Treating disordered feeding at a young age reduces risks of future feeding problems, but not all children profit equally; can we define predictors of a worse prognosis?In 252 children, with a mean age of 4; 7 years , who had undergone behavioral day treatment in the past, several variables were investigated, retrieved from initial consultation (t1) and re-assessed at follow-up (t2).Logistic regressions were carried out with sex, gastro-intestinal problems, refusal of the first nutrition, syndrome/intellectual disability, Down's syndrome, autism spectrum disorder, comorbidity of medical diseases , restrictive caloric food intake and selective food intake, as the predictor variables from t1, and age-appropriate food intake at t2 as the dependent variable. The potential role of sensory processing problems was reviewed at t2.About 73% had improved towards an age-appropriate food intake. Sex (boys), syndrome/intellectual disability, and a lack of varied nutritional intake at t1 were predictors of a worse prognosis. We found a small, but significant correlation between current selective eating patterns and general sensory processing problems.Feeding disordered children, especially boys, with intellectual disabilities or selective eating patterns are at risk for not achieving an age-adequate food intake at a later age, despite behavioral treatment. Problems may be temporary, but some children are at risk for persisting in disordered feeding/eating. The Diagnostic and Statistical Manual of Mental Disorders DSM-5)  describe  describThe literature indicates multiple factors that may contribute to the development and maintenance of disordered feeding and which could interfere with a successful feeding “career”. In children with intellectual disabilities, cases of disordered feeding as high as 80% are reported –11. CalvN = 95 children) with ASD. These sensory processing problems consisted of hyper- and/or hyposensitivity to tactile stimuli, odor stimuli, texture, color and temperature.ARFID was reported in children with gastrointestinal symptoms, a history of vomiting/choking, and a comorbid medical condition . The oddAn association between sensory hypersensitivity and picky eating behavior in typically developing children was also found by Nederkoorn and colleagues . In thisN = 20; age 1–3 years); outcomes were satisfactory. Improvement was observed in all primary outcome measures  and a high rate of satisfaction was reported at three months follow-up. The other RCT  was developed in The Netherlands , and entAlthough the above studies imply that an intensive behavioral intervention is effective in reducing disordered feeding in young children, clinical practice shows that not all children benefit optimally, and persist in disordered feeding at a later age. Some patients may show a relapse after successful treatment. It is unclear which factors predict who will and who will not achieve an age-appropriate food intake (years) after treatment. Therefore, we aimed to identify predictors from the initial consultation (t1) at a tertiary center for feeding/eating problems that are related to a continuation of disordered feeding at a later age (t2). To this end, we contacted all children who had received an intensive day treatment, several years after finishing this treatment, in order to investigate their present state of feeding/eating and current sensory processing problems. Several variables at t1 were taken from these children's files to investigate their predictive value regarding the present state.Due to the retrospective nature of this study and its start before ARFID was officially included in the DSM-5, participants received no official ARFID diagnosis as measured by standardized measures. Therefore, we decided to describe these problems as “disordered feeding” and to take “age-appropriate food intake” as the outcome measure. Still, the feeding and eating problems that are treated in our last resort treatment center, have always been identical to what we now call (severe) ARFID.in)appropriate food intake. Furthermore, we investigated the relationship of these ten variables with current sensory processing problems by correlating them with the parents' scores on a sensory processing measurement at t2.In this study we investigated the following question: What percentage of children who had received an intensive behavioral day treatment in the past for their disordered feeding/eating patterns did (not) display an age-appropriate food intake, several years post treatment? We hypothesized based on the literature that the following variables might be associated with a worse outcome: (1) an Intellectual Disability (ID) or related syndrome; (2) pre-dysmature birth; (3) Autism Spectrum Disorder (ASD); (4) Down's syndrome; (5) serious gastro-intestinal problems; (6) organic failures/diseases; (7)  sex; (8) cultural background (Caucasian vs. non Caucasian); (9) refusal of the first nutrition after birth, and (10) (older) age. We investigated whether any of these variables were related to a present age-(2.2.1.N = 291) who had entered the intake phase, and obtained treatment between 2009 and 2015, at a specialized treatment center for children with disordered feeding or eating, were invited for participation in this study by letter. The parents of 39 children did not respond or refused participation. The remaining sample consisted of parents of 252 children (109 girls (43.3%) and 143 boys (56.7%)) with a mean age of 4; 7 years  at initial consultation (t1). During the telephone survey (t2), which took place after - on average - 