Background: The coronavirus disease (COVID-19) pandemic is an international public health threat. This study aimed to evaluate COVID-19-related knowledge, preventive behaviors, and risk perception among Saudi Arabian medical students and interns. Materials and Methods: This cross-sectional study was conducted among fourth- and fifth-year medical students and interns between June and August 2020 at three colleges of medicine in Qassim Region, Saudi Arabia. A previously validated questionnaire was distributed as an online survey. Results: The total mean knowledge score was 12.5/15 points; 83.9% achieved a high score. The mean score of self-reported preventive behavior was 8.40; 94.1% achieved a high score. The overall mean risk perception score was 5.34/8 points; 31.6% achieved a high score. Conclusion: Medical students assessed in this study displayed sufficient knowledge and preventive behaviors regarding the COVID-19 pandemic and an average level of risk perception. Lower scores by younger medical students suggest that they must improve their COVID-19 knowledge and risk perception, as they are a potential source of health information in their communities. Coronavirus infections are caused by emerging respiratory viruses that are known to cause diseases ranging from the common cold to illnesses involving severe respiratory distress . In 2002The disease is highly infectious, and further studies identified that the most important route of transmission to humans occurred via respiratory droplets or direct contact, with an incubation period ranging from 2 to 14 days . ClinicaHealthcare providers are the primary individuals in contact with patients who are the main source of infections; thus, they are at high risk of becoming infected themselves. At the end of January, the WHO and the US Centers for Disease Control and Prevention (CDC) published recommendations to help prevent the rapid spread of COVID-19 among healthcare workers ,5. SeverThis knowledge and awareness of the COVID-19 pandemic can influence the risk perceptions of healthcare workers and their ability to engage in preventive strategies. However, some studies have noted a significant gap in information sources available to healthcare workers and have identified low knowledge levels concerning COVID-19 .Despite numerous studies that have addressed this topic worldwide, there are limited data about the levels of knowledge and behaviors pertaining to COVID-19 treatment in Saudi Arabia. We hypothesized that only a little information is available on COVID-19 and that medical students exercise some incorrect preventive behaviors. Medical students are a source of information for their families and communities; therefore, our study aimed to evaluate COVID-19-related knowledge, preventive behaviors, and risk perceptions among fourth- and fifth-year medical students and interns.This cross-sectional, descriptive study was conducted between 1 June 2020, and 30 August 2020. The study population included both male and female fourth- and fifth-year medical students and interns in the multi-colleges of medicine of Al-Qassim Region, Kingdom of Saudi Arabia (KSA), including the College of Medicine at Qassim University in Buraidah, the College of Medicine at Qassim University in Unaizah, and the College of Medicine at Sulaiman Al Rajhi University. The total number of fourth- and fifth-year students and interns in each university was as follows: College of Medicine at Buraidah (333 students), College of Medicine at Unaizah (221 students), and the College of Medicine at Sulaiman Al Rajhi University , and the ratio of male to female students was 2:1. This study was approved by the Committee of Bioethics of the Qassim University, Saudi Arabia .A previously validated questionnaire was administered as an online survey that was distributed to all clinical year students through WhatsApp application . The samTo assess the subjects’ levels of knowledge, preventive behaviors, and risk perceptions, the questionnaire was divided into four parts. The first part assessed demographic information , followed by 26 questions to quantify their knowledge, preventive behaviors, and risk perception. Knowledge was assessed through questions about the nature of COVID-19 transmission, its history, symptoms, incubation period, diagnostic procedures, preventative measures, and treatment options.Medical students’ general knowledge regarding COVID-19 was assessed using 15 questions; the correct response to each question was coded as a “1,” and an incorrect response was coded as a “0.” The total score for knowledge was calculated as the sum of the responses for the 15 questions, which yields a score ranging from 0 to 15. A higher score was indicative of a higher level of knowledge regarding COVID-19. Using 50% and 75% as cutoff points, participants were classified as having low knowledge if their scores ranged between 1 and 7, scores ranging between 8 and 11 indicated average knowledge, and scores ranging between 12 and 15 indicated a high level of knowledge.The self-reported preventive behaviors were assessed by binary responses to nine questions; the “yes” response was coded as “1,” and the “no” response was coded as “0.” The total score for the self-reported preventive behaviors was calculated as the sum of the responses to the nine questions, which yielded a score ranging between 0 and 9; a greater degree of self-reported preventive behaviors was indicated by a higher score. Using 50% and 75% as cutoff points, medical students were considered to have a low level of preventive behavior if their scores ranged between 1 and 4, an average level for scores ranging between 5 and 6, and a high level for scores ranging between 7 and 9.Risk perception was assessed by two questions whose responses were recorded on a 4-point Likert scale, with categories ranging from “strongly disagree” (coded as “1”) to “strongly agree” (coded as “4”). The total risk perception score was calculated as the sum of the responses to the two questions, generating a score ranging from 2 to 8. A higher score was indicative of a greater level of perception pertaining to the risk of COVID-19. Using 50% and 75% as cutoff points, medical students were classified as having low risk perception for scores ranging from 1 to 4, scores ranging from 5 to 6 were considered an average level of perception, and scores ranging from 7 to 8 were considered to be a high level of perception.p-values < 0.05 were considered to be statistically significant. Normality, statistical interactions, and collinearity  were also assessed with the Kolmogorov-Smirnov and Shapiro-Wilk tests, and p-values < 0.05 were considered indicative of skewed data distributions. Correlation analyses were also conducted to assess the linear relationships between knowledge, behavior, and risk perception. All data analyses were performed using Statistical Packages for Software Sciences (SPSS) version 21 software .Data are presented as counts, proportions (%), ranges, means, and standard deviations whenever appropriate. The comparisons of knowledge, behavior, and risk perception among the basic demographic data of medical students were conducted using the Mann Whitney U test (in cases of two categories) or the Kruskal Wallis test (in cases of three categories). Of the 710 medical trainees approached, 323 responded (response rate 45.5%); their basic demographic data are presented in In the assessment of medical students’ knowledge regarding COVID-19 , respondAlmost all medical students (varying from 88.5% to 96.3%) self-reported good preventive behaviors toward COVID-19, as shown in Of the respondents, 38.1% disagreed that they may be infected with COVID-19 more easily than others, while 28.8% agreed. We also observed that nearly half of the medical students (47.7%) agreed that they were afraid of being infected with COVID-19, whereas 26.9% stated otherwise. The level of knowledge, self-reported preventive behavior, and risk perception among medical students are summarized in p < 0.001), suggesting that when knowledge increased, self-reported preventive behavior was also likely to increase. The correlation between knowledge and risk perception was not statistically significant . There was, however, a weak positive correlation between self-reported preventive behavior and risk perception scores , indicating that a higher self-reported preventive behavior score was associated with an increase in risk perception.Knowledge and self-reported preventive behavior scores were weakly positively correlated, and this relationship was highly statistically significant . Furthermore, fourth-year students had significantly less knowledge (p = 0.009) and a lower perception score (p = 0.001) than other academic year students. However, age, sex, and having medical knowledge about COVID-19 were not significant when compared to knowledge, self-reported preventive behavior, and risk perception scores (All p > 0.05).When comparing the knowledge, self-reported preventive behavior, and risk perception score among medical students and interns, we noted that medical students studying at Almleda Qassim University had significantly better risk perception score than those studying at other universities (p > 0.05), which contradicts previous findings. We believe that the source of information (curriculum and extra-curriculum) for both sex and age were similar during the pandemic. Additionally, the number of students who had no medical knowledge (as per the student self-assessment) was extremely low (4.3%).Senior medical students, as well as interns, are on the frontline in the fight against the novel coronavirus, and they have always been at risk of contracting the infectious disease. For these reasons, it is necessary to evaluate their knowledge, assess their preventive