Although cough was more frequent in 46 patients with normal pleural elastance (p < 0.0001), it was associated with significantly higher ∆P in patients with elevated elastance . Cough during TT is associated with small but beneficial trend in Ppl changes, particularly in patients with elevated pleural elastance, and should not be considered solely as an adverse event.Cough during therapeutic thoracentesis (TT) is considered an adverse effect. The study was aimed to evaluate the relationship between cough during TT and pleural pressure (Ppl) changes (∆P). Instantaneous Ppl was measured after withdrawal of predetermined volumes of pleural fluid. Fluid withdrawal (FW) and Ppl measurement (PplM) periods were analyzed separately using the two sample Kolmogorov–Smirnov test and the nonparametric skew to assess differences between ∆P distributions in periods with and without cough. The study involved 59 patients, median age 66 years, median withdrawn fluid volume 1800 mL (1330 ÷ 2400 mL). In total, 1265 cough episodes were recorded in 52 patients, in 24% of FW and 19% of PplM periods, respectively. Cough was associated with significant changes in ∆P distribution ( Although, in general, therapeutic thoracentesis (TT) is thought to be a safe procedure, the withdrawal of a large pleural fluid volume can be associated with some complications, including chest discomfort, pain, pneumothorax and re-expansion pulmonary edema; some of these side effects are at least partially related to pleural pressure (Ppl) fall caused by fluid withdrawal6. In patients with normal pleuro-pulmonary mechanics, a gentle slope of the withdrawn pleural fluid volume−Ppl curve reflects the replacement of the fluid by the expanding lung. However, if lung expandability is limited by, for example, visceral pleural thickening, lung scars, fibrosis, or airway collapse, even a small amount of withdrawn pleural fluid may result in significant Ppl decline, which in turn may produce symptoms such as vague chest discomfort or cough7. Although significant chest discomfort is believed to be an indication for TT termination because it may suggest a potentially unsafe decline of Ppl8, the significance of cough seems to be controversial. Jones et al. recorded cough in less than 1% of patients undergoing ultrasound guided TT and it was not related to post procedure pneumothorax9; however these authors referred to other studies showing a significantly higher cough incidence (9–24%) and stated that cough in the late phase of TT should be regarded as an indication to stop the procedure. On the other hand, cough may be associated with lung re-expansion and thus, if not accompanied by other symptoms, it should not be considered as an indication for TT termination10.Pleural effusion affects approximately 1.5 million patients per year in the United States with the annual number of thoracenteses reported between 127,000 and 173,000pl monitoring during TT and provides new insight into processes occurring during pleural fluid withdrawal, including a better understanding of complications and symptoms reported by the patients11, although its routine use in clinical practice in daily life was recently put into question12. Some previous observations led to the intriguing conclusion that cough during TT may favorably impact Ppl allowing to avoid excessive Ppl decline10. Although that observation was based on three patients only, it suggested that cough during TT need not necessarily be considered as a predictor of forthcoming complications, but may also be viewed as a protective phenomenon during the procedure. In general, we hypothesized that: (a) cough is linked with subsequent Ppl increase or its less significant decrease, (b) cough can be related to increased recruitment rate of atelectatic lung regions. Verification of the first hypothesis was the main purpose of this study.Pleural manometry is a key tool to study different aspects of pleural pathophysiology in patients with pleural effusion. Access to pleural manometers, whether water or electronic, enables Ppl measurements caused by the presence of fibrin membranes and loculations (n = 2) and low quality records (n = 1). One patient was excluded due to unclear and uncertain cough markings in the study documentation. Thus, the records of 59 patients were included in the final analysis. Patients' characteristics are presented in Table Data of 63 patients who underwent TT with pleural manometry between January 2015 and January 2019 were reviewed. Records of 3 patients were excluded due to a questionable reliability of Ppl measurement periods  and in 222 of 927 analyzed fluid withdrawal periods . Characteristics of the changes in Ppl (∆P) distributions for PplM and FW, with and without cough, are presented in Table Cough during TT occurred in 52 patients. The total number of cough episodes was 1265 and the median and maximal number of episodes in individual patients were 11 and 104, respectively. Cough was found in 172 of 926 analyzed PplM and FW. Histograms of ∆P during PplM showed that cough was associated with an increase of the right tail of the ∆P distribution . The median number of coughs in normal and elevated Pel group was 22 and 1, respectively; ΔP was greater in patients with increased Pel, particularly during PplM with cough  an increase of Ppl due to lung re-expansion and (2) no significant changes if cough was not associated with additional lung re-expansion. It can be supposed that the withdrawal of the first several portions of pleural fluid in patients with large volume pleural effusion may not significantly reduce lung compression. Therefore cough in the initial phase of pleural fluid withdrawal may not generate significant Ppl changes until the space for lung re-expansion appears. Then, the increase in Ppl may be observed if cough helps to open the compressed alveoli and fill them with air. The above hypothesis seems to be confirmed by our results. We found that cough-associated increase in Ppl was more pronounced in measurements performed after withdrawal of 1 L of pleural fluid  applied during TTion Fig.  is also ues Fig. .Figure 29, cough is a symptom presented by 9–24% of patients undergoing TT (while 88% in our study), its mechanism remains unclear. It has been documented that the main receptors responsible for cough are present in the larynx, trachea and main bronchi19. The vagal afferents are also present in small bronchi and lung parenchyma (juxtapulmonary receptors), and the new ERS chronic cough guidelines mention potential existence of cough receptors also in the alveolar septa and parenchyma of the lungs20. However, there is no proof that their irritation results in cough, despite the fact that cough is one of the symptoms in patients with heart failure, pulmonary edema or altitude sickness. It is supposed that cough in those cases appears only when sputum moves to larger bronchi and irritates the cough receptors or when there is bronchial compression21. The presence of cough receptors in the pleura is also considered4. Some authors suggested that negative Ppl caused by fluid withdrawal stimulates cough receptors on the visceral pleura, particularly in patients with nonexpendable lung 22. The results of our study do not seem to support these opinions since cough was significantly less common in patients with elevated Pel. Importantly, although less common, cough in patients with high Pel resulted in higher increase in Ppl than in patients with normal Pel  sustainably elevated Pel, and (b) elevated Pel which can be overcome by additional maneuvers e.g. cough or CPAP.Although, according to the literatureel Table . Perhapsgh Table . Thus, c25. The results of our study do not support this opinion. This is because our study showed that in some patients a large fluid volume could have been withdrawn without significant Ppl fall despite episodes of cough which appeared even in the early phase of the procedure (data not published). Therefore, we agree with the opinion of Feller-Kopman et al. that TT should not be terminated solely because of cough5. Moreover, our findings may even support a hypothesis that cough can even be a beneficial factor preventing an excessive Ppl drop during TT in some cases. To confirm this view and to evaluate the clinical application of voluntary cough during thoracentesis, further, well-designed prospective studies are mandatory. It also seems necessary in the future to have a closer look to cough episodes characteristics in terms of Ppl increase; namely to check whether all types and episodes of cough can be construed as beneficial, especially cough attacks or severe excessive cough during the procedure.Some authors believe that cough is a criterion for TT termination as it is considered a sign of complete or near-complete drainagepl for each breath as a surrogate of Ppl at FRC, whereas this value depends on the Ppl at FRC and possible intrinsic positive end-expiratory pressure  and by the Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences (the IBBE PAS own research fund). The study protocol was approved by the Institutional Review Board of Medical University of Warsaw (KB 105/2012) and registered at ClinicalTrials.gov (NCT02192138) on 16/07/2014. The study conformed to the standards set by the Declaration of Helsinki. All medical procedures were performed in patients hospitalized in the Department of Internal Medicine, Pulmonary Diseases and Allergy, Medical University of Warsaw, and all patients signed an informed consent to participate in the study.Sixty three patients who underwent TT with pleural manometry between January 2015 and January 2019 were included in the analysis; consecutive subjects were enrolled to avoid selection bias. The inclusion criteria were as follows: (1) age 18–85 years; (2) symptomatic pleural effusion occupying at least 1/3 of the hemithorax (in posteroanterior chest radiograph); (3) symptoms severity (dyspnea) warranting TT; (4) no contraindication for TT; (5) signed informed consent for participation in the study. The exclusion criteria were: (1) poor performance status requiring maximal shortening of the procedure; (2) unstable hemodynamic or respiratory status unrelated to pleural effusion; (3) respiratory failure requiring mechanical ventilation.29. Pleural fluid was evacuated through a small-bore pleural catheter . Pleural pressure was measured with a digital pleural manometer  and recorded for one minute directly after catheter insertion and then during the procedure. There were interchanging periods of Ppl measurement (PplM) and periods of fluid withdrawal (FW) , and then after the withdrawal of each 100 mL (frequent Ppl measurement phase). The procedure was terminated when no more fluid could be aspirated, a significant pleural pressure decline was observed or chest pain occurred. Ppl was displayed on a monitor during the procedure and its instantaneous values were recorded on a portable computer for further analysis. Each cough episode, both during PplM and FW, was marked in the computer record at the corresponding place on the time vector and noted in the patient's individual study documentation ) should ideally be used for analysis. This is because at FRC  respiratory muscles are fully relaxed and Ppl depends entirely on the volume of pleural fluid and the relationship between the outward pull of the thoracic cavity and the inward elastic recoil of the lung. Since, however, the exact time points of expiration ends were impossible to determine without airflow measurement, the maximal value of Ppl (Pplmax) in a breathing cycle was assumed to reflect Ppl at FRC. The median values of Pplmax at the beginning (PplmaxB) and the end (PplmaxE) of PplM were used to quantitatively characterize Ppl changes during PplM and FW. Thus, ∆P equal to the difference between PplmaxE and PplmaxB (PplmaxE–PplmaxB) was considered as the index characterizing the Ppl change during the corresponding PplM, whereas ∆P equal to the difference between PplmaxB for the subsequent PplM and PplmaxE for the previous PplM was used to quantify the Ppl change during FW  the number of Pplmax values used in determination of the median should be as large as possible to avoid random errors, and thus the interval for analysis should be relatively long; (b) PplmaxB and PplmaxE should reflect the Pplmax values at the actual beginning and end of each PplM, and thus the intervals should be short and limited to extremes of the PplM.In each patient, all P FW Fig. . The firel), i.e. the ratio of Ppl fall during the whole procedure to the total volume of withdrawn fluid. The first group included patients with normal Pel (< 14.5cm H2O/L)30, while the second included those with elevated elastance (≥ 14.5cm H2O/L).Additionally, to examine whether a possible link between cough and ΔP depends on lung expandability, all patients were classified according to the total pleural elastance . Data were presented as median and quartiles. Since the majority of analyzed variables had non-normal distributions, non-parametric statistical tests were used. The differences between groups were tested with the Mann–Whitney U-test. To analyze the statistical significance of cough influence on the ΔP distribution shape, the two-sample Kolgomorow-Smirnow test was used. The nonparametric skew was used to determine the nature of this influence.