4; 8 years after t1, their mean age was 9; 3 years .The parents of all children , Maastricht University (ERCPN_20180222_0490).2.3.All parents of the children involved were referred to the specialized treatment center by a pediatrician. At initial consultation, a multidisciplinary team consisting of a pediatrician, dietician, and psychologist examined the child and obtained a history of the child's feeding and several other relevant variables. During the initial consultation (t1), the child's feeding pattern was determined and a treatment advice was provided. The children in the present study all had serious feeding/eating problems and participated in the behavioral day treatment program . The treatment involved a (cognitive) behavioral procedure, named SLIK, which is described in the introduction section. In addition to the behavioral therapists, a dietician, speech therapist and a pediatrician were also involved to monitor each child's treatment steps towards an adequate food intake, oral motor skills, body weight and other health issues.The treatment had a varying duration, depending on pre-defined  treatment goals taking into account the severity of the feeding/eating problems, child's age, cognitive abilities and underlying problems, and parents' potential. The treatment ended when these goals were achieved. If possible, a more varied diet and a healthy weight were pursued, and the use of medical and/or tube feeding was phased out. The average duration of the inpatient behavioral treatment was 7 months, (range 4–14 months).At a second time point (t2) which occurred, on average, 4; 8 years after t1, the parents of the children whose treatment had ended in that previous period, and had agreed to participate in the study were approached to take part in a telephone survey, to discuss their child's current feeding/eating status and to take the sensory profile questionnaire.2.4.in)appropriate eating pattern at t2.The study was a retrospective correlational study, with measurements taken at two time points (t1 and t2). We aimed to establish which, if any, variables measured at t1 could successfully predict an age-(2.4.1.(a)Sex .(b)Ethnicity .(c)Prematurity/dysmaturity Medical comorbidities at t1 .(e)Presence of gastrointestinal disease at t1 .(f)Down's Syndrome (DS).(g)Having a delineated genetic syndrome .(h)Autism Spectrum Disorder (ASD).(i)Refusal of the first nutrition after birth .(j)Age at t1.(k)Latency time between t1 and t2.Next, we reviewed the child feeding performance on a 12-item rating scale derived from the Seyshuizen Food Refusal Questionnaire (SFRQ appropriate food intake (code 1), or age-inappropriate (code 0) at t1. If the child scored “no” (code 0) on all these binary variables  rated at t1, this was ranked as “age-appropriate” (referred to as “AAFI”). If the code on at least one of these variables was “1”, this was ranked as age-inappropriate food intake. Logically, all children classified as age-inappropriate at t1.First, all binary variables pertaining to a characteristic were coded “0” if absent and “1” if present .ire SFRQ ;, which 2.4.2.via a standardized flow-chart. Besides that, the interviewer rated the child's behavior on the Sensory Profile (The aim of the telephone interview (t2) was to collect information about the children's current status to determine the child's food intake, with similar qualifications as at t1,  Profile  by questThe independent interviewer was well experienced in conducting telephone surveys, and she was blind with regard to the child's specific feeding problems and background variables. The survey and the SP rating took 30 min, on average.inappropriate food intake (code “0”), or not (code “1”) at t2. In addition to the observed t2 variables, the latency time (in years/months) between t1 and t2 was calculated.Afterwards, it was determined by two researchers, independently, whether the child could be classified as having an age-2.5.2.5.1.To compile a representative group, 7 (t2) surveys corresponding with each year of consultation (between 2009 and 2015), were randomly chosen by an employee who was not involved in the research. Forty-nine surveys  were randomly selected and independently rated by 2 observers. For every survey, each observer rated the (1) SFRQ scale score (score range 1–12) and the binary variables (2) “RCFI”, (3) “LOVNI”, (4) “STC” and (5) “AAFI” (score 0 or 1) which amounts to a total of 245 (49 × 5) observed variables, each which were compared afterwards on agreement. Inter-rater reliability was determined by calculating Cohen's Kappa.2.5.2.t-test and determined the effect sizes using Cohen's d for differences between t1 and t2 outcome variables. Then, we computed correlations between all t1 and t2 variables followed by a binary logistic regression to assess whether one or more of the covariates proved predictive of an age (in)adequate food intake at t2. Due to the large number of predictors, significance was judged using an α level of 10%. This adjustment of the conventional significance level, which is common in exploratory backwards regression analyses, was chosen to decrease the probability of type II errors. The last step was to compute correlations between the predictor variables as well as the outcome variables RCFI, SFI, LOVNI, STC at t2 with the total score on the “Sensory Profile” at t2 to determine any relation between these variables and sensory processing problems.All statistical analyses were performed using SPSS 27. To examine the sample characteristics, we first conducted univariate analyses by calculating frequencies, means