behaviors, and measure their risk perception in regards to the COVID-19 pandemic. The outcomes of this research could be useful for health policymakers and medical educators to design a systematic plan to ensure that medical students are aware of the COVID-19 pandemic and the necessary preventive measures. A literature search conducted in Saudi Arabia revealed limited available data from studies that measured medical students’ knowledge, preventive behaviors, and risk perception during the COVID-19 pandemic. In this study, nearly all students 83.9%) exhibited a high level of knowledge regarding COVID-19; the rest exhibited either an average 14.6%) or a low level (1.5%) of knowledge. The overall mean knowledge score was 12.5 SD 1.47). Consistently, the current literature indicates that many people, including healthcare workers, medical students, and the general public, demonstrated a high level of knowledge regarding the pandemic, varying from 69% to 91% [. Consist3.9% exhi.6% or a Moreover, it is important to note that academic level was the only significant factor associated with the level of COVID-19 knowledge, as the 4th-year students exhibited significantly lower knowledge scores than those at the 5th-year level or interns. This is comparable to the results of other studies conducted in Saudi Arabia , Egypt , and UgaThe data revealed that medical students exhibited an overwhelmingly high degree of preventative behaviors to protect against COVID-19 (94.1%); only a few students exhibited average (5.3%) and low-level preventive behaviors (0.6%). This finding mirrored that of a study conducted by Taghir et al., , who fouApart from measuring the knowledge and behavior of medical students related to the COVID-19 pandemic, we also investigated their levels of risk perception, which was at an average level among medical students. This is also consistent with the average level of risk perception that was reported by Taghir et al. . In contOne of the highlights of our results was that there was a positive, significant correlation between the knowledge and self-reported preventive behavior scores, suggesting that as medical students’ knowledge of COVID-19 increased, so did their self-reported preventive behaviors. On the other hand, there was no significant correlation between knowledge and risk perception scores. These results were not consistent with those of Kim and Choi , who repTo learn more about the current pandemic, it is necessary to provide everyone with access to sources of information. In this study, the most frequently noted source of COVID-19 information was material provided by the local Ministry of Health (75.7%), followed by the CDC (68.9%), and the WHO (63.9%). Due to the influence of social media, many people also sought information on the internet. This has also been consistently reported in the literature, where the majority of individuals read information about the pandemic on public health websites ,16,17,18This study had some limitations. In particular, the study was conducted in only a single region, which may not be representative of all university students in Saudi Arabia. Additionally, the questionnaire was conducted online due to the mitigation measures in place in response to the COVID-19 pandemic, which might have affected the response rate and generalizability among the university students. Nevertheless, this study provides an estimate of medical students’ current COVID-19 knowledge and risk perception in Saudi Arabia during the pandemic.Medical students play an important role as volunteers during emergency, including pandemics. This study revealed that current knowledge and self-reported preventive behaviors of medical students pertaining to the COVID-19 pandemic were sufficient, although risk perception was average and could be improved. Medical students studying at the Almleda Qassim University had a better outlook in terms of risk perception. However, younger medical students need to improve their Knowledge of COVID-19 and risk perception since they are a potential source of health information within their communities. Thus, more education must be provided to fill the gaps, especially for those in the earlier academic levels of medical training, as they are a vulnerable group that may underestimate the risks of COVID-19.


The literature has recognized premature birth as a risk factor for infant development and maternal anxiety. This study investigated the impact of the severity of birth weight, as well as of maternal anxiety at 3 months of infants’ corrected age, on infants’ outcomes during the 1st year postpartum. Moreover, it described the longitudinal trajectories of developmental outcomes, additionally exploring the impact of anxiety.The study compared 147 mothers and their 147 newborns, differentiated in 25 Extremely Low Birth Weight (ELBW), 41 Very Low Birth Weight (VLBW), and 81 Full-Term (FT) infants. At 3, 9, and 12 months (corrected age in the case of preterm infants) the level of infants’ development was investigated according to the 5 quotients  of the Griffiths Mental Development Scales (GMDS-R). During the assessment of 3 months, mothers fulfilled Penn State Worry Questionnaire (PSWQ) to evaluate the presence of generalized anxiety.Among the 5 GMDS-R quotients, significant effect of severity of birth weight emerged only for Performance quotient: preterm infants  showed lower scores than FT ones. Moreover, this quotient decreased from 3 to 9 and to 12 months for VLBW and FT infants, while it was stable for ELBW ones. A significant interaction between severity of birth weight and maternal anxiety emerged for Hearing and Language and Locomotor quotients. In the first case, scores for ELBW infants, independently from maternal anxiety, decreased from 9 to 12 months. The same results emerged for VLBW infants, in the case of non-anxious mothers. Regarding Locomotor quotient, mean scores decreased from 3 to 9 and to 12 months for all groups in the case of non-anxious mothers. Conversely, when mothers were anxious, this decrease emerged only for VLBW infants. Lastly, ELBW, VLBW and FT showed difference in the growth and slope of the trajectories of different quotients.The severity of birth weight for preterm infants, also in interaction with maternal anxiety, had significant and specific impact on different dimensions of infants’ development. Clinical implications of these results underline the need for individualized interventions. Prematurity is an unexpected and traumatic event during childbirth  and it rA preterm birth also negatively influences the transition to parenthood , as pareAccording to an estimation by the World Health Organization, each year approximately 15 million babies are born prematurely, worldwide . In ItalLow Birth Weight-LBW) and “high-risk” preterm infants, with birth weight less than 1500 grams (Very Low Birth Weight-VLBW), and specifically less than 1000 grams (Extremely Low Birth Weight-ELBW)  .The risk of sequelae, including neurodevelopmental delays, is inversely proportional to infant birth weight  and is sRecent studies and reviews on ELBW or VLBW sequelae report evidence regarding different developmental domains, such as: neurosensory , motor , linguisFurthermore, most studies have often focused on one specific population (VLBW or ELBW), neglecting the comparison between the 2 groups. It is reasonable to speculate that ELBW and VLBW infants may show specific and somewhat different profiles concerning impairments, needs and resources, as we have already discovered in previous studies , 2017. SIn fact, due to the influence of multiple and heterogeneous variables, the trajectories of preterm infant development may show a wide range of variability from child to child.Other than considering the role played by the biological and neurodevelopmental factors, we have to include environmental variables, which may interfere with/or positively influence preterm baby growth. For example, we can acknowledge the quality of the care provided by both the hospital environment and the staff, as well as the way in which the preterm baby’s parents react to the unexpected birth of their infant.Indeed, many studies have, in the last years, focused on the investigation and description of emotional reactions and stress experienced by the mother after a premature childbirth. One of the most frequent consequences for maternal mental health is a heightened risk of experiencing different kinds of symptoms, such as traumatic stress symptomatology, depression, anxiety and acute stress disorder . In factConcerning the premature babies, the degree of severity of prematurity seems to be related to a high risk of maternal symptomatology. In fact, VLBW has been recognized as a relevant risk factor for preterm babies’ mothers, increasing the risk of being postnatally depressed from 4 to 18 times . Also, VPerinatal anxiety has been defined as anxiety experienced during the antenatal and/or postpartum period (first 12 months after birth) . DespitePostnatal anxiety, in association with the possible negative consequences of a preterm birth, may impact on the child development. Indeed, we may hypothesize that maternal anxiety is expected to be particularly intense in the first postpartum months, usually corresponding to the period of NICU stay. Some empirical evidence would support this; for example, It is notable that a debate on the specificity and characterization of perinatal anxiety, with obvious implications on the tools to use for the assessment, has received a growing interest from the scientific literature in the last few years . Based oThis is the case, for example, with the studies on “pregnancy-specific anxiety” , 2016, oTo sum up, despite the evidence of the influence of postnatal anxiety on parent-infant-relationships and child outcomes, the