Bradyrhizobium japonicum  and Bacillus megaterium (newly isolated strains) as a single inoculant and co-inoculant during seed bio-priming to improve seed germination and initial seedling growth of two soybean cultivars. The treated seeds were subjected to germination test (GT), cold test (CT) and accelerated aging test (AAT). B. megaterium significantly improved all parameters in GT and CT; final germination, shoot length, root length, root dry weight, and seedling vigor index in AAT, as compared to control. In addition, co-inoculation significantly increased all parameters except shoot dry weight in GT; all parameters in CT; germination energy, shoot length, root length, and seedling vigor index in AAT, in comparison to the control. Moreover, Br. japonicum significantly improved the germination energy, shoot length, shoot dry weight, root dry weight, and seedling vigor index in GT; all parameters in CT; shoot length, root length, and seedling vigor index in AAT, compared with non-primed seeds. Thus, B. megaterium strains could be used in soybean bio-priming as a potential single inoculant and co-inoculant, following proper field evaluation.Bio-priming is a new technique of seed treatment that improves seed germination, vigor, crop growth and yield. The objective of this study was to evaluate the effectiveness of  Glycine max (L.) Merrill) is one of the most important leguminous crops, with the highest protein content—around 40%, and the second highest oil content—around 20% [Soybean  and identified based on 16S rDNA sequencing (quencing .B. megaterium strains are presented in Bacillus strains were able to produce indole-3-acetic acid (IAA) in medium with L-tryptophan (8.00–15.65 µg mL−1). Among the seven B. megaterium strains, five were able to solubilize phosphorus, seven were positive for phosphate mineralization and able to grow on N-free medium, and six strains were capable of producing siderophores. Similarly, Haque et al. [B. megaterium expressed production of IAA, solubilization of nutrients , production of siderophores, and other PGP activities. Moreover, Nascimento et al. [B. megaterium that participate in the biosynthesis of auxins and cytokinins, stress resistance, antagonistic activities, and other PGP traits.Multiple plant growth-promoting (PGP) properties of the e et al.  reportedo et al.  found geB. megaterium: B8, B12, B15 and B17 were selected due to the highest potential for plant growth promotion. Dual culture assay demonstrated that the chosen B. megaterium strains were compatible with each other. Furthermore, compatibility assay did not show any antagonistic actions among Br. japonicum and B. megaterium strains.Although the newly-isolated bacterial strains tested in this study generally had good PGP abilities, four strains of Seed germination, together with seed vigor, represents a key factor in determining crop yield . Seed quB. megaterium or co-inoculation and the control were noted in final germination, abnormal seedlings, and root length, while single treatments had a significantly higher effect compared to control on shoot dry weight. Bacillus treatment led to the highest increase in final germination (3.5%), root length (8.9%), shoot dry weight (18.9%), and seedling vigor index (12.5%) compared to the control. The highest increase in germination energy (3.4%), and shoot length (8.2%) in comparison to the control was recorded with Br. japonicum. However, Br. japonicum did not improve final germination and abnormal seedlings. The highest decrease in abnormal seedlings (−2.8%), and increase in root dry weight (70.2%) related to the control was observed after co-inoculation. Nevertheless, the mentioned treatments, that showed the highest and significant effect compared to the control, did not differ significantly in relation to certain individual treatments. Only Bradyrhizobium had significantly lower effect compared to Bacillus and co-inoculation on root length and seedling vigor index, and single treatments had significantly higher effect compared to co-inoculation on shoot dry weight  and decrease in abnormal seedlings (−4.8%) in comparison to the control was obtained from inoculation with Br. japonicum, while the highest increase in root length (43.7%) and root dry weight (49.1%) was observed after inoculation with B. megaterium. A combined treatment with Br. japonicum and B. megaterium led to the highest improvement in shoot length (35.1%), shoot dry weight (9.3%), and seedling vigor index (48.5%) compared to the control , root length (43.8%), root dry weight (17.2%), and seedling vigor index (32.4%) was obtained from inoculation with B. megaterium. Co-inoculation had the highest impact on germination energy (3.1%), and shoot length (19.8%) in comparison to the control . Stressful conditions, such as high or low temperatures and high humidity, can decrease germination and extend germination time, primarily due to cell membrane damage and electrolyte leakage. Low temperatures, especially during the first few days of the germination period, have a major effect on seed germination and initial growth, as shown later in soybean development and yield. According to Szczerba et al. , low temBradyrhizobium treatment in cv. Teona, due to its lower effect on the examined soybean parameters. Additionally, PCA showed that cultivars were separated from each other within the same group. Hence, it was confirmed that Bacillus and co-inoculation treatments had a better effect than Bradyrhizobium on both cultivars.Furthermore, the association between bacterial treatments and cultivars in optimal and adverse conditions is illustrated by the biplot of the principal component analysis (PCA) . In optiBradyrhizobium was clearly separated from Bacillus and co-inoculation, being the optimal treatment in cv. Teona.According to the PCA for bacterial treatments and cultivars under cold stress , bacteriBased on the PCA for bacterial treatments and cultivars under double stress , bacteriB. megaterium strains have the ability to grow at a temperature range 3–45 °C [Br. japonicum strains tolerate 15–20 °C and below well, although less frequently [B. megaterium as a single and/or co-inoculant compared to the sole application of Br. japonicum, especially under high temperature and humidity conditions, can be attributed to their ability to produce spores, grow and remain active under stress. Concurrently, survival and efficacy of bacterial strains, particularly on aged seeds, indicates the possibility for prolonged storage of bio-primed seeds.The results emphasized the role of inoculants in establishing soybean tolerance to stressful conditions such as low or high temperature and high humidity. However, the different response of cultivars to inoculation , especia 3–45 °C , whereasequently . HoweverAdditionally, correlation analysis confirmed positive effects of seed bio-priming treatments under both optimal and unfavorable conditions, with the largest impact under cold stress. Overall, a positive interrelationship was established between germination and other parameters, with an exception of abnormal seedlings . In optiBr. japonicum, in order to improve plant growth under adverse conditions. Namely, in a meta-analysis of studies from 1987 to 2018, Zeffa et al. [Bradyrhizobium and PGPR  improves plant development, increases nodulation, and facilitates nutritional limitations and potential stresses during plant growth. Moreover, co-inoculation of soybean with Br. japonicum and Bacillus strains, which were shown to have PGP activity, increased nodule number, total biomass, total nitrogen, and yield, under the conditions of low root zone temperatures [Bacillus strains tested in this study, produce IAA. It was found that PGPR use this hormone to interact with plants as part of their colonization strategy. Being the first contact between the bacteria and the seed, IAA penetrates the seed coat with water, promotes seed germination, enhances and regulates vegetative growth and development, affects biosynthesis of various metabolites, resistance to stressful conditions, photosynthesis, etc. [Bacillus strains improved biomass and stress tolerance of soybean plants, while modulating abscisic acid (ABA) and salicylic acid (SA) levels. Additionally, PGPR have the ability to solubilize/mineralize phosphorus, and produce siderophores, which was also confirmed for B. megaterium strains used this study. This study confirmed that single inoculation and co-inoculation of soybean with B. megaterium improved seed germination and initial seedling growth, probably due to the production of IAA and other PGP activities. Similarly, Bacillus velezensis increased germination, root length and root surface of soybean compared to control, while strain genome analysis revealed the presence of genes linked to root colonization and PGP ability [Br. japonicum and Bacillus amyloliquefaciens promoted early seedling growth and significantly improved nodulation, probably due to the production of high levels of auxin, gibberellins and salicylic acid by the Bacillus strains [The current study, as well as several other studies, reported the positive effects of using PGPR as co-inoculants of soybean with a et al.  summarizeratures . Cardareeratures . It initeratures . The beneratures . Diverseis, etc. . As a reis, etc. . For insis, etc.  found th ability . Single  strains . Br. japonicum (BJ) and B. megaterium (BM) strains from the collection of the Laboratory for Microbiological Research of the Institute of Field and Vegetable Crops, Novi Sad (IFVCNS), Serbia. Strains of Br. japonicum are commercially used as microbiological fertilizer in the production of soybean in Serbia. Strains of B. megaterium were isolated in 2015–2016 from soil samples collected from different locations in northern Serbia . Soil samples included the agricultural soil (rhizosphere and bulk soil), as well as non-agricultural soil, which differed in chemical and physical properties. Isolation was performed using serial dilution and streak plate methods on nutrient agar (NA). Bacillus strains were morphologically characterized and identified by 16S rDNA sequencing , while the amplification of 16S rDNA gene fragments of Bacillus strains was performed using primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) [Bradyrhizobium strains were cultured in yeast extract mannitol broth (YEMB) and incubated at 28 ± 2 °C and 120 rpm  for 72 h. Bacillus strains were grown in nutrient broth (NB) and incubated at 30 ± 2 °C and 120 rpm for 24 h. Each strain of Bradyrhizobium and Bacillus was grown individually, and then mixed in equal proportions to form the mixtures for further examination. A culture suspension of each mixture was adjusted to have a final concentration of 109 colony forming units per mL (CFUs/mL).This study used quencing . MorpholACGACTT) . AmplifiACGACTT) . BradyrhBacillus strain was inoculated in NB supplemented with 500 µg/mL of L-tryptophan  and incubated at 30 ± 2 °C for 24 h. The supernatant was mixed with Salkowski reagent (FeCl3-HClO4: 1 mL of 0.5 M ferric chloride solution in 49 mL of 35% perchloric acid) in a ratio of 1:2 (supernatant/reagent v/v). Development of a pink color after 20 min at room temperature indicated the production of IAA. Indole production was measured by spectrophotometric absorption  at 530 nm [Br. japonicum vs. B. megaterium, and/or B. megaterium strains with each other was tested by dual culture assay on yeast extract mannitol agar (YMA) and/or NA plates. All determinations were performed in triplicate  production, 1 mL of overnight grown culture of each t 530 nm . The phot 530 nm . The comiplicate .Glycine max L.), namely Atlas and Teona, developed at the Soybean Department, IFVCNS were used in the present study. Seeds were surface disinfected in 2% sodium hypochlorite , washed with sterile distilled water four times, and then dried back on sterile filter paper under aseptic conditions to their original weight. In addition, seeds were soaked in 150 mL of bacterial culture suspension per each test and treatment (4 × 100 = 400 seeds) for germination and accelerated aging tests, and 75 mL per treatment (4 × 50 = 200 seeds) for cold test. Three bacterial treatments were tested: Br. japonicum (BJ), B. megaterium (BM), and Br. japonicum + B. megaterium (BJ + BM), with the inoculum rate 2 × 109 CFUs/g. The ratio of the strains in their mixtures in single inoculation treatments, as well as the ratio of the Bradyrhizobium and Bacillus mixtures in the co-inoculation treatment, was 1:1 (75 mL: 75 mL per 150 mL and 37.5 mL: 37.5 mL per 75 mL). Bradyrhizobium mixture contained 6 strains of Br. japonicum: the amount of each strain in single and co-inoculation treatments was 25 mL and 12.5 mL, respectively, per 150 mL (germination and accelerated aging tests), and half less milliliters per 75 mL (cold test). Bacillus mixture contained 4 strains of B. megaterium: the amount of each strain in single and co-inoculation treatments was 37.5 mL and 18.75 mL, respectively, per 150 mL (germination and accelerated aging tests), and half less milliliters per 75 mL (cold test). Non-primed seeds were used