and standard deviations of the background variables, and presence and absence of all t1 variables, followed by frequencies and percentages of the dependent variables: restrictive caloric food intake (“RCFI”), selective food intake (“SFI”), lack of varied nutritional intake (“LOVNI”), selective texture choices (“STC”) and age-appropriate food intake (“AAFI”) at t1 and t2. We calculated significance for differences by a paired samples 3.3.1.We first determined the inter-rater agreement concerning the assessment of the survey results at t2 of the coded variables. A Kappa of 0.98 was obtained, indicating excellent agreement. Of the observed 245 ratings over the 49 surveys, the two independent raters disagreed about 3 ratings. These ratings were subsequently discussed, to reach agreement.3.2.The presence of gastro-intestinal diseases at t1 was remarkably high: in 73.4% (185 children). In addition, 97 children (38.5%) were diagnosed with comorbid organic diseases other than gastro-intestinal disease. Fifty-five children (21.8%) were born prematurely and/or dysmaturely. A delineated genetic syndrome (excluding Down's Syndrome) or intellectual disability (IQ < 70) was present in 67 (26.6%) children. DS was seen in 16 children (6.3%), and 34 (13%) of the children were diagnosed with ASD by a previous care provider. The occurrence of comorbid mental and organic involvement are displayed in not acquired an age-appropriate eating pattern. Cohen's d showed an effect size of −1,607 which is considered to be a (very) large effect size. The variable “restrictive caloric food intake” (RCFI t1:65%/t2:14%) showed also a great improvement with a Cohen's d of 0,995 which is considered to be a large effect size. The variables “lack of varied nutritional intake”,  with a Cohen's d of 0,712 and “selective texture choices”,  with a Cohen's d of 0,689 show somewhat smaller rates of improvement, but are still considered as medium to large effect-sizes. Overall, selective food intake , showed a substantial decrease, with a large effect size (Cohen's d = 0.866) but had still the highest presence (21%) at t2 of all variables regarding an age-inappropriate food intake. Restrictive caloric food intake (“RCFI”) was observed in 164 (65%) of the sample, while selective food intake (“SFI”) was seen in 179 (71%). Within the “SFI” group, 39% (98 children) were familiar with a lack of varied nutritional intake (“LOVNI”) and another 47% (119 children) with  selective texture choices (“STC”). At t1, none of the 252 children showed an age-appropriate food intake (AAFI). At t2, 27% of the children had It appears from the findings on the SFRQ that feeding skills (related to the need for tube feeding and texture differentiation), in general, improved over time. The mean score at initial consultation (t1) was “6”, as opposed to “11” (range 1–12) at t2. This score suggests that there was, in general, a decrease in (partly) tube feeding dependency, indicating an improvement in oral acceptance and, largely, in texture differentiation. At t1, a score of “1”  was observed as the most common observation, namely in 67 (27%) of the children, while at t2 this score was observed in just 6 (2%) of the children. An opposite shift was observed with regard to item 12 . At t1, score “12” was present in only 58 children (24%), as opposed to 198 children (79%) at t2.3.3.r = −0.335, p = 0.01), indicating that boys more often had a lack of varied food intake than girls at initial consultation. Another significant but weak correlation was found between the variable “LOVNI” and “age” at t1 (r = 0.307 p = 0.01) which means that children with “LOVNI” on t1 were, on average, older than children without “LOVNI”. A third (negative) significant weak correlation was found between “age” at t1 and “gastro-intestinal problems” , indicating that children with these problems were, on average, younger than children without these diseases. Between the two main selective and restrictive eating variables representing an age-inappropriate food intake at t1 namely: “SFI” and “RCFI”, a significant weak negative correlation  was found meaning that children with a typical selective eating representation do not automatically display a restrictive caloric food intake and vice versa. Especially the selective food intake (SFI) representation “LOVNI” seems responsible for this weak to moderate correlation  relative to “STC” . This suggests that the above found negative correlation between “RCFI” and “SFI” might especially be true for children with a lack of a varied intake and less for children with texture problems. A similar negative but almost negligible correlation  was found between these selective eating representations (“STC” and “LOVNI”) at t1 which might indicate that being selective in texture choices does not automatically imply having or developing a lack of varied food intake. Another weak but interesting (negative) correlation between “Syndrome/Intellectual Disability (Syn/ID)” and AAFI at t2 was found  which indicates that children without a syndrome and/or intellectual disability more often achieved an age-appropriate food intake at t2 than children with a syndrome and/or ID. However, given that the strength of significance is below 0.3, this correlation is negligible.We conducted bivariate correlations on all t1 and t2 variables including scale scores of the SFRQ t1 and t2 and the “Sensory Profile” at t2. In r = 0.200, p = 0.002) as well as sensory processing problems and selective food intake . However, also these relatively low associations should be interpreted with