literature still shows a lack in investigating this issue. Moreover, the above-mentioned studies on preterm birth and maternal anxiety did not compare different subpopulations of preterm infants. In a previous study , we analThe literature has developed knowledge and findings on macro areas of child development in the case of preterm infants, such as language, attention and motor skills. However, there still is a lack of investigation regarding specific developmental dimensions, considering the severity of prematurity and specific maternal symptoms, especially in a longitudinal perspective.Based on this, we developed a longitudinal study considering 3 specific time points that represent significant steps  for the progress of infant development during the 1st year of life. Three months represent an important step for the detection of the early skills of the baby . At 9 months, new skills are supposed to emerge, like crawling and joint attention. At the end of the 1st year of life (12 months), the infant’s autonomy may be observed by the development of deambulation and/or the occurrence of the first words.The first aim of this study was to investigate the impact of severity of preterm birth on specific areas of infant development , at 3, 9, and 12 months of age, corrected for preterm infants. We hypothesized that, according to a higher degree of severity of premature birth (that is the case of ELBW), infant development would be worse compared to VLBW and FT. According to previous literature, we supposed that in ELBW infants, but not in VLBW and FT ones, scores in all dimensions would significantly decrease across the 1st year.Secondly, we investigated whether maternal anxiety, at 3 months of the infant’s corrected age, could influence infant development in the different dimensions considered. Specifically, we hypothesized lower quotient scores according to the presence of both low birth weight and maternal anxiety.Thirdly, we aimed at giving a description, through growth trajectories, of the different areas of development in ELBW, VLBW and FT infants from 3 to 12 months, considering also the effect of maternal anxiety.This study was part of a wider longitudinal research aimed to assess the course of infants’ development from 3 to 24 months postpartum.All mothers were recruited according to the following exclusion criteria: presence of previous or present psychiatric illness, lack of fluency in Italian, presence of infants’ chromosomal abnormalities, cerebral palsy, malformations, fetopathy, severe complications . In the case of twin birth, only the first-born one was included.At the end of the recruitment, our sample included 147 mothers and their 147 newborns.The Preterm (PT) group, recruited at Neonatal Intensive Care Unit (NICU) of Bufalini hospital , was composed by 66 mothers and their 66 preterm infants (44.9% of the infants’ sample), with a birth weight under 1500 g and gestational age < 32 weeks. This group was differentiated in two groups: 41 mothers and their 41 babies with weight between 1000 and 1500 g and gestational age < 32 weeks (27.9% of the infants’ sample) constituted the VLBW group; 25 mothers and their 25 babies with weight under 1000 g and gestational age < 28 weeks (17% of the infants’ sample) constituted the ELBW group.The Full Term (FT) group, recruited at the antenatal classes held in Cesena  during the third trimester of pregnancy, was composed by 81 mothers and their 81 full term healthy infants (55.1% of the infants’ sample), that had a birth weight > 2500 g and gestational age > 36 weeks.All the assessments took place at “Anna Martini” University Laboratory  at 3 months (T1), at 9 months (T2), and at 12 months postpartum (T3) (corrected age for preterm infants). During all the assessments, the level of infant development was evaluated by a trained psychologist according to the 5 quotients of the Griffiths Mental Development Scales GMDS-R; .ad hoc questionnaire regarding socio-demographic variables  and infant information . They were also asked to complete a self-report questionnaire aimed to assess the level of anxiety, while a trained psychologist assessed their infants’ development.At T1, all mothers, after providing their written informed consent, were asked to complete an The Ethical Committee of the Department of Psychology (University of Bologna) approved the design of the study.The Griffiths Mental Development Scales GMDS-R- is a welThe presence of maternal anxiety was investigated by the Penn State Worry Questionnaire PSWQ; . The PSWThe PSWQ is composed by 16 items, rated on a Likert scale between 1  to 5 . Eleven items are positively worded , while five items are negatively worded . The sum of all items provides a total score that ranges from 16 to 80, where the higher the value, the higher the levels of pathological worry. In the present study, we administered the Italian version of PSWQ that showed good internal consistency (0.85), suggesting a clinical cut-off score ≥ 57 to discriminate anxious from non-anxious subjects .Statistical analyses were carried out using the IBM SPSS statistical package version 25.0.To verify the homogeneity among ELBW, VLBW and FT dyads regarding of socio-demographic and clinical variables, we performed Pearson’s Chi Square Test and Univariate Anova.For the first and second purposes, Repeated Measures Manova were used to investigate the influence of specific factors , and of their interactions, on the 5 quotients of GMDS-R continuous scores at T1, T2, and T3.For the third purpose, growth curve analysis was used to describe trajectories of each GMDS-R quotients from T1 to T3 in ELBW, VLBW, and FT babies as a function of time and maternal anxiety at T1. With three repeated measures  of outcome variables, analyses were limited to linear and quadratic models . TherefoModeling took place in two steps. Model 1 was fit as an unconditional growth model, where only the intercept, linear slope, and curved slope were specified in order to determine the trajectories of each GMDS-R quotients in ELBW, VLBW and FT babies irrespective of maternal anxiety at T1. Model fit was evaluated using the −2 log likelihood difference test (−2LL).Model 2 was fit as a conditional growth model for exploring the effect of maternal anxiety at T1 on trajectories of each GMDS-R quotients in the three birth weight groups.p-values were lower than 0.05.Significant results were considered when 2(2) = 18.11; p < 0.0001), level of education (χ2(2) = 13.12; p < 0.0001), and anxiety (χ2(2) = 6.36; p = 0.042). In particular, FT mothers, compared to VLBW and ELBW ones, were primiparous, had graduated and were non-anxious in a higher percentage  = 36.19; p < 0.0001), twinning (χ2(2) = 28.60; p < 0.0001), gestational age  = 1066.80; p < 0.0001), days of hospitalization  = 325.30; p < 0.0001), and small gestational age (χ2(2) = 4.26; p. 039). Specifically, in FT mothers, cesarean section delivery and twinning were less frequent, compared to VLBW and ELBW mothers  = 6.413; p = 0.002): FT infants had significantly higher mean score than those observed in VLBW and ELBW infants   .F = 3.365; p = 0.038). Looking at the differences between groups, at T1, ELBW infants showed a significantly lower mean score than that reported by FT and VLBW infants ; while, at T3, VLBW infants showed a significantly lower mean score than that observed in FT infants (Bonferroni post hoc test p = 0.033). Looking at the differences within groups, mean scores of both FT and VLBW groups significantly decreased from T1 to T2  and from T1 to T3 . No differences emerged in the ELBW group  = 5.052; p = 0.008): ELBW infants showed a significantly higher mean score at T2 than that observed at T3 (Bonferroni post hoc test p < 0.0001)  .No significant differences emerged for the other GMDS-R quotients .The majority of VLBW and FT infants did not show delays at T1, while a small percentage was observable at T2 and T3. Conversely, ELBW showed delays in all three assessments.When specific quotients are observed, a low number of cases emerge in Hearing and Language scores, while a high rate emerges in Locomotor ones.In line with the second aim, we investigated the interaction between birth weight, maternal anxiety and time of assessment on infants’ GMDS-R quotients. All results are shown in Regarding the interaction between birth weight and maternal anxiety, no significant differences emerged.F = 3.274; p = 0.041) and Hearing and Language quotients  = 3.255; p = 0.042).When the interaction among birth weight, maternal anxiety and time of assessment was considered, significant results emerged on Locomotor (post hoc test p = 0.022)  (post hoc test p < 0.005)  . Lookingctively) . In the < 0.005) .post hoc test p = 0.017). Considering the differences within groups, ELBW infants showed a mean score at T2 significantly higher than that reported at T3 in both cases of non-anxious and anxious mothers  , though only in the case of non-anxious mothers  ; moreove mothers .In line with the third aim, we explored the trajectories of each GMDS-R quotient in ELBW, VLBW and FT infants as a function of time (model 1) and maternal anxiety (model 2).2(1) = 10.72; p < 0.01), VLBW (χ2(1) = 24.18; p < 0.01) and FT infants (χ2(1) = 34.85; p < 0.01) were characterized by a significant negative linear slope, followed by a positive quadratic (curved) slope, indicating a U-shaped pattern , the −2 log likelihood model comparison tests indicated that the average trajectories in ELBW , results showed that in the case of FT infants, maternal anxiety, even if it did not predict change of the intercept, showed a significant main effect on the linear slope: FT infants with non-anxious mothers had a significantly greater negative linear slope than those with anxious mothers. No significant effect emerged on the quadratic curve.No significant