as the control. Seed bio-priming was conducted using the aforementioned liquid bacterial suspensions at 25 °C for 5 h, under dark conditions. After priming, seeds were rinsed thoroughly with distilled water, and dried on sterile filter paper at room temperature for 72 h [Seeds of two cultivars of soybean  and high relative humidity (≈95%) for 72 h . The gerSamples consisted of 25 randomly selected soybean seeds per replicate to measure shoot and root length. Samples were germinated in rolled filter paper for eight days in the germination chamber. Afterwards, 10 normal seedlings per replication were randomly selected for further assessment. In the cold test, samples were first exposed to a low temperature (10 °C) for seven days and then transferred into a germination chamber for six days. In the accelerated aging test, seeds were exposed to a high temperature and humidity for 72 h and then placed into a rolled filter paper for eight days in the germination chamber at 25 °C. The length of the shoot and roots were measured using a ruler on the day of germination. The fresh shoot and root weight of 10 seedlings were determined using analytical balance on the day of seed germination. For determination of dry weight, samples were oven dried at 80 °C for 24 h. The seedling vigor index was determined according to Abdul-Baki and Anderson , using tp ≤ 0.05). The relationship between germination and other parameters was determined by Pearson’s correlation analysis. Principal component analysis (PCA) was performed to determine the effect of seed bio-priming treatments on the examined parameters of soybean cultivars. The data were statistically processed by STATISTICA 10 programme .Four treatments were arranged in a complete randomized design (CRD) for laboratory tests, with four replications. The obtained data were processed statistically, using analysis of variance (ANOVA), followed by mean separation according to Tukey’s HSD test (Bacillus and co-inoculation improved the majority of the investigated soybean parameters as compared to Bradyrhizobium. The greatest improvement was obtained for the shoot length, root length, and seedling vigor index in cold-treated seeds, followed by aged and normal seeds, as well as for shoot and root weight recorded in germination, cold and accelerated tests, respectively. Bio-priming treatments led to the highest increase in final germination in cold-treated seeds, whereas the effect was lower in normal and aged seeds. The present study represents the first experimental evidence of using newly-isolated, indigenous B. megaterium strains from soil, for seed bio-priming of soybean under different conditions of laboratory tests. This technique can be recommended for priming seeds prior to field planting as an environmentally friendly strategy to improve seed germination and initial seedling growth. Extensive field trials under different environmental conditions, using several seed lots of each soybean cultivar, will be necessary in order to establish the efficiency of these strains as potential bio-priming seed treatments for improving soybean productivity.Overall,


Food selectivity is among the most common problems for children with Autism Spectrum Disorder (ASD). The present study aims to validate the Brief Autism Mealtime Behavior Inventory (BAMBI) in an Italian population of children with ASD. BAMBI was translated and cross-culturally adapted following international guidelines, then we investigated internal consistency as measured by Cronbach’s alpha and test–retest reliability, as measured by the Intraclass Correlation Coefficient (ICC) in a sample of both children with ASD and with typical development (TD). A total of 131 children were recruited in a clinical and community sample. Internal consistency revealed significant data for both TD and ASD children, with a Cronbach’s Alpha of 0.86 and 0.71, respectively. Test–retest reliability showed excellent values for each item of the BAMBI (range 0.83–1.00). Furthermore, we investigated differences in gender and body max index; however, no significant differences were found among groups. In conclusion, the Italian version of the BAMBI showed good internal consistency and test–retest reliability and it can be used for clinical and research purposes. Autism Spectrum Disorder (ASD) is an early onset neurodevelopmental disorder primarily affecting two main areas: ‘social communication’, and ‘restricted, repetitive and/or sensory behaviors or interests’ . It affeFeeding and eating problems are common problems that affect persons with ASD across all ages and cognitive abilities. It is well known that children with typical development, especially at preschool age, show an attitude of preference or rejection toward some foods; in these cases, children are referred to as “picky eaters”. Scientific literature points out that this behavior declines around the age of 6 years, when children have more opportunities to eat outside the family context and are exposed to a greater variety of foods that promotes the extinction of dietary restrictions . In the Therefore, the assessment of feeding problems in ASD should be included in routine screenings, and it would be appropriate for health professionals to have a greater awareness of this topic . Food seThe need to have validated tools to measure a specific construct is well noted. Considering the lack of specific assessment tools on this topic, no measurement tools have been developed or adapted to measure the mealtime and feeding problems of individuals with ASD or other neurodevelopmental disorders in Italy; therefore, the present investigation aims to translate the BAMBI into Italian and to validate its psychometric properties in an Italian population of children with ASD. The study was conducted from June 2021 until September 2021. The present investigation was carried out by a research group of Italian rehabilitation and healthcare professionals within the Child Neuropsychiatry Unit of the Department of Human Neurosciences of Sapienza University of Rome, together with the collaboration of a non-profit organization R.O.M.A.—Rehabilitation and Outcome Measures Assessment. The research group has great experience with validation outcome measures in children ,20,21,22BAMBI  is an asFollowing the recommendation of the Beaton and colleagues , the traAn expert committee, comprising all translators and the research group, reviewed the translated versions, and discussed discrepancies and difficulties during the process. This led towards a pre-final version of the BAMBI that was administered to the first five participants as a pre-test. There were interviewed and answered questions concerning the wording and comprehensibility of the tool. Consensus was achieved and the final Italian version of the BAMBI was produced see .We recruited children aged between 6 and 10 years, with parent who demonstrated good ability to communicate in Italian. For the objective of the present study, we opted to include a convenience sample of both children with typical development and children with ASD. Children with a certified diagnoses of ASD were recruited in the outpatient clinic of the Institute of Child Neuropsychiatry of the Policlinico Umberto I University Hospital in Rome, while children with typical development were recruited in a primary school nearby the aforementioned institute. Participants with other neurodevelopmental disorders and genetic syndromes  were excluded. Before starting the study, the research group participated in an internal training course to increase confidence in administering BAMBI. A speech therapist with significant experience using the assessment tools led this training. The recruitment period lasted four months, from June to September 2021. Before administering the BAMBI, parents were asked about the socio-demographic information of the family  and information on the child, in particular age, weight, height, and the presence of gastric disorders or eating problems. Data for children with ASD were obtained from clinical records and parents’ interviews, while for children with typical development, information was collected by two speech and language therapists . Once inclusion and exclusion criteria were verified, the research team administered the BAMBI. Data were summarized and analyzed using frequency tables, means, and standard deviations. We used Cronbach’s Alpha to measure and investigate internal consistency. Cronbach’s Alpha expresses the correlations between different items on the same tool; a coefficient greater than 0.7 indicates acceptable internal consistency ,29. AnotWe recruited 47 individuals with ASD and 90 individuals with typical development. Of these, six were excluded due to lack of consent to data processing. Therefore, the final sample was composed of 84 children with typical development with a mean age of 6.73 (SD 2.5) and 47 children with a diagnosis of ASD with a mean age of 7.45 (SD 1.8). Sample characteristics are summarized in Internal consistency analysis showed statistical significance for both children with typical development and children with ASD, with a Cronbach’s Alpha of 0.86 and 0.71, respectively. To better understand differences and how items work in both groups, we reported the data of Items—total statistics. The findings are summarized in For test–retest reliability, we reported a descriptive analysis of mean (SD) scores of the two repeated measurements and ICC values with 95% CI, resulting in a range of 0.83–1.00. We also investigate differences in mean scores among gender and BMI. From the analysis, we can assert that there are no differences in gender and correlation between BMI and total BAMBI score. Data are reported in The present study aims to preliminarily validate the Italian version of the BAMBI in a sample of children with both ASD and with typical development. Recently, the DSM-V included diagnostic criteria for Avoidant and Restrictive Food Intake Disorder (ARFID) , charactOne of the most frequently cited feeding problems for patients with ASD is the limited variety of food intake , which cThe BAMBI questionnaire also allows for ASD characteristics to be assessed through some questions . Children with ASD, compared with controls, are found to be more aggressive at mealtimes, have difficulty remaining seated until the meal is finished, and exhibit less flexibility in mealtime routines . FurtherThe BAMBI is a simple and quick-to-use Observer Reported Outcome, which is a measurement based on an observation by someone other than the patient or a health professional in general. The original version of the BAMBI was translated into Italian following international guidelines. Equivalence between the Italian version of the BAMBI with the original was investigated on a semantical domain. Only a few modifications were suggested; for example, Item 8 was literally translated using the Italian verb “chiudere” (close in English), but the research group opted to insert the synonym verb “serrare” because it indicated closing the mouth as a refusal behavior. Also, Item 18 was modified because the original version reported as an example “fried foods, cold cereals, raw vegetables”, while the research group opted to insert “pasta” because more appropriate for the Italian culture and eating habits of families; therefore, the examples of item 18 were modified as “cibi fritti, pasta fredda e verdure cruda” . These changes were also discussed with participants during the pre-test phase. Participants’ observation allowed us to gain cross-cultural validity and proved to be strictly related to the meaning of the original items.For internal consistency, as measured with Cronbach’s coefficient alpha, our findings showed significant values for both children with typical development (0.86) and children with ASD (0.71), in line with the Turkish version (α 0.79) , the VieDespite these encouraging results, several limitations can be acknowledged. First of all, we did not investigate differences in the ASD population according to severity of symptoms, while emerging evidence suggests an overlap between the severity of ASD symptoms and eating problems, like food selectivity . Second,Since food and, in general, mealtime is a convivial event with high social and cultural value, it would be interesting to assess the correlation that may exist between food selectivity and the development of communication and social skills of people with ASD, and how this characteristic is delineated in different cultures. Finally, it would be desirable to train rehabilitation professionals who can develop individualized programs and strategies that consider the specific difficulties of these children to reduce and extinguish food selectivity, thereby promoting the well-being of the affected individuals and the entire family unit.In conclusion, the BAMBI has proven to be a reliable, quick, and easy-to-use tool and can be used for clinical and research purposes. The flexibility and specificity of the scale identified in this study also allow it to be used in various clinical and research settings depending on the purpose and participants.