caution.Among the t2 variables, significant but weak correlations were observed between the general outcome scale score of the “SP” and the variables “STC” and “SFI”, that could indicate the possible expected relation between sensory processing problems and selective texture choices , RCFI-t2 , STC-t2 , and SFI-t2 . These correlations were expected and indicate that the SFRQ is an adequate measure to determine age-appropriate food intake.The SFRQ-scale score at t2 showed moderate to strong levels of correlation with the disordered eating outcome measures AAFI-t2 (3.4.p = 0.077) and appeared to be significant. This implies that boys more often are at risk for not achieving an age-appropriate food intake at a later age than girls. Second, “Syndrome/Intellectual Disability” had an OR of 0.424 (p = 0.011) which indicates that children with a syndrome and/or intellectual disability are more at risk for not achieving age-appropriate food intake than children without. A third significant predictor was found in “LOVNI” with an OR of 0.474 (p = 0.081), meaning that a less varied nutritional intake at a younger age predicts age-inappropriate food intake at t2. A fourth predictor was a lower score at the SFRQ-scale at t1 with an OR of 1.151 (p = 0.048); We used a logistic regression model to examine the predictive value of all t1 variables related to the outcome measure age-appropriate food intake at t2. An age-appropriate food intake implies no significant disturbances in the necessary caloric food intake, a varied nutritional food intake and a presentation of all textures in daily meals. In As displayed in 3.5.r = 0.267, p = 0.000) and STC , indicating that children with selective eating patterns and selective texture choices scored higher on the SP than children without these eating patterns. Further, a weak but significant negative correlation was found between SP and SFRQ-t1 , meaning that lower SFRQ scores at t1 were associated with higher SP scores at t2; however, due to their low strength, these could be interpreted as negligible. Unexpectedly, there was no significant association between autism spectrum disorder (ASD) and the “Sensory Profile” score  at t2.To test the hypothesis that sensory processing problems might influence reaching an age-appropriate food intake, we calculated correlations between SP total scores and all variables at t1 and t2. Unexpectedly, we found only weak positive, though significant correlations of SP with selective eating at t2: SFI  at a later age (t2), namely (1) having a syndrome and/or an intellectual disability (excluding Down's syndrome), and (2) being a boy. “Sex” was found to be the variable with the highest odds ratio  which means that boys are almost twice as much more likely to be at risk as girls. Despite boys in general being somewhat underrepresented in ARFID population appropriate food intake (AAFI) at t2. Regarding the eating measures the children with a lack of a varied nutritional intake (LOVNI) at t1 were predictive for not achieving an age adequate at t2. Also, the Seyshuizen Food Refusal Questionnaire (SFRQ)-score at t1 turned out to be predictive, indicating that a lower score at t1 was predictive for not achieving an age-appropriate food-intake at t2, which means that children who are fully tube fed seems to be more at risk. Concerning the Sensory Profile (SP) total score, we only found several weak, but significant correlations, all below a size of r = 0.3. Given that the relationship between sensory processing problems and eating difficulties is observed more often in the literature (inappropriate food intake (at t2) when behavioral treatment had preceded.In addition, we investigated whether restrictive (RCFI) and selective eating measures , and the SFRQ-scale score at t1, predicted an age- follow-up effectiveness post behavioral treatment intervention, are in line with the levels found in Sharp's studies , 19, 26 In children with a syndrome and/or severe intellectual disability, physical and neurological comorbidities are more common than in typically developing children. This may also explain why they are less able to meet the criteria of an age-appropriate food intake (AAFI) as stated in this study. Afterwards we can discuss whether this outcome measure is relevant for this group. Thus, feeding intervention may significantly improve intake and achieve developmentally appropriate targets, but falls short of age expectations. However, we also see children with these limitations who do meet this criterion.Having a lack of varied nutritional intake (t1), which also was predictive for not developing an AAFI, could possibly be a better subject of prevention. Confronting children in early stages of life with more varied foods, textures or tastes, if only by systematic touching and tasting, might prevent (increased) eating problems later in life .Limitations of the study concern the use of binary variables instead of Likert type scales which might show more variability. Because of the available chart review data, we chose for these types of variables, which is also inherent to this type of research. In addition to the observed binary variables from the chart reviews, we found supported measures on categorical variables for disordered feeding patterns in the SFRQ and the SP. The SFRQ showed good validity and reliability for the Dutch population  and onlywho refrained from behavioral treatment, they found that after an average follow-up period of 6; 3 years, only 37% of the participants had developed an age-appropriate eating pattern and 63% still had a restrictive