change of the intercept, as well as of the linear and the quadratic slope emerged in the case of VLBW and ELBW infants.2(1) = 19.63; p < 0.01), VLBW (χ2(1) = 19.47; p < 0.01) and FT (χ2(1) = 52.77; p < 0.01) infants were characterized by a significant negative linear slope and a positive quadratic (curved) slope, indicating a U-shaped pattern  = 9.86; p < 0.01) and VLBW infants (χ2(1) = 15.19; p < 0.01) were characterized by a significant positive linear slope followed by a negative quadratic (curved) slope, indicating an inverted U-shaped pattern; while the trajectory of FT infants (χ2(1) = 0.33; p < 0.05) was characterized by a non-significant negative linear slope, indicating a linear pattern  = 10.67; p > 0.05) was best described by a significant negative linear slope, indicating a linear pattern, while those of VLBW (χ2(1) = 4.08; p < 0.05) and FT infants (χ2(1) = 9.43; p < 0.01) were characterized by a significant negative linear slope and a positive quadratic (curved) slope, indicating a U-shaped pattern  = 0.01; p > 0.05) was best described by a non-significant negative linear slope, indicating a linear pattern, while those of VLBW (χ2(1) = 23.62; p < 0.01) and FT infants (χ2(1) = 58.03; p < 0.01) were characterized by a significant negative linear slope and a positive quadratic (curved) slope, indicating a U-shaped pattern . No significant change emerged in the case of VLBW and FT infants.This study aimed at assessing preterm infants’ outcomes in different developmental areas  during the 1st year of life, exploring the impact of severity of birth weight, also in relation to postnatal maternal anxiety. A further aim was to describe trajectories of these developmental dimensions in ELBW, VLBW and FT infants from 3 to 12 months postpartum. One of the main strengths of this study was to explore the impact of the severity of prematurity on each GMDS-R quotient in order to highlight possible areas of vulnerability in specific phases of development.Different results emerged in relation to specific developmental areas, as measured by GMDS-R quotients.Regarding the Performance quotient, which measures skills in manipulation, speed of working and precision, as well as the ability to apply them in novel situations , a firstThe presence of maternal anxiety did not seem to have a significant impact on infants’ mean scores of performance quotient, independently from the birth weight classification.Furthermore, growth trajectories analyses underline that ELBW infants showed a non-significant negative linear pattern of growth, while VLBW and FT infants demonstrated a U-shaped pattern of growth. These findings suggest that, during the 1st year postpartum, VLBW and FT infants had a similar trend of development, even if VLBW infants showed lower scores across time. The only significant result in the case of maternal anxiety emerged on the intercept of ELBW infants: ELBW with anxious mothers showed significantly lower scores than those without anxious mothers.The decrease of quotients observed in the study is somehow unexpected. However, a possible explanation could be given by the increase of the complexity of task demands required by the GMDS-R . Indeed,ad hoc interventions; in particular, in the case of ELBW babies, interventions aimed at promoting very simple and general abilities should start since the first postpartum months of life, while in the case of VLBW babies, the therapeutic interventions, aimed at building more complex abilities, could start later.These results may suggest that the development of ELBW and VLBW infants could benefit from To our knowledge, no previous studies have explored ELBW, VLBW and FT infants’ performance development across time, nor their trajectories of growth, by also considering the role of maternal anxiety. Therefore, further studies are recommended.Regarding the Hearing and Language quotient, it is relevant to note that most of the infants did not present an index of delay, defined as a score < 1 DS. This result is unexpected and is not in line with previous studies , in whicA first consideration is about the significant decrease in scores from 9 to 12 months shown by ELBW infants. This effect emerged both in the case of anxious and non-anxious groups. Conversely, we observed this decrease in VLBW only if their mothers were non-anxious and it never emerged for FT infants. These results could be explained considering the steps of the acquisition of hearing and language skills during the 1st year: the period between 9 and 12 months is a sensitive and pivotal time in which a baby should show more complex linguistic abilities, such as using gestures  and vocalizations . For this reason, during the first postpartum months of an infant’s age, items of GMDS-R scales are mainly focused on hearing skills, while at the end of the 1st year the quantity and quality of infant vocalizations are deeply assessed. So, the decrease in the scores from 9 to 12 months could show how the adaptation to new skills is highly demanding in the case of preterm infants, especially for high-risk babies like ELBW ones, as we found in a previous research . This trConversely, full-term infants might have already found an adjustment on these abilities, thus showing more stability in their scores throughout all the assessments.At 9 months postpartum, VLBW infants showed a higher quotient compared to the FT group and this result emerged only in the case of non-anxious mothers. This somehow unexpected result may possibly be explained considering the fact that the VLBW group represents a low risk sample and it could have taken advantage of the supportive interventions realized in NICU and during the follow-up programs , aimed aFurther studies including the role of maternal anxiety are recommended.To sum up, our results suggest that more evident difficulties for hearing and language development manifest at the end of the 1st year postpartum, when a baby should start to use gestures, vocalizations and single words to communicate. These findings are important because they highlight the importance of planning early language-focused interventions in order to limit these subsequent difficulties .Regarding the Locomotor quotient, ELBW, VLBW and FT babies had similar scores, independently from time of assessment. This result is not in line with previous studies, highlighting that preterm infants, compared to FT ones, had significantly more difficulties in acquiring gross motor skills in the 1st and 2nd year of life . RegardlWhen maternal symptomatology was also included, specific patterns relating to anxious and non-anxious groups emerged. When mothers were non-anxious, the mean scores observed in ELBW, VLBW and FT infants significantly decreased from 3 to 9 months, followed by relatively stable scores from 9 to 12 months. In this case, the Locomotor quotient tends to show a specific trend of development across time, independently from the severity of birth weight.Conversely, when mothers were anxious, ELBW and FT infants showed similar scores from 3 to 12 months postpartum, while only VLBW infants maintained the decrease observed from 3 to 9 and 12 months. It is unexpected for VLBW infants to reach good performance at T1 despite maternal anxiety. Previous literature has suggested that, in the case of preterm birth, anxiety could function as an adaptive response to traumatic condition , helpingThis result confirms the way in which ELBW and VLBW may show different profiles in the acquisition of Locomotor skills, with different time of improvement and different resources to environmental stimulations.To our knowledge, no previous studies have explored the differences between ELBW, VLBW and FT infants on locomotor development at 3, at 9 and at 12 months of life, considering the presence of maternal anxiety and this study represents a first step in this direction.Lastly, considering Eye-hand Co-Ordination and Personal and Social quotients, significant results did not emerge according to severity of birth weight and maternal anxiety.In the case of the Eye-hand Co-Ordination quotient, ELBW infants presented a small prevalence of delays in all the assessments and a negative linear pattern, different from the U-shaped pattern reported by VLBW and FT infants. This result is in line with previous studies , reportiIn the case of the Personal and Social quotient, no significant differences emerged when parametric analyses were run; nevertheless, relevant changes were observed in the rate of delay in the 3 groups. In particular, during the assessment at T2, 9 months, a very high rate of delay was present in the preterm group, especially in the case of ELBW infants (44%). Nine months represent a sensitive period for development, as babies more actively interact with the surrounding environment and new skills are learned, especially in the food area; e.g., the consolidation of weaning leads to increasing autonomous behaviors, like taking foods by hands, attempting to drink from the bottles alone, etc. The acquisition of these skills could be more difficult for severely preterm infants and for their parents; indeed, memories of previous experiences during hospitalizations  could interfere with the scaffolding role that parents could play. Therefore, the clinicians need to pay specific attention to the meaning that feeding has for these families. Furthermore, results of growth curve analysis showed that, despite not significant, preterm infants obtained lower mean scores than FT ones, with a U-shaped pattern.A relevant consideration is required about the use of developmental scales for the assessment of relational skills, rather than the evaluation by interactive scales, as in previous literature . As suggTo our knowledge, this is the first study that longitudinally explored the impact of the severity of birth weight along with maternal anxiety on each GMDS-R quotient, in order to understand whether specific areas of development are more exposed to impairments across the 1st year postpartum.Taken together, the results may suggest a discrete instability of the scores at 3-, 9-, and 12-months, as assessed by GMDS-R, strengthening the evidence from previous research that the assessment of infant development by developmental scales would show lower sensitivity in the case of high risk infants , compareThough this may suggest a limit regarding the methodology of the study, some clinical implications for intervention may arise. First, the assessment of high-risk infants and first signs of delay in the context of preterm birth should benefit from the inclusion of a series of diagnostic and observative instruments , have significant impact on infant development across the first postpartum year.ad hoc tailored interventions should be promoted to assess the risk of preterm infants’ delay and anxiety symptoms with adequate tools, to offer special support and treatment for symptomatology and to enhance parental functioning. This could help to implement more accurate interventions, as suggested by Developmental Care . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.EN prepared the study design, organized the sample recruitment, collected data, performed statistical analysis, and contributed to the writing of all the sections of the manuscript. FG performed statistical analysis and contributed to the writing of all the sections of the manuscript. FM, ET, AB, and MS contributed to the preparation of the study design and supervised data collection and the research team. FA prepared the study design, supervised all the phases of the research study and contributed to the writing of all the sections of the manuscript. All authors reviewed and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The search for an active, stable, and abundant semiconductor-based bifunctional catalysts for solar hydrogen production will make a substantial impact on the sustainable development of the society that does not rely on fossil reserves. The photocatalytic water splitting mechanism on a  However, some of these materials suffer from poor absorption of solar light, and photo-corrosion. Therefore, finding an efficient photocatalyst for water splitting is still a challenging problem. At present, the best-known catalyst for oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) is ruthenium oxides, iridium oxides, and platinum-based materials9. However, the scarcity and high cost of noble metals and precious metal oxides impose severe limitations for large-scale applications. The development of efficient and stable materials for OER and HER, by modifying chemical composition, structure, morphology, and support, is therefore of largest importance.Fossil resources, such as oil, gas, and coal are depleting rapidly, which urge the research community to find alternative energy technologies to be able to solve the demands of global energy issues. One of the promising alternative technologies is solar hydrogen production through water splitting with the help of semiconductors. The past few decades have seen tremendous efforts in the discovery and fabrication of highly efficient semiconducting photocatalysts for water splitting, including metal oxides, metal sulfides, metal nitrides, and metal oxynitrides14. Graphene-based materials, like N-doped graphene16 and N-containing co-doped graphene17, have been reported as excellent catalysts for HER and OER of high activity. Transition metal dichalcogenides (TMDs)19, metal monochalcogenides20, MXenes22, g-24, and 27, have also been identified as potential catalysts for water splitting.Two-dimensional (2D) materials have attracted increasing attention in numerous research fields during the past few years. This is owing to their superior properties, which are completely different from their bulk counterparts. The prospect of using these 2D materials in the field of energy conversion and storage, is very promising due to their various intrinsic properties, such as the presence of many surface-active sites, enhanced electron-hole separation, fast mobility of photogenerated charge carriers, and reduced recombination rate. After the graphene’s breakthrough, a wide range of 2D materials for HER and OER have been extensively examined28. This is a theoretically predicted material that possesses high mechanical, thermal, and lattice dynamical stability. The reported carrier mobility at room temperature is higher than that of many other 2D materials31. All these exceptional properties make the 32 elucidated the oxygen reduction reaction mechanism for the 33 studied the photocatalytic water splitting activity onto this monolayer through the band edge position with respect to the water redox potentials. However, the reaction mechanism for the catalysis of HER and OER have not yet been reported for this material.The The purpose with the present study has been to get a deeper understanding about the reaction mechanisms of HER and OER that takes place on a 33. Moreover, the band decomposed charge density, corresponding to the valence band maxima (VBM) and the conduction band minima (CBM) at the 34, phosphorene (3.48)35, and 36. This indicates the bonding in The optimized structure of the mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimIn order to gain more insight into the thermal and dynamical stability of the 37.It is also essential to investigate the stability of the mentclass2pt{minimmentclass2pt{minim38. Therefore, the Next, to evaluate the corrosion resistance or electrochemical stability of The electronic properties of the see Fig. b, was al39. For the situation with an atomic O adsorbate, the oxygen atom was found to break the Be−N bond and form a chemical bond with both Be and N. As can be seen in Fig. p-states are strongly hybridized near the Fermi level. Due to this circumstance, the Be and N atoms will buckle up from its xy−plane, which is a clear indication of a spBefore investigating the OER and HER mechanisms, the adsorption of the reaction intermediates O, OH, OOH, H, mentclass2pt{minimxy−plane, indicating the occurrence of sp40. Moreover, the OH adsorption also introduced unoccupied defect states in the bandgap region, as can be seen from the PDOS spectrum in Fig. For the situation with an OH adsorbate, it was shown to prefer to bind on top of the Be atom, and the Be−O bonding distance was found to be 1.63 Å . The Be atom is thereby somewhat buckled up from its entclass1pt{minimasee Fig. b indicatAs can be seen in Fig. entclass1pt{minimaAtomic hydrogen was also observed to prefer to adsorb strongly onto N 42,4342,43\docThe water molecule adsorption on a The calculated reaction free energy profile, with different electrode potentials, is displayed in Fig. The first and third steps in the OER mechanism involves a proton extraction from water, and these two steps are more energy demanding than the other reaction steps. In order to estimate their associated barrier energies, the minimum energy path (MEP) for the dissociation of a water molecule into hydroxide and a proton were identified .8\documFigure 45. If The hydrogen evolution reaction mechanism on the 48:This reaction can proceed in two possible ways, either by hydrogen or by a water molecule. Both situations have here been looked at. The HER reaction steps are as follows (see Eqs. –11))46–)46–11))4Figure 50, 51, and Pt52, etc. However, the 53 . This material can only facilitate HER. g-CN is another example54, which shows good activity for OER (but not HER). In addition, the CdS nanotube55 has larger HER and OER overpotentials  strongly affect the catalytic activities. The adsorption strength should not be too strong, or too weak. Usually, in OER, the rate-determining step would be O* The reaction mechanism of the photocatalytic water splitting onto a Vienna Ab initio Simulation Package (VASP)57. The generalized gradient approximation proposed by Perdew, Burke, and Ernzrhof (GGA-PBE)58 with van der Waals(vdW) correction proposed by Grimme, was used for the geometrical relaxation (due to its good description of long-range vdW interactions)59. Moreover, the Heyd-Scuseria-Ernzerhof (HSE06) hybrid functional was used in calculating the electronic structures 60. The Brillouin zone was sampled by using the Monkhorst-Pack scheme, with an employed k-mesh of 61. In addition, a plane-wave basis set, with a cutoff energy of 450 eV was used. A large vacuum of 18 Å was used to avoid spurious interactions. All structures were optimized until the total energy converged to less than 1062. Moreover, the PHONOPY code was employed to calculate the phonon dispersion curves of the 63. In addition, 64 was used to control the temperature. The transition state was searched using the climbing image nudged elastic band (CI-NEB) method66, as implemented in QuantumATK-2019.03.All calculations were performed using the projector augmented wave (PAW) method, implemented in the 67 and co-workers. By using this model, free energy changes of electrochemical reactions have been calculated by using the expression shown in Eq. , and e is the number of transferred electrons. Here, kB is the Boltzmann constant, the temperature T has been taken as 300 K, and pH=0. Zero-point energy correction and entropies for adsorbed intermediates were calculated from vibrational frequencies analysis using density functional perturbation theory, whereas the entropies of the species in the gas phase were taken from the NIST database68.Free energies for the intermediates in the oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) have been computed by using the computational hydrogen electrode (CHE) model developed by Nørskovn in Eq. 13\documeSupplementary Figures.