Macrophages are multifunctional immune system cells that are essential for the mechanical stimulation‐induced control of metabolism. Piezo1 is a non‐selective calcium channel expressed in multifarious tissues to convey mechanical signals. Here, a cellular model of tension was used to study the effect of mechanical stretch on the phenotypic transformation of macrophages and its mechanism. An indirect co‐culture system was used to explore the effect of macrophage activation on bone marrow mesenchymal stem cells (BMSCs), and a treadmill running model was used to validate the mechanism in vivo for in vitro studies. p53 was acetylated and deacetylated by macrophages as a result of mechanical strain being detected by Piezo1. This process is able to polarize macrophages towards M2 and secretes transforming growth factor‐beta (TGF‐β1), which subsequently stimulates BMSCs migration, proliferation and osteogenic differentiation. Knockdown of Piezo1 inhibits the conversion of macrophages to the reparative phenotype, thereby affecting bone remodelling. Blockade of TGF‐β I, II receptors and Piezo1 significantly reduced exercise‐increased bone mass in mice. In conclusion, we showed that mechanical tension causes calcium influx, p53 deacetylation, macrophage polarization towards M2 and TGF‐β1 release through Piezo1. These events support BMSC osteogenesis. Piezo1‐mediated mechanical tension stimulates macrophage polarization and secretion of transforming growth factor‐beta1, promoting osteogenic differentiation. Acetylation and deacetylation of p53 play a major role in this process. Piezo1 is found in a variety of tissues, including macrophages.Piezo1, a non‐selective CaPrevious research has shown that stretching can influence macrophage function and polarization towards M2. It has been demonstrated that 10% stretch intensity is optimal for activating M2‐type macrophages strongly boosting suture stem cells (SuSCs) osteogenic differentiation.2+, and controlled the acetylation or deacetylation of p53 in response to mechanical stress, which caused macrophages to switch from a pro‐inflammatory to an anti‐inflammatory phenotype. M2‐type macrophages produced TGF‐β1, which induced BMSC migration, proliferation and osteogenic differentiation. Running mice were given TGF‐β receptor inhibitors or Piezo1 inhibitors, which decreased the increase in bone mass brought on by exercise. In summary, we found that Piezo1‐mediated changes in calcium inward flow, as well as p53 acetylation and deacetylation, caused macrophages to polarize towards M2, secret TGF‐β1 and promote BMSC osteogenesis.Despite numerous studies showing the osteogenic effects of macrophages on mesenchymal stem cells (MSCs), there is no agreement on which macrophage phenotype is favourable for MSC under mechanical stretch. Here, we created a cellular model of tension to examine how mechanical stretch affected the phenotypic transformation of macrophages. We revealed that the optimal mechanical stretch exhibited strong immunomodulatory effects and encouraged M2 macrophage polarization, the latter of which was a key regulator driving bone marrow mesenchymal stem cells' (BMSCs) osteogenic differentiation in an indirect co‐culture system. Mechanistically, we identified Piezo1 ion channels as tensile stress sensors in macrophages. Piezo1 triggered inflammatory responses, activated Ca22.12O and then administered to mice as a 10 μL (5 μM) intraperitoneal injection.Male C57BL/6 wild‐type mice aged 6 weeks  were divided into five groups, each with five animals: the blank control group, the simple running group, the negative control group and the remaining two groups were administered drugs that were TGF‐β receptor I, II and Piezo1 inhibitor. Each mouse was raised in a separate cage to prevent fights that might mask the benefits of exercise. We used operating circumstances from other labs as references.2.22. After 3 days of incubation, the culture media was changed to remove non‐adherent cells. The adhering cells were washed with PBS when the confluence reached 80%–90%, and the medium was changed every 3 days while they were being grown. The cells were then cultured once more after being passaged at a ratio of 1:3. The cells from passage 3 were employed in the subsequent studies. The murine‐derived macrophage cell line RAW264.7 was purchased from the Cells Resource Center of Shanghai Institutes for Biological Sciences, the Chinese Academy of Science. It was then cultured in Dulbecco's modified Eagle's medium  containing 10% FBS , 100 IU/mL penicillin and100μg/ml streptomycin at 37°C in a humid atmosphere with 5% CO2.Male C57BL/6 wild‐type mice aged 3–4 weeks had their femurs and tibias thoroughly separated and washed with phosphate‐buffered saline (PBS). Following the removal of the bone marrow from these bones' cavities, cells were cultured in alpha minimal essential medium  with 10% foetal bovine serum  and 1% penicillin–streptomycin . The cells were kept alive at 37°C in a humidified incubator with 5% CO2.3To apply mechanical stimulation to macrophages, RAW264.7 cells were uniformly seeded into six‐well collagen I‐coated BioFlex culture plates with flexible silicon membrane bottoms . A Flexcell® FX‐5000™ Tension System  was used to apply cyclic sinusoidal continuous tensile strain for 0, 1, 2, 4 and 6 h . Cells were applied this optimal mechanical stretching  throughout the in vitro series. Cells were cultivated in the same plates but were not stretched as controls.2.4In accordance with the manufacturer's recommendations, RAW 264.7 cells were transiently transfected in six‐well plates with Piezo1 siRNA at a final concentration of 100 nM using Opti‐MEM (Gibco) supplemented with Lipofectamine 2000 Reagent (Invitrogen). We refer to the sequences of other subject groups2.5−ΔΔCt method. The messenger RNA (mRNA) expression levels were normalized with β‐actin. Table Using Trizol reagent , total RNA from RAW264.7 cells was extracted for quantitative real‐time PCR analysis. HiScript III Q RT SuperMix was used to reverse‐transcribe RNA (Vazyme). Next, qPCR was performed in ABI QuantStudio7 (Applied Biosystems) using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The relative gene expression was quantified using the 22.6Total proteins from macrophages were extracted using RIPA lysis buffer. The protein concentration of the cell lysates was determined using a BCA protein assay  and separated using 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) before being transferred to a polyvinylidene fluoride membrane. After blocking with 5% skimmed milk or BSA blocking solution in Tris‐buffered saline containing 0.05% Tween 20 (TBST), the membranes were incubated overnight with the following primary antibodies: anti‐Runx2, anti‐OSX, anti‐OPN, anti‐Col1, anti‐iNOS, anti‐Arg‐1, anti‐p53, anti‐ac‐p53, anti‐β‐actin and anti‐GAPDH. After primary antibody incubation, membranes were washed, incubated with a secondary anti‐rabbit or anti‐mouse antibody  for 1 h at room temperature and then visualized using ECL detection kits . The dilutions and suppliers of antibodies used in this study are listed in Table 2.7Flow cytometry was used to analyse the expression of the M1 marker CD86 and the M2 marker CD206 in order to describe the M1 and M2 macrophage phenotypes. 0.25% trypsin in ethylenediaminetetraacetic acid was used to separate the cells, and they were then thrice washed with PBS. The cells were incubated in PBS with APC‐F4/80, PE‐CD86 antibody and FITC‐CD206 antibody for 60 min at 4°C in the dark. The labelled cells were measured using a flow cytometer . The negative control cells were those with no additional antibodies.2.8Upon the cell density reached roughly 70%, we washed the cells three times in PBS before fixing them for 30 min in 4% paraformaldehyde. We used goat serum  to block the samples for 30 min at room temperature. After that, we chose the primary antibodies against F4/80, CD206, Ac‐p53 and Piezo1 for overnight incubation at 4°C. On the second day, the samples were washed in PBS three times. Subsequently, the species‐matched secondary antibodies  were applied, and the nucleus was stained with 4′,6‐diamidino‐2‐phenylindole as a counterstain . Cells were similarly fixed in 4% paraformaldehyde for 15 min and then soaked in 0.5% Triton‐X for 5 min prior to phalloidin staining. According to the manufacturer's instructions, DAPI staining and phalloidin staining  were carried out. For 5 or 30 min, cells were fixed in DAPI and phalloidin staining solution. The images were captured using an immunofluorescence microscope .2.9Cell migration was performed in 6.5 mm Transwell® with 8.0 μm Pore Polycarbonate MembraneInserts . The system's lower chamber contained 600 μL