and or selective eating pattern.The results of this study show a substantial improvement of feeding/eating problems in the long run and we can only substantiate that behavioral treatment may serve as a serious contribution to solving feeding/eating problems in children. We base this assumption also on another study by Dumont and colleagues . In a sinot achieving an age-appropriate food intake. Now that we know more about potential risk factors, we can look more specifically at targeted interventions for these children.In contrast, in this study we found in 73% of the children an age-appropriate food intake, still years after a behavioral intervention; a selection of the sample did not benefit optimally, being at risk for a disordered eating pattern, as with ARFID may be the case. Having a selective eating pattern at a young age, suffering from a syndrome with an intellectual disability and being a boy were found predictive for


Prenatal depression is one of the most common risks during pregnancy. This study examined the prevalence and likelihood of prenatal depression association with sociodemographic factors, paid sick leave, and place of care among U.S. pregnant women. We conducted bivariate Chi-square tests to assess the statistical difference and multivariable logistic regression models to assess the association of prenatal depression using the National Health Interview Survey, cross-sectional data from 2010 to 2019 of pregnant women aged 18–44 years (N = 957). The prevalence of prenatal depression was 40.6%, 28.5%, and 27.2% among White, Black, and other racial pregnant women, respectively. Pregnant women with no regular/routine place of care had a prenatal depression prevalence rate of 58.1%, and those without access to paid sick leave had 46.9%. Also, pregnant women without access to paid sick leave were found to have an increased likelihood of reporting prenatal depression , as well as those without a regular place of care . The findings identify factors that need to be addressed to minimize depression among U.S. pregnant women and establish the need for tailored interventions to address prenatal depression. HoweverAccording to a study by Risk factors such as adverse childhood experiences , relatioAccording to a Pew research report, the percentage of women giving birth in the past year was higher among immigrants  than among U.S.-born  . SeveralSocioeconomic status and health behaviors are important determinants of health. Previous studies have reported that low SES increases the risk of antenatal depression . Another22.1https://www.cdc.gov/nchs/nhis/index.htm). The NHIS is a cross-sectional household interview survey targeting the civilian noninstitutionalized U.S. adult and child population. The analyses conducted in this study were restricted to U.S.-born and non-U.S.-born pregnant women aged 18–44 years with depression. Although individuals born in U.S. territories are citizens, they are not U.S.-born. Hence, we included them as non-U.S.-born. Participants with missing responses for any variables of interest were excluded from the analysis  to minimize any statistical errors, resulting in a final sample of 957 pregnant women. See Data for this study came from the National Health Interview Survey (NHIS), 2010–2019  for depressed and “No.”  as not depressed. The second question was “How often do you feel depressed?” Response options were: “1 = daily; 2 = weekly; 3 = monthly; 4 = a few times a year; and 5 = never.” Similar to the first question, we recategorized the response options  as “Yes” for depressed and “No” for not depressed. The third question was “Do you take medication for depression?” with responses “Yes” and “No.” All “Yes” recategorized responses to questions 1 and 2 as well as question 3 were combined to indicate the presence of depression in pregnant women, and all “No” as not depressed.The primary outcome of interest was self-reported prenatal depression status among pregnant women. Individuals were identified as depressed based on three questions: First, “18–30” and “31–44” years, while race was categorized into “White,” “Black,” and “Other.” Household/family size was combined into two groups of “1–4” and “≥5”. Family income levels were classified based on the ratio of family income to poverty guidelines. Class of work was categorized as “Private” and “Government” and employment status as “Employed” and “Unemployed.” Independent variables such as diabetes and hypertension status were classified as “Yes,” having the disease and “No,” not having the disease. Regular place of care was dichotomized as 0= “No,” not having a regular place of care and 1+ “Yes,” having a regular place of care. Further, access to paid sick leave status was categorized into “Yes” and “No.” Alcohol and smoking status were dichotomized into “Never” and “Ever,” whereas leisure-time physical activity as “Inactive/Insufficient” and “Physically Active.” Finally, individual’s region of residence was grouped into “Northeast,” “North Central/Midwest,” “South,” and “West.”The independent variables included in our analysis were sociodemographic , socioeconomic , health characteristics , diabetes, hypertension, regular place of care), behavioral characteristics , access to paid sick leave, and region of residence. Age was dichotomized into 2.3We estimated the prevalence of prenatal depression and conducted a bivariate analysis using a Chi-square test to ascertain the differences in prenatal depression between the various categories of sociodemographic , socioeconomic , health factors , behavior factors , paid sick leave, and region of residence. See 33.1The between-group comparison showed that U.S.