These systems were subjected to a robust intra-laboratory qualitative validation process prior to their application, by DNA extraction and fast real-time PCR, on some real market samples to reproduce a potential allergen contamination along the food chain. The developed system results were specific and robust, with a sensible limit of detection . The performance and the reliability of the target systems were confirmed on commercial food samples. This molecular approach could be used as a screening or as a support tool, in association with the other widespread monitoring techniques (such as ELISA).Food allergy is a worldwide health problem that concerns infants to adults. The main health risk for sensitised individuals is due to the presence of traces of allergens as the result of an accidental contamination during food processing. The labelling of allergens such as sesame, pistachio, and macadamia nut on food products is mandatory according to Regulation (EU) N. 1169/2011; therefore, the development of suitable and specific analytical methodologies is advisable. The aim of this study was to perform a multi-allergen real-time PCR system that works well in fast mode at the same annealing temperature and with the same thermal profile. The real-time PCR was developed designing new, specific, and efficient primer and probe systems for the  Food allergies are considered a relevant public health problem that affects over 220 million people worldwide ,2. They genes of the target as an indirect method [On this basis, the development of suitable and specific analytical methodologies to ensure consumers protection and compliance with food labelling regulation is strictly necessary. The available approaches for the detection and quantification of food allergen are immunological, proteomics, and DNA-based tests, especially enzyme-linked immunosorbent assay (ELISA) and lateral flow device, mass spectrometry (MS) and polymerase chain reaction (PCR), respectively ,10,11. Et method .The aim of the study was to implement the panel of food allergens already investigated in our control laboratory (soybean and peanut) . TherefoFrom an accurate literature investigation, the real-time PCR systems for sesame, pistachio, and macadamia nut did not satisfy the established parameters.(Sesamum indicum L.) belongs to the Pedaliaceae family. Roasted sesame seed could be found in different types of food such as bakery products , processed meat, fast foods, vegetarian food, and ethnic dishes, or it could be consumed in the form of oil used for cooking above all in Oriental, Chinese, and South American cuisines [Sesame cuisines ,20,21. Scuisines ,23,24, acuisines .2S albumin gene (NCBI acc. No. AF240005), obtaining variable limit of detection (LOD) values [oleosin gene (NCBI acc. No. U97700), while Zhang et al., 2018 [2S albumin in SYBR green assay. Furthermore, Lopez-Calleja et al., 2015 [To date, some of the allergenic proteins of sesame have been identified ,26, some) values ,32,33. K) values ,35 condul., 2018  studied l., 2015  used a ll., 2015  describe(Pistacia vera), is an edible tree nut; the kernels can also found in ice cream, confectionery, and other gourmet products or can often be eaten whole, either fresh or roasted and salted. The fraudulent substitution of pistachio nuts is not very frequent because of the high price and its presence also in the form of a lower-quality variety in processed products is usually due to adventitious contamination [dehydrin (Cor gene) (NCBI acc. No. Y07600) [genes (NCBI acc. No. AY677201) was the target gene of two real-time PCR with the hydrolysis/TaqMan probe method [gene in three methods: a multiplex PCR coupled to capillary electrophoresis [Pistachio belongs to the Anacardiaceae family together with cashew and mango, and allergenic cross-reactivity between these species could be registered. Pistachio nut, the fruits of the pistachio tree mination . To datemination , and themination . Pis v1,mination . Furthermination . To datemination  with a lmination , and a m Y07600) , with a e method ,44, obtaphoresis  with a rphoresis . For theMacadamia integrifolia (Maiden and Betche) and Macadamia tetraphylla (L.A.S Johnson), of commercial importance. Macadamia nuts are appreciated for their organoleptic properties and are largely consumed either directly, roasted, or as a component of confectionery products such as filled chocolates or nougats [vicilin precursor gene (NCBI acc. No. AF161883) as the target for TaqMan real-time PCR and LPA systems, respectively; López-Calleja et al., 2015 [gene in real-time PCR and TB green  singleplex/tetraplex PCR, respectively. All the methods exhibited a 100% of specificity, even if Brežná et al., 2009 [At last, macadamia nuts, belonging to Proteaceae family, are shelled kernels of the fruits of two species, namely  nougats . Fraudul nougats  and Ehle nougats  used thel., 2015  applied l., 2009  tested al., 2009  to 0.1 ml., 2009 .2S albumin gene for sesame and pistachio and the vicilin precursor gene for macadamia nut, rather than internal transcribed spacer (ITS), which is characterised by species variability. To meet the project aims, it was necessary to develop and validate new systems for pistachio, sesame, and macadamia nut allergens, which were extracted with an efficient and “food field-universal” extraction protocol [From the bibliographical investigation, we decided to adopt systems based on allergenic targets, in particular, the protocol . Another® QUANTARD Allergen 40  and Allergen RM 800  were used as positive controls for both the DNA extraction and the real-time PCR.Commercial sesame seeds, pistachio, macadamia nut, and the other samples used in the experimental tests were purchased in a local supermarket. Some samples were milled with Grindomix GM200 , adding dry ice to avoid melting and aggregation in high-fat products. All the ground samples were conveniently homogenised using the plastic bag technique . Nuts, s®, Duren, Germany) for DNA purification. The starting material for DNA extraction was 2 ± 0.2 g for not homogenised matrices and 0.2 ± 0.02 g for uniform ones. To obtain a higher yield, DNA was concentrated and eluted in 50 µL of elution buffer (DE), as also reported in Pierboni et al., 2018 [gene [DNA was extracted twice according to the protocol published in Pierboni et al., 2018 , combinel., 2018 . DNA qual., 2018 ,53 in fa18 [gene . The tes®, Foster City, CA USA), which is a specific tool to provide the best qPCR assay design on the basis of different considered parameters and of the lower penalty. For pistachio, two sets of oligonucleotides, designed on two different regions of the target gene and with differently labelled probes, were assessed. The amplicon length for sesame, pistachio (a), pistachio (b), and macadamia nut were 77 bp, 62 bp, 59 bp, and 78 bp, respectively.The primers and probes used in this study were synthesised by Invitrogen  and Metabion  for BHQ1 quencher; details are reported in ® Fast Universal PCR Master Mix (2X) No AmpErase UNG , 2 µL of DNA, and primers/probes at concentrations obtained in optimisation test. Data analysis was performed by SDS 2.4 software . Each PCR run included not template control (NTC) and positive amplification control.Real-time PCR was run in fast mode by a 7900HT Fast Real-Time PCR System , using the following cycling conditions and thermal profile: 95 °C for 20 s, 40 cycles with 95 °C for 3 s, and 60 °C for 30 s. The reaction volume was 20 µL, with 1X of TaqMan® Universal PCR Master Mix.For the optimisation of sesame primer and probe concentrations, about 200 hge , corresponding to about 0.2 ng of DNA, was tested assuming that the haploid genome size (1C) is 0.97 pg ,56. For During the design, all primers and probes were successfully checked in silico for relevant homologies by the Blast program on the Nucleotide Collection (nr/nt) sequence database, (BLASTN) within the GenBank databases in the NCBI . In vitro, the specificity was checked experimentally in a wide exclusivity panel of a total of 31 samples, including also allergen and botanical closely related species; these samples were tested in duplicate with concentrations adjusted on Cq 25–28 for the most of the samples and on Cq 29–32 for those DNA that were more difficultly extracted , whose Cq values were obtained by actin fast real-time PCR inhibition assay . Each sy10 and was verified in 60 replicates together with the upstream or downstream dilution to define LOD95 or the absolute LOD of the method, corresponding to a dilution point with at least 59/60 amplification signals. The amplification efficiency and linearity were represented respectively by slope and correlation coefficient R2 of the regression line obtained with the first five dilutions used in the LOD10 test [The LOD of sesame, pistachio, and macadamia nut methods was evaluated testing 9 concentrations series of DNA in decreasing ng and/or hge, each in 10 replicates. The first point of dilution corresponded to 320 hge or 0.31 ng, 0.12 ng, and 0.8 ng, respectively for sesame, pistachio, and macadamia nut systems. The last dilution, where all 10 results gave amplification, was defined LODD10 test ,57,58.