conditioned medium while the upper chamber contained 200 μL of BMSCs suspended in serum‐free medium. The transwell filter system containing the BMSCs was subsequently placed in a humidity incubator. After 12 h of incubation at 37°C, BMSCs were fixed with 4% paraformaldehyde, and the non‐traversed cells that remained on the filter's upper surface were carefully removed with a cotton swab. Migrated (traversed) cells on the lower side of the filter were stained with crystal violet for 30 min, and then counted in five randomly selected fields under an inverted microscope .2.102. The plate was subsequently processed with Cell Counting Kit 8 (CCK8) solution  for specified durations  at 37°C. Optical density was measured at 450 nm to calculate the percentage of cell viability on a microplate reader .For the CCK8 proliferation assay, BMSCs (3000 cells per well) were seeded in 96‐well plates and incubated at 37°C with 5% CO2.114 BMSCs was evaluated using 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation assay reagent  in a 96‐well plate. Simply put, the cells were exposed to the EdU reagent for 4 h once cell growth in the plate reached 80% confluence. Cells were fixed, permeabilized and dyed with 4′,6‐diamidino‐2‐phenylindole (DAPI) solution before being observed under a fluorescent microscope (Leica).The proliferation of 5 × 102.126 cells/well (in six‐well plates), and a scratch was formed on the cell monolayer 6 h later. The cells were placed in a humidified incubator with 5% CO2 at 37°C after being washed three times with serum‐free media. They were then monitored for a further 24 h. New scratch width/original scratch width was used as the unit of measurement for cell migration.For the scratch wound‐healing assay, cells were seeded at a density of 1 × 102.13The cells were PBS‐washed before being fixed in 4% paraformaldehyde at room temperature for 30 min. Following that, the samples were stained with the BCIP/NBT Alkaline Phosphatase Color Development Kit  according to the manufacturer's instructions. The images were captured with a scanner (GE Image Scanner III). The staining was simultaneously viewed under a microscope. We measured the alp enzyme in three randomly chosen fields using Image‐J software.2.14BMSCs were cultured in plates with different conditional mediums for 21 days. Cells were rinsed three times in PBS after a 21‐day osteogenic induction, and then paraformaldehyde was used to fix the cells for 30 min at room temperature. The samples were then washed and stained at room temperature with Alizarin Red S . The images were captured with a scanner (GE Image Scanner III). The staining was simultaneously viewed under a microscope, and after that, Hexadecylpyridinium chloride monohydrate  was employed for quantification.2.152+ fluorescence images were captured with a fluorescent microscope (Leica).RAW264.7 cells were cultured in BioFlex culture plates after Piezo1 silence or Yoda1 injection. Cells were then loaded with Fluo‐3 AM  and simultaneously treated to mechanical stretch  for 0–6 h. The macrophages were washed with PBS three times after mechanical stretch. Ca2.16TGF‐β1 expression levels were quantified for inflammatory cytokine measurement. The supernatant was utilized to identify macrophages after they had been stretched. ELISA was performed as directed by the manufacturer's instructions .2.17Tibia bones were extracted, fixed in 4% paraformaldehyde solution and scanned with a high‐resolution micro‐ct  at a resolution of 15.6 μm. Three‐dimensional images were reconstructed for analysis , and cross‐sectional images of the distal tibia were employed. Based on the selection of the volume of interest (VOI), which was generated in accordance with the previous report,2.18After being fixed in paraformaldehyde overnight, the samples were decalcified in 14% EDTA and embedded in paraffin. The tibias were cut into 4‐μm‐thick sections. The slices were stained with haematoxylin and eosin (H&E). The trabecular structure and osteocyte lacunae were examined to assess bone alterations. Femurs were first deparaffinized and heated to facilitate antigen retrieval for immunohistochemical tests. Goat serum was used to block tissue slices for 30 min at room temperature. TGF‐β1 and F4/80 primary antibodies were added and incubated overnight at 4°C.The sections were then treated with fluorescein Cy3‐conjugated secondary anti‐body (Beyotime) and fluorescein CoraLite488‐conjugated secondary antibody (Proteintech) on the second day. Nuclei were stained with DAPI. A fluorescent microscope was used to obtain the images (Carl Zeiss).2.19p‐Values were calculated using a two‐way analysis of variance (ANOVA) with Bonferroni's correction or multiple t‐tests. A p‐value of <0.05 was judged statistically significant.To limit experimental error, all tests were repeated three times. The data were recorded as mean ± SD. Graphpad Prism 8.0  was used for statistical analysis. 33.1+ cells in RAW264.7 cells, further demonstrating that stress alters the phenotype of macrophages , or TGF‐β receptor I, II inhibitor (LY‐364947) were injected. Running significantly increased bone mass in mice, but we could see that mechanical force transmission was also stopped when Piezo1 was inhibited, and bone mass in the GsMTx‐4‐injected group was almost the same as in the control group . TGF‐β1 released by macrophages failed to play a function after TGF‐β receptor I, II was blocked, resulting in severe bone repair in the tibia of mice , we found that the p53 axis is important in the mechanical force‐dependent Piezo1 that leads to macrophage polarization.+F4/80+ macrophages sense mechanical signals via Piezo1 and secrete TGF‐β, which encourages the production of periosteal bone. Recent research has also demonstrated the advantages of exercise on M2 macrophage polarization and energy homeostasis.2+ inward influx mediated by macrophage‐sensitive ion channels.Bone consists of cortical bone and bone trabeculae.Although the current work confirmed that Piezo1 mediates mechanical signalling in macrophages in vitro and in vivo, numerous issues remain. Actually, detected TGF‐β1 may also originate from MSCs.5In conclusion, we found that mechanical forces mediated changes in calcium inward flow, p53 acetylation and deacetylation through Piezo1, inducing macrophages to polarize towards M2, secreting TGF‐β1 and promoting BMSCs osteogenesis Figure . This deConceived and designed the experiments: Hua Wang and Wei‐Bing Zhang. Performed the experiments: Guanhui Cai, Yahui Lu, Weijie Zhong, Ting Wang, Yingyi Li and Xiaolei Ruan. Analysed the data: Hongyu Chen, Lian Sun, Zhaolan Guan, Gen Li, Hengwei Zhang, Wen Sun and Minglong Chen. Wrote the paper: Guanhui Cai and Yahui Lu.The authors declare no conflict of interest.Figure S1. Morphology of macrophages before and after mechanical stretching. (A) Phalloidin staining (Red) of RAW264.7 cells under the tension of 0–6 h. Blue indicates DAPI staining of nuclei. Scale bar, 10 μm.Click here for additional data file.Figure S2. Effect of different concentrations of conditioned medium on osteogenesis of BMSCs. (A) After 7 days, bone marrow mesenchymal stem cells treated with conditioned mediums of macrophages under 2 h of tension were induced into osteogenesis in different concentrations of osteogenic differentiation medium. ALP expression in BMSCs was measured by the ALP staining method. The top are gross scanning images , and the lower are enlarged images . (B) Cell Counting Kit 8 (CCK8) was performed to explore BMSCs proliferation following treatment with different concentrations of conditioned medium.Click here for additional data file.Figure S3. Expression changes of Piezo1 and calcium influx under mechanical tension. (A) The intensity of Ca2+ fluorescence marked by Fluo‐3 AM probe under fluorescence microscope after 0–6 h of tension. (B) Immunofluorescent staining of Piezo1 (red) in RAW264.7 cells under the tension of 0–6 h. Blue indicates DAPI staining of nuclei. Three random fields from each time period of the slides of cells were examined. Scale bar, 10 μm. The Piezo1+ area were qualified as area values of overlapping fields. (C) mRNA expression of Piezo1 in RAW264.7 cells after tension.Click here for additional data file.Figure S4. Drug stimulation of Yoda1. Piezo1 in RAW264.7 cells with different concentrations of Yoda1‐administration were measured by real‐time RT‐PCR. GAPDH was used for normalization. Data are presented as three biological replicates from three independent experiments.Click here for additional data file.Table S1. List of antibodies used in this research, along with their dilutions and suppliers.Table S2. List of primers used for this study.Click here for additional data file.


