biodegradability is an attractive feature for both PGS and APS membranes, their reusability was not deteriorated via a long-term stability test on ethanol/water pervaporation for a period of one month. Furthermore, the pervaporation for the simulated ABE fermentation solution using the two biodegradable polymeric membranes resulted in greater normalized flux and comparable solvent recoveries with the PDMS-based mixed matrix membrane and the HTPB-based PUU membrane in the literature. These positive results expand the feasibility of PGS and APS membranes for practical pervaporation applications.In this study, two biodegradable polymeric membranes PGS and APS were successfully applied in the pervaporation of five organic solvent/water systems. In all the cases, the PGS membrane exhibited higher permeation flux and larger separation factor than APS. In comparison with the popular PDMS membrane, the two biodegradable membranes showed more impressive effectiveness for ethanol/water and acetic acid/water separations. Moreover, a positive relation between the DS