®, Foster City, CA USA), with different master mix concentrations (−10%), different primer and probe concentrations (−30%), different reaction volumes , and with an annealing temperature farther from the Tm (melting temperature) of primers [In robustness assessment, the target sequence copy numbers or ng corresponds to the LOD value of the method multiplied by three . A multi primers .gene, testing one of each extract, after the inhibition test.In order to test and verify the feasibility of the validated systems for sesame, pistachio, and macadamia nut, some different products were collected at a local supermarket, selecting samples with PAL ; samples declaring the presence of allergen on the label; and samples not declaring the presence of allergen on the label. As no commercial products stating the presence of macadamia nut on the label were found in the local supermarket, spiked samples were prepared. The different products were ground with Grindomix GM200, homogenised, and then 0.2 g of macadamia nut were added to 1.8 g of starting sample. The matrix effect was also evaluated, co-extracting 0.2 g of macadamia nut as control. DNA extracted twice from the samples were assessed by real-time PCR for the allergen target gene revealed that the mean difference (ΔCq) between the measured mean Cq of undiluted DNA and its dilution 1:4 was in accordance with the parameters [DNA extracted from sesame, pistachio, and macadamia nut and from the collected commercial food samples were evaluated by the fluorometer, showing a final concentration in the range between about 40 and 100 ng/μL. The DNA concentration of the other samples used for the specificity test was always measured with a fluorometer and varied from about 10 to 200 ng/μL, depending on the type of matrix, where the lower values were obtained for leaves, seeds, and dried fruits. The lower concentrations concerned only a few species belonging to the various categories , which was probably due to the different variety, variable genome size, and components. The inhibition test carried out by fast real-time PCR for the actin rameters ,53. As d2S albumin), pistachio  gene , and macadamia nut target (vicilin-AMP) was selected according to the best parameters reported in the Materials and Methods section, and these were 900/900 nM, 900/150 nM, and 900/900 nM respectively for primer forward and reverse. The probe concentrations of sesame, pistachio , and macadamia nut were respectively 150 nM, 200 nM, and 250 nM. For probe selection, among the comparable different concentrations, also the reagent cost factor was taken into account. Data and the results relative to mean Cq, a standard deviation of repeatability (SDr), and ΔRn are listed in Variation of the primers and probe concentrations was tested in order to provide optimal assay performance. The primers concentration of sesame target  system revealed 59/60 amplifications in a concentration of 0.004 ng but only 54/60 for 0.002 ng; in the same way, for the pistachio (b) system, 60/60 amplification signals were registered in a concentration of 0.004 ng, but there were only 52 for 0.002 ng.The obtained limits of detection of sesame, pistachio , and macadamia nut systems are reported in Summarising, the practical LOD expressed in percentage was 0.005% for sesame, 0.004% for the two systems of pistachio, and 0.006% for macadamia nut. Considering these LOD values, it is possible to state that the developed real-time PCR systems are adequate, sensitive, and efficient also to detect very low quantities of the involved allergen.The linearity of sesame, macadamia nut, and pistachio  was above 0.99. Concerning PCR efficiency, the best percentage was obtained for pistachio (a), specifically about 98%, and then around 90% for the macadamia system, about 86% for sesame, and 80% for pistachio (b). The differences in PCR efficiency may depend on the sequence position where the oligonucleotide set anneals, as demonstrated by pistachio system (a) compared to (b).2S-albumin and vicilin-AMP genes appeared to be robust, with especially a concordance of 100% for the considered and analysed multi-factorial parameters [As shown in rameters ,59.Therefore, the behaviour of the two pistachio systems  was very similar, showing to be specific, sensitive, and robust at the same time. The only difference was observed in terms of efficiency, which was lower for the b system as also confirmed by a further experimental assessment.From the analysis of samples spiked with macadamia nut, no relevant matrix effects were observed: indeed, the same extracted matrices showed similar mean actin Cq, except for the “Tarallucci Salt snack”, where 3 cycles of difference were obtained.The spiked samples had lower repeatability than the relative matrices without macadamia nut, as highlighted by the higher standard deviation.genes codifying allergen proteins present in single copy; however, they were not present in multi-copy in the cell, such as the ITS region, which could interfere with the uniformity of the results obtained for different varieties and/or cultivars of the same species. This approach can be useful both to overcome cross-contamination problems related to multi-copy targets and possibly to translate these methods in digital PCR. This new molecular approach allows providing a precise and accurate quantification of nucleic acids without the need of a standard curve, especially for low concentration and/or complex samples, reducing susceptibility to inhibitors [The aim of the work was to implement the panel of food allergens already investigated in our control laboratory (soybean and peanut) with new target systems that work at the same conditions. Therefore, the study presented new fast real-time PCR assays designed for sesame, pistachio, and macadamia nut. The developed system results were robust, specific, and sensitive, and the systems were able to work well in a fast mode, at the same annealing temperature and with the same thermal profile. These advantages allowed performing a multi-allergens assay and verifying different targets in the same analytic session, saving time and protecting consumer safety. With the adoption of a “food field-universal” in-house DNA extraction procedure, it was possible to extract DNA of good quality from a large variety of matrices, even complex ones, resulting in adequate performance. This methodology could be easily implemented in any analytical laboratory that performs routinely real-time PCR, and it could be used in food control, safeguarding both producers and consumers against the presence of hidden allergens. Last but not least, we focused this study on hibitors . Furtherhibitors . In conc


Hemorrhoidal disease is one of the most common and frequently occurring benign anorectal disorders, presented with bleeding and prolapsed, and surgery is the main and effective method for severe prolapsed hemorrhoids. Yet, the recurrence rate after procedure for prolapse and hemorrhoids (PPH) is significantly higher. To reduce the recurrence rate and protect the anus function, we try to carry out a randomized, controlled, prospective study to compare the efficacy and recurrence rate of tissue selecting technique (TST) with mega-window stapler (TST-MS) combined with anal canal epithelial preservation operation and PPH combined with external hemorrhoidectomy and inferior internal hemorrhoid ligation operation for the treatment of severe prolapsed hemorrhoids.This study is a single-center, evaluator-blinded, randomized, controlled clinical trial. Participants meet the inclusion and exclusion criteria in this RCT will be randomly divided into treatment group  and control group  in a 1:1 ratio according to a computer-generated randomization list. The outcomes of recurrence, anal function, intraoperative variables, and postoperative complications will be recorded at different follow-ups.The findings of the study will help to explore the efficacy and recurrence rate of TST-MS combined with anal canal epithelial preservation operation on the treatment of severe prolapsed hemorrhoids.This study protocol was registered in open science framework (OSF). (Registration number: DOI 10.17605 / OSF.IO / 4JYNF). By removing the hemorrhoid tissue with pathological changes, it could largely damage the anal cushion and the anal canal epithelium, associated with postoperative pain, and a 10% recurrence rate.Hemorrhoidal disease is one of the most common and frequently occurring benign anorectal disorders, presented with bleeding and prolapsed. The incidence is extremely high in adults according to different studies. procedure for prolapse and hemorrhoids (PPH), also known as stapled hemorrhoidectomy, is an effective alternative by removing the prolapsed rectal mucosa on the dentate line to recover the anatomical position of internal hemorrhoids instead of just removing hemorrhoids. Several studies have shown it is an effective, safe, far less painful, and relative complication free procedure with fewer days off work.–10 However, a large number of studies also show that the recurrence rate after PPH is 3.4% to 45%, which is significantly higher than the recurrence rate of conventional hemorrhoidectomy.,11,12 Therefore, on the basis of the advantages of PPH technique, it is important to further reduce its postoperative recurrence rate.Since first described by Longo in 1998,–16 At the same time, patients with severe prolapsed hemorrhoids are often accompanied by protruding external hemorrhoids, and approximately 12.7% of patients still suffer from residual external hemorrhoids after PPH stapler surgery. The combined method of PPH technique with the external hemorrhoidectomy and inferior internal hemorrhoid ligation could significantly reduce postoperative edema and residual external hemorrhoids, which expands the indications of PPH technique. However, there is still insufficient tissue removal for severely prolapsed hemorrhoids, which makes internal hemorrhoids prone to prolapse again. Also, external hemorrhoidectomy and inferior internal hemorrhoid ligation could partially damage the dentate line and the anal canal epithelium, which can easily cause stricture of anus and influence the anus function.Other than that the pathological changes of the hemorrhoids were not removed, the primary reason for the high recurrence after PPH is that for patients with third-grade hemorrhoids with severe internal rectal prolapse, the limited capacity of the PPH stapler cannot remove enough prolapsed tissue.To reduce the recurrence rate and protect the anus function, we proposed a novel operation of tissue selecting technique (TST) with mega-window stapler (MS) combined with external hemorrhoid arc resection and suture to preserve the anal canal epithelium for the treatment of severe prolapsed hemorrhoids. The operation could