In audio transduction applications, virtualization can be defined as the task of digitally altering the acoustic behavior of an audio sensor or actuator with the aim of mimicking that of a target transducer. Recently, a digital signal preprocessing method for the virtualization of loudspeakers based on inverse equivalent circuit modeling has been proposed. The method applies Leuciuc’s inversion theorem to obtain the inverse circuital model of the physical actuator, which is then exploited to impose a target behavior through the so called Direct–Inverse–Direct Chain. The inverse model is designed by properly augmenting the direct model with a theoretical two-port circuit element called nullor. Drawing on this promising results, in this manuscript, we aim at describing the virtualization task in a broader sense, including both actuator and sensor virtualizations. We provide ready-to-use schemes and block diagrams which apply to all the possible combinations of input and output variables. We then analyze and formalize different versions of the Direct–Inverse–Direct Chain describing how the method changes when applied to sensors and actuators. Finally, we provide examples of applications considering the virtualization of a capacitive microphone and a nonlinear compression driver. The transduction process that characterizes such devices involves different physical domains , which not only are affected by different nonlinear behaviors but they do interact in a nonlinear fashion. For instance, piezoelectric loudspeakers are impaired by hysteretic phenomena which do increase the Total Harmonic Distortion (THD) incident matrices ,17, it hcompendium for the design of inverse circuital models of audio transducers in different vitualization scenarios. In fact, we describe both the case of actuator virtualization and of sensor virtualization, making appropriate adjustments to the employed Direct–Inverse–Direct Chain [In this paper, we discuss the task of audio transducer virtualization from a general theoretical perspective, by analyzing different scenarios and combinations of input/output signals. Our aim is to provide a ct Chain ,26. In dct Chain ,27. FinaThe manuscript is organized as follows. In this section, we first provide background knowledge on nullors, and, then, we present the four major classes of inversion scenarios, supplementing the overview that is available in the literature which only comprises two cases out of four .nullator, which has both port voltage and port current equal to zero, and norator, which is characterized by unconstrained port variables [Nullors are theoretical two-port elements composed of other two theoretical one-ports: a ariables . Figure rs, etc. ,29,30.Nullor is the fundamental element for carrying out the inversion of circuits according to Leuciuc’s theorem ,18,19. AIn the following subsections, we will reword the original theorem considering the four possibile combinations of input and output signals of the system to be inverted, providing proofs and a complete overview over the method.Direct System, are Inverse System, are Direct System, we can write down the following system of equationsx of the network shown in Before presenting the nullor-based inversion theorem, let us consider the two linear-time-invariant (LTI) non-autonomous circuits shown in quationsv1=z11i1+x of the network shown in In Equation , v^x andHereafter, we first introduce a reworded version of Leuciuc’s theorem which also generalizes the above approach for the inversion of linear circuits to the case of nonlinear circuits, and then we go through possible inversion scenarios characterized by pairs of input/output variables of different kinds.Theorem 1.Let us consider a nonlinear non-autonomous circuit containing at least one nullor, as the one shown in Proof. v as the output, we can describe such a system according to the state-space formalism as followsU and u and t. If we re-introduce the nullator, we constrain voltage v to be zero leading the system into an unphysical condition. In order to avoid such an unphysical state, one of the two sources u and u on the entire state space, we can write y is a bounded solution for Equation  driven by the output voltage of the Direct System. The inverse of Network A is Network B of Let us consider Network A of  theorem , and lat theorem ,17 for dDirect System  driven by the output current of the Direct System. The inverse of Network C is Network D of Let us consider Network C of uciuc in . Let us Direct System, this can be augmented with the parallel connection of a nullator and a norator. In fact, this adjunct does not modify the behavior of the circuit given that a nullator and a norator in parallel are equivalent to a short circuit [Inverse System will be similar to the one shown in In the case in which no nullors are present into the  circuit . Such a  circuit . The resDirect System features as input a current signal and as output a voltage signal, namely, Direct System. For this particular case, the Inverse System is obtained by replacing the input current source of the Direct System with the norator and the norator with a VCVS driven by the output voltage of the Direct System. The inverse of Network E is Network F of Let us consider Network E of Direct System, this can be augmented with the series connection of a nullator and a norator. As for the VIVO case, such a series connection must be inserted between the very same nodes where the output voltage is taken. The result will be a circuit similar to the one shown in In the case in which no nullors are present into the Direct System features both as input and output a current signal, namely, Direct System. For this particular case, the Inverse System is obtained by replacing the input current source of the Direct System with the norator and the norator with a CCCS driven by the output current of the Direct System. The inverse of Network G is Network H of Let us consider Network G of Direct System ∑n=1∑n=1adjoiPassive elements are kept without any changes.Nullators are replaced with norators, while norators with nullators.The input voltage is replaced with a short circuit . The output of the adjoint circuit will be then the current flowing through such a short, where the positive direction follows the element convention, i.e., from the positive to the negative terminal.A current source is connected to the output port. This will be the input of the adjoint circuit. In this case, the direction of the current follows the source convention, i.e., from the negative to the positive terminal.Controlled sources are replaced with their dual .The procedure for deriving the adjoint of a given circuit, whose input is a voltage signal, can be summarized as follows:Similar considerations can be drawn for current inputs. The interested reader is referred to ,33 for aDirect System or directly on the Inverse System. For example, Inverse System of Direct System shown in Direct System and applying then the rules presented in the previous subsections for deriving the Inverse System.The adjoint transformation can be carried out either on the In the next section, we will present the inversion-based virtualization algorithm providing details on how to apply it for both the cases of actuator and sensor virtualization.Direct–Inverse–Direct Chain (DIDC) and was proposed in [Direct Systems, and one Inverse System. The Inverse System is always implemented in the digital domain, whereas, according to the considered actuator or sensor application, only the first or the last Direct System is implemented in the digital domain since the other is the actual physical transducer. Moreover, in real scenarios, amplifiers could be present in-between blocks. Hence, gains should be considered at different stages of the processing chain for the algorithm to properly work.We now present a general block chain to perform virtualization of transducers. Such a chain, shown in posed in  for addrInverse System and the Physical Direct System is equivalent to the identity. This means that the digital processing chain allows us to somehow cancel out the behavior of the transducer such that the target behavior, i.e., the behavior of the digital Direct System, can be imposed. Hence, the proposed processing chain can be employed to accomplish the task of transducer virtualization .The DIDC working principle is based on the assumption that the cascade of the In the following two subsections, we will present application-specific DIDCs targeting both the cases of actuator and sensor virtualization.Target Direct System is the digital implementation of the actuator circuital model which we would like to obtain, whereas the Inverse System is the inverse circuital model of the Physical Direct System, which is the transducer itself. Hence, given that we are considering actuation, we may call this chain Target-Inverse-Physical Chain (TIPC), since the target behavior must be imposed in a pre-processing phase and thus before driving the actuator, i.e., the Physical Direct System.Let us consider the particular DIDC shown in Target Direct System) can be a linearized or equalized version of A, or a different speaker. It follows that the more accurate the considered electrical models, the higher the performance of the algorithm. Nonetheless, in [For the case of loudspeaker virtualization, the input ectively ,16,17,27Direct System, and the derivative of the displacement in the Inverse System.It is worth adding that other variables aside currents and voltages could be taken into account to obtain the inverse of a given circuit. For example, for the case of loudspeakers, it might be convenient to consider the displacement of the diaphragm  as the output variable. In this case, another stage should be inserted into the processing chain for performing the integral of the velocity  in the Target Direct System). In this case, the purpose of virtualization is to improve the performance of the transducers by reducing the Total Harmonic Distortion (THD) imposing somehow the acoustic behavior of an ideal version of the transducer under consideration. Such a discussion is also valid for the specific DIDC that we introduce in the next subsection.Finally, note that according to the type of virtualization, the blocks could be either linear or nonlinear. For example, all the three blocks could be nonlinear if the chain is exploited for imposing the nonlinear sonic behavior of a target transducer. Instead, if linearization is envisaged, one out of the three blocks will be linear , since first the audio signal is acquired by means of the sensor, and then, after compensating for the physical behavior by means of the Inverse System, the signal is processed to impose the target acoustic response. For the case of microphone virtualization, the input signal Let us now consider the DIDC shown in Once again, input and output variable different from voltages and currents  can be considered by introducing integrators and derivators into the green blocks of the processing chain.In this section, we provide an example of sensor virtualization by taking into account a capacitive microphone as a case study. For this application, we employ a linear model, while an example of transducer virtualization based on nonlinear models will be provided in the next section. The microphone is described by means of the circuit shown in The two transformers model the transduction between the physical domains, where The implementation of the microphone circuital model in the digital domain can be carried out by employing different techniques ,36,37. IIn order to test the accuracy of the WD implementation, we compare the Discrete Fourier Transform (DFT) of the impulse response obtained by simulating the reference circuit in the WD domain with the DFT of the impulse response obtained by simulating the same circuit in Mathworks Simscape (SSC), for both BK4134 and BK4146 microphones. The curves are then normalized with respect tot the pressure at 1 kHz as it is typically done to describe the microphone sensitivity. The results are shown in Direct System. By applying the theorem presented in Inverse System circuit shown in Inverse System, once augmented the Direct System with a parallel connection of a nullator and a norator as explained in Inverse System can be implemented in the WD domain in a fully explicit fashion. In order to validate the Inverse System implementation, we consider the processing chain in Direct System and the Inverse System, and we verify that the output of the cascade is equal to the input of the same cascade, i.e., Direct System to be an impulse and we compute the response of the microphone, which is shown in Inverse System with the obtained voltage signal and we compare the output Inverse System is indeed an impulse. In order to further remark the accuracy of the Inverse System implementation, we compute the Root Mean Square Error (RMSE) between the input and output of the processing chain, obtaining a result below the machine precision. Finally, a similar test is carried considering the circuit equivalent parameters of microphone BK4146; even in this case, the RMSE is numerically zero.In this subsection, we refer to the digital implementation of the microphone BK4134 equivalent circuit as Physical Direct System is microphone BK4134 and that we would like to obtain a voltage signal Target Direct System. It follows that the Inverse System is the circuital inverse model of microphone BK4134. The circuit parameters of both BK4134 and BK4146 are, once again, those listed in k the sample index, and Physical Direct System when the PITC-based algorithm is not active, the dashed red curve represents the target behavior that we would like to obtain, while the continuous blue curve represents the output of the PITC, i.e., the output of the system when the algorithm is active. The overlap between blue and red curve is perfect given that the RMSE is below machine precision. The algorithm is thus able to impose the response of microphone BK4146 even if the pressure signal is acquired by means of BK4143, being characterized at the same time by real time capabilities. In fact, the algorithm implemented in a MATLAB script is able to process, on average, one sample in Physical Direct System is simulated by means of the WDF shown in In this subsection, we provide an example of sensor virtualization. In particular, we employ the PITC-based algorithm presented in Finally, we would like to stress the fact that the circuital model shown in x is the displacement of the diaphragm in millimeters obtained integrating velocity We now consider a compression driver as an example of audio actuator. Such a transducer can be described by means of the circuit shown in  follows 15)Bl nicely disappear, leading to a perfect match between With the purpose of validating the WD implementation, we consider a processing chain similar to that shown in Target Direct System is the linear version of the circuit shown in Target Direct System, the Inverse System, and the Physical Direct System. Contrary to what done in the microphone case, the desired behavior is imposed at the beginning of the processing chain since the physical transducer is an actuator. In order to test the chain, we set the input A is the amplitude, k is the sample index, Direct System output (“Non Compensated”) and of the TIPC output (“Compensated”), together with the values of THD. Physical Target System is simulated but, in real scenarios, it represents the actual physical transducer.In a last application scenario, we show how the transducer linearization task can be accomplished as a particular case of the proposed virtualization algorithms. We aim, in fact, at eliminating the distortion effect introduced by the nonlinear behavior of the loudspeaker SEAS. In order to reach this goal, we employ the TIPC-based algorithm presented in n Bl=Bl0 . The parThe TIPC-based algorithm can be thus promising for improving the acoustic response of the loudspeaker on the fly by pre-processing the electrical signal driving the loudspeaker itself. Finally, it is worth stressing that the tested virtualization algorithm can be exploited not only to accomplish linearization but also to impose a desired nonlinear behavior, similarly to what already shown in .Direct System with a theoretical two-port called nullor, exploiting nullor equivalent models of short and open circuits [Physical Direct System, which is the responsible for the actual transduction process, an Inverse System, which is the circuital inverse of the Physical Direct System, and a Target Direct System, which is the transducer characterized by the behavior that we would like to obtain. We exploited WDF principles to implement the digital blocks of such processing chains in a fully explicit fashion, i.e., without resorting to iterative solvers. Finally, we tested both the PITC-based and the TIPC-based algorithms for addressing microphone virtualization and linearization of a loudspeaker system with nonlinear compression driver.In this paper, we described a general approach for the virtualization of audio transducers applicable to both sensors, like microphones, and actuators, like loudspeakers. We defined virtualization as the task of altering the acquired/reproduced signal by making it sound as if acquired/reproduced by another ideal or real audio sensor/actuator. In order to accomplish such a task, we started by reformulating Leuciuc’s theorem and proof for circuit inversion, providing case-specific guidelines on how to derive the inverse circuital model for all combinations of input and output variables. In particular, circuital inversion is achieved by augmenting the circuits . We thenFuture work may concern first the extension of circuital inversion theory to the Multiple Input Multiple Output (MIMO) case, and then, by exploiting this new theory, the development of refined DIDC-based algorithms for addressing the case of array virtualization, both in sensing and actuation scenarios.