-born individuals with any history of smoking status had a higher prevalence of prenatal depression (47.3%) than non-U.S.-born individuals (34.0%). Also, the highest prevalence of prenatal depression was observed among U.S.-born individuals with a BMI ≥30 (47.1%), followed by 25–29.9 (39.2%), and <18.5 – 24.9 (30.0%). A similar pattern was observed among non-U.S.-born with 42.6%, 32.5%, and 29.6% for a BMI ≥30, 25–29.9, and <18.5 – 24.9, respectively, although there was no statistically significant difference in prenatal depression between these categories (p = 0.51).The prevalence of prenatal depression among pregnant women without access to paid sick leave was reported to be higher among U.S.-born than non-U.S.-born (47.9% vs. 42.9%). Pregnant women without a regular place of care reported a higher prevalence of prenatal depression among both US-born (55.4%) and non-US-born (54.5) than those with at least one regular place of care . In the total sample, the highest prevalence of prenatal depression by region was reported in the West (48.5%), followed by Midwest/North Central (37.2%), South (34.8%), and Northeast (32.2%). Among non-U.S.-born, the highest prevalence of prenatal depression was reported in the West (49.6%), followed by Midwest/North Central (33.4%), South (25.8%), and Northeast (17.0%) regions of the U.S. While a significant difference in prenatal depression was observed among the combined sample by region of residence, there was no difference among the separate subgroup of U.S.-born and non-U.S.-born. See 3.2The multivariable logistic regression analysis is presented in 4To the best of our knowledge, this is the first study that utilized the NHIS to investigate the prevalence and factors associated with prenatal depression among U.S.-born and non-U.S.-born pregnant women. As shown in our study, we found several factors associated with prenatal depression among U.S. pregnant population. Factors like age, BMI, access to paid sick leave, regular place of care, and region of residence were found to impact the prenatal depression status of pregnant women. A lack of access to paid sick leave, for instance, may impact prenatal depression among pregnant individuals in the U.S., as they are more likely to experience prenatal depression regardless of their origin of birth. Our results further show that pregnant women who lack a routine place of care had an increased likelihood of reporting prenatal depression. This means they are more likely to miss follow-up for prenatal and depression treatment, which may increase their morbidity and mortality during the postpartum period. Yet, access to paid sick leave improves healthcare access and timely treatment of health conditions, including mental health problems, for pregnant women and ultimately reduces their risk of experiencing postpartum depression. These risk factors for prenatal depression, therefore, could lead to an increased morbidity risk. As shown in this study, a significant majority of pregnant women  who did not have a place of routine care had a higher likelihood of reporting being depressed. This finding could also be due to a lack of access to prenatal and postnatal services for these populations. A similar problem is observed nationwide, with young adults and minorities less likely to have a primary care provider . The curThe finding that U.S.-born individuals without access to paid sick leave were more likely to experience prenatal depression is concerning. Paid sick leave is essential for pregnant women to access treatment and have improved health. As a result, a lack of access to paid sick leave could delay treatment and make health conditions and pregnancy complications worse. A previous study by Goodman and Schneider found that workers with access to paid sick leave have better sleep quality and less hunger than those without . SimilarMoreover, the lack of access to paid sick leave may increase the burden of prenatal depression among pregnant women, as shown in this study, especially if they already have other children under their care. Past studies have revealed that employed parents relied on paid sick leave to care for their children, especially those with chronic conditions . ConsequFurther studies that evaluate the prevalence of prenatal depression among pregnant individuals with access to paid sick leave, type of routine place of care, occupation, and/or industry type by birthplace or citizenship would be vital in gaining a broader understanding of depression among these populations. Such an understanding would aid in devising strategies for follow-up on chronic conditions such as diabetes, postpartum depression and hypertension that often contributes to maternal morbidity and mortality in the prenatal and postpartum period. Moreover, studies focusing on the impact of sociodemographic factors and depression among pregnant women locally could provide a better understanding of effective patient/person-centered medical care and public health collaborative strategies to mitigate the prevalence of prenatal depression and other chronic medical conditions.We observed differences in the region of residence and prenatal depression. Women residing in the West were more likely to report prenatal depression, whereas those in the South and North Central/Midwest were less likely compared with their counterparts in the Northeast. With a stratified analysis of U.S.-born and non-U.S.-born, only non-U.S.