make it better to treat internal hemorrhoids and hemorrhoid mucosa prolapse, reduce the incidence of internal hemorrhoids re-prolapse, completely excise the external hemorrhoids, preserve the anal cushion, dentate line, and anal epithelium, and protect the anal sensation and control bowel control. Therefore, we try to carry out a randomized, controlled, prospective study to compare the efficacy and recurrence rate of TST-MS combined with anal canal epithelial preservation operation and PPH combined with external hemorrhoidectomy and inferior internal hemorrhoid ligation operation for the treatment of severe prolapsed hemorrhoids.22.1 and the results will be reported according to the CONSORT Statement extension for trials . This trial has already been registered in open science framework (OSF) (Registration number: DOI 10.17605 / OSF.IO / 4JYNF). The protocol of the clinical trial has been approved by the Ethics Committee of Changshu Hospital of Traditional Chinese Medicine, and it will be carried out in accordance with the Declaration of Helsinki. The study protocol conforms to the Standard Protocol Recommendations for Interventional Trials (SPIRIT) 2013 Statement,2.22.2.1Participants meet the inclusion and exclusion criteria in this RCT will be mainly recruited from inpatients of proctology department in Changshu Hospital of Traditional Chinese Medicine.According to the previous report, the recurrence rate of PPH in the treatment of hemorrhoids was 9.6%. According to the preliminary experiment, the recurrence rate of severe prolapsed hemorrhoids by the TST-MS combined with anal canal epithelial preservation operation was 0%. Taking α = 0.05 and β = 0.2, 85 samples will be needed in each group. Considering the withdrawal rate of 20%, the total number of patients is determined as 204, that is 102 in each group.2.2.2Patients are included if they meet the following inclusion criteria:1.Patients meeting the diagnostic criteria for Circumferential mixed hemorrhoids, with internal hemorrhoids grade III–IV;2.Patients aged from 18 to 70 years’ old;3.Patients without obvious deformity of anus before the operation;4.Written informed consent.2.2.3Patients are excluded if they present the following exclusion criteria:1.Patients who have undergone mixed hemorrhoids surgery, or combined with other operations.2.Women in pregnancy, lactation, and menstruation.3.Patients with rectal cancer, rectal polyps, tuberculosis, Crohn disease, and other anorectal diseases.4.Patients with severe diseases of respiratory, digestive, circulatory, and blood systems, and obvious abnormal liver and kidney functions.5.Patients with acute inflammatory or thrombotic external hemorrhoids.2.2.4The patients will be randomly divided into treatment group  and control group  in a 1:1 ratio according to a computer-generated randomization list. Due to the limitation of treatment, the surgeons and patients are not blinded, and the statistical analysis will be carried out by an independent professional researcher who will not know the identification of the groups.2.3The preoperative and postoperative treatment of the 2 groups is the same. Before the operation, the patients will get blood routine test, urine routine test, liver and kidney function, blood glucose, coagulation, infectious disease indicators, electrocardiogram, chest x-ray examination, and abdominal ultrasound, colonoscopy, pathological biopsy, if necessary, to exclude surgical contraindications. The circumference of the anal canal will be measured before the operation. Intraspinal anesthesia will be used in all cases, and the left lateral decubitus will be taken during the operation. After the operation, the total amount of infusion in 6 hours is controlled within 500 mL to reduce the incidence of urinary retention, with 5 consecutive days of routine antibiotics.2.3.11. Internal hemorrhoid surgery will be performed with TST-33-MS  with the following procedure:① Routinely disinfect the perineum area;② Appropriately expand the anus, insert an anoscope, and remove the inner tube to reveal the hemorrhoid tissue to be removed;③ Purse—string suture the hemorrhoid with “0” silk thread 3 to 5 cm from the dentate line only on the mucosa and submucosa;④ Unscrew the tail wing of the stapler counterclockwise, put the head of the stapler into the anal expander after the head and the body of the stapler are completely loosened, tighten the 2 ends of the purse string around the central rod, lead the suture string out from the symmetrical hole of the stapler through the string lead-out rod, tighten the stapler clockwise, and pull the drawn rectal tissue into the staple groove. At this time, when feel the resistance of the knob, fire the stapler to achieve the cutting and anastomosis. After waiting for 30 seconds, unscrew the tail 3/4 lap counterclockwise and take out the stapler;⑤ Observe the anastomosis, suture to stop the bleeding if there is active bleeding.2. Surgery of external hemorrhoids will be performed with the operation of arc resection and suture to preserve the anal canal epithelium with the following procedure:① After the successful anastomosis, carefully check the remaining external hemorrhoids outside the anus;② Clamp the 2 ends of the external hemorrhoids with a vascular clamp, and make an arc-shaped incision in the anal canal skin line with a dermatome; peel from the incision and trim the skin edge to smooth if there are severe varicose veins;③ Perform mattress suture using 3-0 absorbable thread as the skeleton of the incision, and 4-0 absorbable thread for both ends and the suture gap;④ Examine the resected specimens and submit for pathological examination.2.3.2PPH stapler will be performed for internal hemorrhoids with the same procedure with that in the treatment group; the external hemorrhoids will be stripped to the vicinity of dentate line and ligated at a low position.2.4Follow-up of visits to the hospital is required at week 2 and 4 after surgery, and telephone follow-ups are conducted at week 3, 8, and 1 year after the operation, to report and record the following variables  method will be used, and 0 means no pain, 10 means the most pain. The VAS scores will be observed at 12 hours, 1, 2, 3, and 5 days postoperatively. Record the types and frequency of analgesics.② BleedingObserve the bleeding at 12 hours, 1, 7, and 14 days after the operation. Mild bleeding: The bleeding can spontaneously cease; Moderate bleeding: The large bleeding should be stopped after conservative compression; Severe bleeding: The massive bleeding needs suture hemostasis operation when compression is ineffective.③ Urinary retentionObserve and record the patients receiving at least one catheterization during hospitalization.④ Anal edge edemaObserve the edema around the anal at 12 hours, 1, 7, and 14 days postoperatively. Grade I: no edema; Grade II: mild edema occupies less than one-fourth of the perianal region; Grade III: edema occupies more than one-fourth and half or more of the perianal area; Grade IV: edema occupies more than half of the perianal area.2.4.3All possible adverse events (AEs) will be recorded on the Serious Adverse Event Report form, such as bleeding, anal stenosis, anal fissure, pain, urinary retention, anal fistula, prolapse, difficulty in defecation, urgency of stool, pelvic sepsis, systemic complications, and itching. In addition, AEs forms will record all deaths due to any cause during the course of the study.2.5P < .05 is considered statistically significant. Efficacy outcomes are analyzed on an intent to-treat (ITT) basis who has at least 1 follow-up, and the last observation carried forward rule will be applied to manage missing data. Considering the homogeneity, group T test for normally distributed data or Mann–Whitney U test for non-normally distributed data will be used to compare variables between groups. For differences within each group, repeated measured analysis of variance will be used for assessments in different time points, whereas Wilcoxon signed-rank test will be selected for non-normally distributed data.Statistical analyses will be performed using the Statistical Package for the Social Sciences (SPSS) v22.0 software . Statistical testing is 2-sided and 3The single PPH technology for all prolapsed hemorrhoids is no longer in line with current clinical needs, which makes it an important issue to improve the surgical instruments and operation methods, as far as possible to solve the contradiction between curative effect and complications, and to expand the indications. It has become an important problem to be solved in the operation of severe prolapsed hemorrhoids. Therefore, to improve the clinical efficacy reduce the recurrence rate, and protect the anal function for severe prolapsed hemorrhoids, we try to improve PPH technique and expand the indications of stapler to completely preserve the anal cushion and epithelium. Compared with the conventional MMH technique, our novel operation could reduce the damage to the tissues, which will make it faster to heal and cause fewer complications, and compared with current PPH operation, it can significantly reduce postoperatively prolapsed internal hemorrhoids and residual external hemorrhoids, and expand the indications of the stapler, so as to cure hemorrhoids while protecting the anal function.However, there are still some limitations in this protocol. First, due to the different operation methods, this study cannot be double-blinded, which may lead to certain biases. Second, this study is a single-center study, and the source of patients is relatively single and limited, which may affect the conclusions to a certain extent.Conceptualization: Liping Weng, Hua Huang.Investigation: Liping Weng, and Lei Li,Project administration: Lijiang Ji, Lei Li, Liping Weng.Study design: Lijiang Ji, Lei Li, and Jun WeiSupervision: Yuemeng Hu, Hua Huang, Jun Wei.Validation: Jun WeiWriting – original draft: Lijiang Ji, Lei Li, Liping Weng.Writing – review & editing: Hua Huang, Jun Wei.