Oryza rufipogon species complex (ORSC), the wild progenitor of cultivated Asian rice, to identify genomic regions associated with environmental adaptation characterized by variation in bioclimatic and soil variables. We further examine regions for colocalizations with phenotypic associations within the same collection. EAA results indicate that significant regions tend to associate with single environmental variables, although 2 significant loci on chromosomes 3 and 5 are detected as common across multiple variable types . Distributions of allele frequencies at significant loci across subpopulations of cultivated Oryza sativa indicate that, in some cases, adaptive variation may already be present among cultivars, although evaluation in cultivated populations is needed to empirically test this. This work has implications for the potential utility of wild genetic resources in pre-breeding efforts for rice improvement.Crop wild relatives host unique adaptation strategies that enable them to thrive across a wide range of habitats. As pressures from a changing climate mount, a more complete understanding of the genetic variation that underlies this adaptation could enable broader utilization of wild materials for crop improvement. Here, we carry out environmental association analyses (EAA) in the  This has enabled their adaptation across diverse environments, and as a result, wild gene pools represent rich repositories of novel alleles that can be utilized for plant improvement . HoweverIn recent years, there has been increased efforts in using ex situ germplasm collections as sources of traits to help adapt crops to climate change , soybeanOryza rufipogon species complex (ORSC) is the wild progenitor of cultivated Asian rice, Oryza sativa, a staple crop particularly important for feeding the world's poorest populations. The ORSC's geographic range is distributed widely across South, Southeast, and Eastern Asia, and the species occupies diverse tropical and subtropical habitats often found near or within human-disturbed areas such as cultivated fields, ditches, and irrigation channels  in the Philippines and the National Institute of Genetics (n = 3) in Japan. Environmental data were sourced from WorldClim for precipitation and temperature variables (2) and the 10 soil variables at a resolution of 2.5 arcmin (approximately 12 km2). The soil data were available at 6 depths: 0–5, 5–15, 15–30, 30–60, 60–100, and 100–200 cm. These data were aggregated to 2 layers (topsoil and subsoil) using a weighted mean approach, whereby the weights were proportional to the size of the original layer depths (n = 240) was recently phenotyped by All data analyzed in this study were retrieved from public sources. Genotype data  on a panel of 286 ORSC accessions were originally published by ariables  and Inter = 0.3. Communities of highly connected clusters of environmental and phenotypic variables were detected using group_infomap in the R package ggraph  on the 16 phenotypic variables that clustered with bioclimatic and biophysical variables, first standardized to a mean of 0 and standard deviation of 1, was conducted using the R library FactoMineR (K) using the R package rrBLUP v. 4.6.1  and minor allele frequency (≥0.05) using Plink1.9 . GWA was also carried out using scores from the first 5 components (n = 5 variables) that resulted from the phenotype PCA. Using the P-value inflation factor (lambda), we initially compared 3 GWA models: naïve (no polygenic background effect or adjustment for population structure), K (polygenic background effect only), and K + Q . The K + Q model with 2 PCs was identified as the best model based on having a high median lambda value but low variance; this model was used for all subsequent analyses (Genomewide association (GWA) analyses were run using the GWAS function in analyses . For envhttps://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-55/gff3/oryza_sativa/; last accessed 2022 December 12). We limited our search to those predicted genes within 150 kb of a given associated SNP. This threshold was based on the average distance at which linkage disequilibrium (LD) decayed to 0.2 across the 6 identified ORSC subpopulations , which houses data on 3,000 cultivated rice genomes . Out of the 41 environmental variables (19 bioclimatic and 10 soil × 2 depths) explored from these georeferenced locations of the ORSC panel, we found that soil organic carbon and precipitation of the driest month/quarter (bio14/bio17) had the greatest dispersion based on the coefficient of variation, while soil bulk density, mean temperature of the warmest quarter (bio10), and mean temperature of the wettest quarter (bio8) were found to be the least variable . In geneO. sativa (n = 259), a medium panel , and a small panel . Overall, we found that the medium panel yielded the most GWA runs that passed QC, resulting in 37 significant SNPs detected across 11 variables , followed by the small panel, which yielded 16 significant SNPs detected across 2 temperature and 5 soil variables. The large panel, despite having the greatest number of individuals, only yielded 9 significant SNPs detected across 4 soil variables , we carried out genome-wide association analyses on 19 bioclimatic variables and 10 soil variables, each at 2 depths, leveraging published genotype data and available georeference information on the panel. Previously, subpopulation-specific GWAS has been carried out with success in the highly stratified subpopulations of cultivated Asian rice, ariables . This maariables . BecauseMaterials and Methods). Chromosomes 7 and 12 harbored 3 significant loci each, chromosomes 1–6 and 9 contained 2 significant loci each, while chromosomes 8 and 11 had one each  previously shown to be inducible by abiotic stress in rice  that encode for MT-like type 1 proteins. MTs in plants play known roles in the scavenging of reactive oxygen species (ROS). In rice, dehydration has been demonstrated to induce expression of an MT type-1 gene, and overexpression of the same gene improved drought tolerance  as well as a superoxide dismutase (SODCC1), whose expression has been reported to be inducible by cold temperatures and ABA . In addition to 10 SNP colocalizations identified between highly similar environmental variables [e.g. clay in topsoil with clay in subsoil or precipitation of the driest month (bio14) with precipitation of the driest quarter (bio17)] that would be expected, we detected 2 SNPs that colocalized across precipitation, temperature, and/or soil variables. One was located on chromosome 3 at 13,114,933 bp and was shared between one temperature variable [mean temperature of the warmest quarter (bio10)] and 2 related soil variables (pH of the topsoil and pH of the subsoil) . Within  and ABA . As soilLSY1; RSR1; OsRAD51B involved in DNA repair). Also among the named genes were 3 ATP-binding cassette (ABC) proteins: one was a cloned gene involved in aluminum tolerance . Found in both prokaryotes and eukaryotes, ABC proteins are primarily transmembrane transporters that also contain a conserved nucleotide binding domain. In plants, these transporters are documented to play varied and critical roles in cellular detoxification , growth and development (e.g. hormone transport and cuticle development), and pathogen defense (e.g. via secretion of plant secondary metabolites)  and various phytohormones such as abscisic acid (down-regulation in root), gibberellic acid (down-regulation in root), and jasmonic acid (up-regulation in both shoot and root) (−1), and had higher diurnal temperature fluctuations relative to annual temperatures [average of 72.4 vs 49.9% for isothermality (bio3)] ], one soil variable (nitrogen in the topsoil), and 2 precipitation variables [precipitation of the driest month (bio14) and precipitation of the driest quarter (bio17)] . These c (bio3)] . Based oTo investigate whether loci significant for environmental variables also colocalized with regions of the genome associated with plant phenotypes, we leveraged trait data published recently on this panel by To reduce dimensionality of the phenotype dataset, a principal components analysis was carried out on the 16 phenotypes that did cluster with environmental variables. The first 5 principal components explained 78% of the total variation, and scores of these PCs were used as composite traits in conjunction with the SNP data to carry out genome-wide scans for associations. Results from GWA on scores from 2 of the components, PC2 and PC4, passed QC and yielded significantly associated SNPs . We founO. sativa. Except for the W5 subpopulation for the QTL on chromosome 3, the 2 minor alleles were rare in the wild populations (0–0.2). In contrast, we observed very high frequencies in the cultivated populations based on a database of 3,000 re-sequenced rice genomes . In the cultivated species, O. sativa, the frequency of the minor allele for the QTL on chromosome 5 was 0.67 in the tropical japonica subpopulation and fixed or nearly fixed in all other subpopulations while the minor allele of the chromosome 3 QTL was fixed across cultivated subpopulations , which were geographically associated (indica (indica-3 (at 0.77); this population was localized to South Asia . We note, however, that the presence of these alleles in cultivated populations does not necessarily indicate they have the same effects as in wild populations. More experimental work in O. sativa is needed to test this.We next cataloged the frequency of the minor alleles at the QTLs identified on chromosomes 3 and 5 across the W1, W2, W4, W5, and W6 subpopulations of ORSC as well as in its cultivated relative, ulations . Previou . Nonetheless, back-introgression may still be the most plausible explanation. The W5 subpopulation is small, geographically constrained in the mountainous region of Nepal, and highly differentiated at a whole-genome level from the O. sativa  detected in wild rice and discuss potential candidate genes within LD of their significant SNPs. Documentation of allele frequencies at these SNPs across the subpopulations of cultivated jkad128_Supplementary_DataClick here for additional data file.