-born living in the West were found significant, with increased odds of prenatal depression compared with those residing in the Northeast. Future research is necessary to examine the regional variations of reported depression and factors contributing to these differences.5Despite the significant contributions of this study, it has some limitations. First, this study uses cross-sectional data; therefore, no causal inferences can be made on the effect of prenatal depression on the independent variables among such a unique population as pregnant women. Second, although we used sampling weight for the analyses, the small samples within some categories likely affected the estimation of prenatal depression among some populations such as the non-U.S.- born. Furthermore, to increase the sample size of our unique study population, we combined three depression questions. Although all screening indicators for depression used in the NHIS are validated, they were self-reported, which may differ from provider-diagnostic validated tools for depression. Also, the employment variable for unemployed includes individuals seeking work and not working for pay. This factor might have impacted the estimation of the results. Finally, social, emotional, and partner support factors were unavailable in the dataset that could have provided further insights into how pregnant women manage prenatal depression. Notwithstanding these limitations, our findings are significant, considering this is a unique sample and not much work has been done with nationally representative datasets.6We recommend providers use tailored, validated screening tools in care processes for these populations. Given the complexity of health care needs during pregnancy, it is imperative to ensure adequate care coordination with mental health providers. The lack of a regular place of care by the significant number of pregnant populations further shows the decreased likelihood of receiving timely and adequate treatment and follow-ups for conditions like gestational diabetes, preeclampsia, and other maternal and mental health services. Efforts to ensure access to routine care and culturally appropriate mental health services are imperative to addressing depression during pregnancy and postpartum.Saanie Sulley: Conceptualization, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. David Adzrago: Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Lohuwa Mamudu: Conceptualization, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Emmanuel A. Odame: Methodology, Writing – original draft, Writing – review & editing. Paul H. Atandoh: Writing – original draft, Writing – review & editing. Ishmael Tagoe: Writing – original draft, Writing – review & editing. David Ruggieri: Data curation, Formal analysis, Visualization, Writing – review & editing. Lisa Kahle: Data curation, Formal analysis, Validation, Visualization, Writing – review & editing. Faustine Williams: Project administration, Methodology, Resources, Visualization, Writing – review & editing, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


STXBP1-Related Disorder Patient Neurons,” by van Berkel et al. .Erratum to: “Reduced MUNC18-1 Levels, Synaptic Proteome Changes, and Altered Network Activity in STXBP1 mutation pathogenic for this patient is c.704G>A, p.Arg235Gln, and not c.[922A>T], p.[Lys308(∗)]. According to the authors, the reporting mistake in the previous article was caused by a mix-up between the mutations of two different patients affected by the same condition.A correction for this article has become necessary, as the mutation originally reported as K308X for patient 5 should instead be R235Q. Specifically, authors of a previous study, on which this study was partially built, recently found that the All references to this mutation have now been corrected to R235Q.


At the same time, the presence of Li+ or Na+ makes these solutions compatible with intercalationmaterials for the development of rechargeable alkaline-ion batteries.Although there is a growing interest in these systems, a fundamentalunderstanding of their physicochemical properties is still lacking.Here, we report and discuss the physicochemical and electrochemicalproperties of a series of solutions based on 20 mol kg–1 CH3COOK with different concentrations of CH3COONa. The most concentrated solution, 20 mol kg–1 CH3COOK + 7 mol kg–1 CH3COONa, gives the best compromise between transport properties andelectrochemical stability, displaying a conductivity of 21.2 mS cm–1 at 25 °C and a stability window of up to 3 Vin “ideal” conditions, i.e., using a small surface areaand highly electrocatalytic electrode in a flooded cell. Careful Ramanspectroscopy analyses help to address the interaction network, thephase evolution with temperature, and the crystallization kinetics.Highly concentrated aqueous binary solutions of acetatesalts arepromising systems for different electrochemical applications, forexample, energy storage devices. The very high solubility of CH In fact, among polar solvents, watershows optimal trade-off between a high dielectric constant and lowviscosity so that electrolytes can achieve higher conductivities thantheir organic counterparts. However, the use of water in high-specific-energybatteries is limited by its modest electrochemical stability window(ESW), especially on the cathodic side, which is affected by the presenceof protons or positively polarized hydrogen atoms.Water-in-salt electrolytes (WISEs) area new class of ionic solutions,which belong to the wider category of solvent-in-salt (SIS) electrolytes,9 allowing the developmentof high-energy aqueous devices.11 In