Background: To estimate associations of sulfur-containing amino acids (SAAs) in the early trimester of pregnancy and gestational diabetes mellitus (GDM) and estimate associations of maternal SAAs with adverse growth patterns in offspring. Methods: We established a 1:1 matched case-control study (n = 486) from our cohort of pregnant women, and 401 children were followed up at ages 1 to 8 years. We conducted binary conditional logistic regression to estimate the risk associations of serum SAAs with GDM. Multinomial logistic regression was implemented to explore associations of maternal SAAs with adverse growth patterns in the offspring. Results: High serum methionine and cystine were independently associated with increased GDM risk . Conversely, a low level of serum taurine was independently associated with increased GDM risk . Maternal high cystine and low taurine were also associated with an increased risk of persistent obesity growth pattern (POGP) in offspring  and the effect was largely independent of GDM. Conclusions: High serum methionine, cystine and low serum taurine in the early trimester of pregnancy were associated with a greatly increased risk of GDM. Maternal high cystine and low taurine were associated with elevated risk of offspring POGP, largely independent of GDM. Gestational diabetes mellitus (GDM) is one of the most common complications of pregnancy and has affected approximately 14.0% of pregnant women globally in 2021, according to an estimate by the International Diabetes Federation ,4,5 and Sulfur-containing amino acids (SAAs), including methionine, cysteine, cystine and taurine, play major parts in the transmethylation and transsulfuration pathways ,10. DysrChildhood obesity is one of the concerns in GDM. Undue insulin resistance, hyperglycemia during pregnancy and/or DNA methylation in the fetus are potential risk factors for obesity during childhood. A small study including 64 prepubertal children (5–9 years old) showed that the level of plasma cystine was elevated in healthy obese children while plasma cysteine was not different compared to healthy lean children . The stuWe established a case-control study from our cohort of pregnant women and its follow-up data to examine (1) associations between the serum SAAs concentrations in early trimester of pregnancy and subsequent GDM risk; (2) the associations between maternal SAAs levels in early trimester of pregnancy with obesity-related growth patterns if any, in offspring at 1 to 8 years of age; and (3) whether GDM mediates the risk associations between maternal SAAs concentrations in early trimester of pregnancy and adverse growth patterns in offspring.The design and methods of this study have been reported previously ,22. In sAt 24–28 weeks of pregnancy, all pregnant women took a 50 g 1 h glucose challenge test (GCT). Thereafter, pregnant women with positive GCT were transferred to the GDM center, where they underwent a standard 75 g 2 h oral glucose tolerance test (OGTT) for diagnosis of GDM. GDM was diagnosed based on the International Association of Diabetes and Pregnancy Study Group’s (IADPSG) criteria .During the fieldwork, 2991 pregnant women out of 22,302 participants were invited and agreed with the provision of overnight fasting blood samples in the first trimester of pregnancy. Among them, we excluded 227 pregnant women who did not have GCT results or whose GCT results were positive but did not have OGTT results. Finally, we included 243 women with GDM and 243 women free of GDM matched by maternal age (±1 year) in the current analysis.At postpartum, 486 children of the included women were asked to attend the follow-up study and were offered health examinations, including measurement of body height and weight, each year from 1 to 8 years. Finally, 401 children (a response rate of 82.5%) participated in the postnatal follow-up and completed body weight and height measurements at 1 to 8 years of age.The nurses or obstetricians with uniform training measured maternal weight, height, and systolic/diastolic blood pressure (SBP/DBP) at the first antenatal appointment and offspring height and weight at each follow-up visit aged 1 to 8 years. We also collected other demographic information on maternal age, parity, ethnicity, family history of diabetes, the habits of smoking and drinking, and child gender. We calculate BMI by dividing weight (kg) by squared height (m).Measurement of serum sulfur-containing amino acids was previously described . Brieflyt-test if normally distributed or a Wilcoxon signed-rank test otherwise. Categorical data were evaluated by the McNemar test or Fisher’s exact test.We performed all statistical analyses using SAS version 9.4 . Quantitative data were expressed as mean ± standard deviation (SD) or median (interquartile range) where appropriate. Differences in quantitative data between the two groups were tested using a paired Student p < 0.05 at entry and exit).Binary conditional logistic regression was conducted to obtain odds ratios (OR) and their 95% confidence intervals (CI) of serum SAAs  for GDM. Restricted cubic spline (RCS) analysis was employed to check the linearity of the associations between SAAs concentrations and GDM risk as before ,26. We sA group-based trajectory modelling method applied to the longitudinal BMI data was performed to identify distinct BMI growth patterns from 1 to 8 years of age . The metThe baseline characteristics of the participating pregnant women are shown in Serum methionine and cystine in the early trimester of pregnancy are positively associated with GDM risk in nonlinear manners, while serum cysteine and taurine are negatively associated with GDM risk in nonlinear manners . Serum mBased on the group-based trajectory modelling approach, four distinct BMI growth patterns were observed in 401 children who participated in the follow-up visits. The four BMI growth patterns can be described as (1) persistent lean growth pattern (PLGP) characterized by a persistent low BMI over time; (2) normal growth pattern (NGP) characterized by middle and “normal” BMI over time; (3) persistent obesity growth pattern (POGP) characterized by a high and persistent increased BMI over time; and (4) late obesity growth pattern (LOGP) characterized by a normal BMI before 5 years of age and rapidly increased BMI thereafter . CompareMaternal cystine ≥ 150 nmol/mL  is associated with markedly increased risk of POGP in the offspring in univariate and multivariate analyses . Further adjustment for GDM slightly attenuates the OR of high cystine for POGP, but its statistical significance persists . However, maternal high cystine is not significantly associated with LOGP risk in offspring. Similarly, maternal taurine ≤ 21.9 nmol/mL  is associated with an elevated risk of POGP, while low taurine is not associated with LOGP risk in offspring . Further adjustment for GDM slightly attenuated the OR of low taurine for POGP, but the statistical significance remained . On the other hand, maternal methionine and cysteine are not significantly associated with the risks of POGP and LOGP in offspring .Our study explored and confirmed that (1) serum methionine and cystine in early pregnancy were positively associated with GDM risk, while serum taurine was negatively associated with GDM risk in the Chinese population; (2) maternal high serum cystine and low serum taurine in the early trimester of pregnancy were also associated with increased POGP risk in offspring, largely independent of the occurrence of GDM.In recent years, a few animal and human studies have surveyed the risk associations of SAAs concentrations with insulin resistance and T2D, but their findings were inconsistent. For instance, an animal study found that plasma levels of methionine and cysteine were markedly decreased, and hepatic methionine, cysteine and taurine were also decreased in Zucker diabetic fatty rats . A cohorBiological links between SAAs and GDM remain unclear. SAAs play a major part in the transsulfuration pathway, which can protect against uncontrolled oxidative stress and inflammation through sulfur’s role in redox biochemistry ,31. One In recent years, much research has surveyed the BMI growth patterns in children. A large longitudinal cohort study implemented in the USA identified three distinct BMI growth patterns in children aged 2–6 years using the group-based trajectory modelling method . The thrThe associations of maternal SAAs and offspring growth patterns had not been explored. A small research implemented in the USA showed that the concentration of plasma cystine was elevated in healthy obese children while plasma cysteine was not different compared to healthy lean children . The uteIt is biologically plausible that maternal high serum cystine and low serum taurine were associated with markedly increased POGP risk in offspring. There is growing evidence that epigenetic changes have been a potential mechanistic link between exposure to the uterine environment and childhood obesity in the offspring . The intGDM can increase the risks of maternal diabetes and cardiovascular disease in later life and childhood obesity risk in offspring ,5,6, wheThere were some limitations in our study. First, the findings of this study were obtained from a case-control study, which was from a single cohort of pregnant women and their offspring in Tianjin. These findings, therefore, need to be verified in other cohorts. Second, the levels of serum SAAs were related to dietary intake, but we did not obtain detailed diet information from the mothers. Third, homocysteine is an important sulfur-containing amino acid and may play a part in GDM. However, in the targeted LC-MS/MS assay, homocysteine was not specifically included in the measurement scheme. We cannot exclude the possibility that it may have confounded associations between other sulfur-containing amino acids and GDM in this report. Fourth, we noticed that the sample size of the POGP group was small, and the 95%CI of the risk association between maternal low taurine and offspring POGP was large. Further replications by other cohorts are certainly needed to reduce the large type 2 error.In summary, our study found (1) high concentrations of serum methionine and cystine and low concentrations of serum taurine in the early trimester of pregnancy were independently associated with greatly increased GDM risk, and (2) maternal high concentrations of serum cystine and low concentrations of serum taurine were also associated with greatly elevated POGP risk in offspring, largely independent of GDM. Further studies are warranted to verify our observations, and biological mechanisms underlying the intriguing findings also need exploring for better comprehension of the pathophysiology of GDM and biological links from early life SAAs exposure to adverse childhood growth patterns.