WISEs, the increaseof ESW is generally attributed to the reduced activity of water atsuch a high concentration, being all of the solvent molecules incorporatedinto the solvation shells,12 which resultsin large overpotentials for the water electrolysis.13 More recent studies, however, have shown that the correctdetermination of E-SW expansion requires special attention to experimentalaspects14 and is influenced by the natureof the species formed at the interface, the study of which requiresadvanced characterization techniques.15Water-in-saltelectrolytes (WISEs) were developed with the aimto increase the ESW,16 lithium (pentafluoroethane sulfonyl)(trifluoromethanesulfonyl)imide(PTFSI), lithium bis(trifluoromethanesulfonyl)imide (LiTFSI),2 sodium trifluoromethanesulfonate (NaOTF),17 sodium bis(fluorosulfonyl)imide (NaFSI),18 and potassium trifluoromethanesulfonate (KOTF).19 Despite the outstanding improvements in thecorresponding ESWs, the environmental and economic concerns remainregarding the use of fluorinated salts. In particular, LiTFSI, whichis widely used in LIBs, exhibits severe oral and dermal toxicitiesand can cause chronic aquatic toxicity.12 Furthermore, the limited availability and very high cost of thesefluorinated salts practically nullify the advantages brought by superconcentration,making the practical implementation of WISEs in energy storage verychallenging.21Many WISEs employ per-fluorinated sulfonyl imide salts, suchaslithium (fluorosulfonyl)(trifluoromethanesulfonyl)imide (FTFSI),3COONa (NaAc), and CH3COOK(KAc). In particular, KAc was recently investigated by our group22 and proposed in potassium-ion batteries (KIBs),and supercapacitors.23 An interesting aspectof KAc, which broadens the scope of its applications, is the possibilityto couple it with Li, Na, and Zn salts to achieve binary electrolyteswith salt concentrations above 20 mol kg–1,24 enabling the use of such systems in differentbattery chemistries. For instance, Chen et al. reported a mixed-cationelectrolyte composed of 32 mol kg–1 KAc and 1 molkg–1 (CH3COO)2Zn, allowinga (2.0 V) Zn-MnO2 aqueous rechargeable cell.25 Another interesting report features an LIB equippedwith 32 mol kg–1 KAc with a 7 mol kg–1 CH3COOLi electrolyte, utilizing C-TiO2 andLiMnO4 as anode and cathode active materials, respectively.12 Even more interesting is the binary solutionwith sodium, given the low cost and wide distribution/availabilityof the raw materials. In this view, a sodium-ion battery was recentlydeveloped based on the use of a 32 mol kg–1 KAcwith an 8 mol kg–1 NaAc solution coupled with NASICON-typeelectrodes.26 Despite their technologicalinterest, the interactions between the components of these liquidphases, which underlie the increase in ESW, are little investigatedboth in the bulk and with respect to the electrochemical propertiesof the interfaces. Interactions among components are also crucialto understand thermal, rheological, and electrical properties, thusaiding the design of new electrolytes. Raman spectroscopy is a usefultool for both the determination of electrolyte structure and the studyof ion/ion and ion/solvent interactions.27Examples of cost-effective,highly soluble, and widely availablesalts are LiCl, CH–1 andincreasing NaAc up to 7 mol kg–1. The physicochemicaland electrochemical properties  of the binary system, which are crucial to correctlyaddress its application in aqueous sodium-ion batteries, were deeplyinvestigated. In addition, the increased ESW and peculiar thermalproperties were rationalized by studying the atomic interactions withRaman spectroscopy measurements performed both at different NaAc concentrationsand in a wide temperature range.Here, we investigated a binary system made of KAc and NaAcby keepingthe KAc concentration constant at 20 mol kg–1) under sonication.Keeping KAc constant at 20 mol kg–1 and varyingthe concentration of NaAc, four different electrolytes were prepared,namely, 1 mol kg–1 NaAc with 20 mol kg–1 KAc (1N20K), 2.5 mol kg–1 NaAc with 20 mol kg–1 KAc (2.5N20K), 5 mol kg–1 NaAcwith 20 mol kg–1 KAc (5N20K), and 7 mol kg–1 NaAc with 20 mol kg–1 KAc (7N20K). All of theelectrolytes were stored and used at room conditions. The solutiondensity was measured by volumetric analysis, and the correspondingmolar concentration was calculated. Appropriate amounts of NaAc and KAc  were dissolved in Milli-Qwater . A parallel plate(50 mm) setup was used to perform shear tests with shear rates inthe range 10–300 s–1, followed by a 5 min isothermat −120 °C, and finally by a heating step from −120to 90 °C at a rate of 1 °C min–1. Initially,all of the samples were equilibrated at 30 °C for 30 min to ensurea completely liquid state. The heating/cooling rate of 1 °C min–1 was selected, following preliminary tests, as thebest compromise to allow the system to undergo structural relaxationwithout excessively sacrificing sensitivity.Differential scanning calorimetry (DSC)was performed using a DSC 1 Star (Mettler Toledo). STARe softwarewas used for the evaluation of the data. The samples were analyzedin a temperature range from −120 to 90 °C. The followingprotocol was used: cooling from 30 to −120 °C at a rateof 1 °C min–1. Impedance spectrawere recorded in a frequency range from 1 Hz to 200 kHz with an amplitudeof 25 mV. All of the samples were first degassed with nitrogen flux,and the measurement was done under N2 flux in the temperaturerange from 5 to 70 °C in a climatic chamber .For all of the samples, the bulk ohmic resistance was recorded viathe x-axis intercept at high frequencies in theNyquist plots.Conductivity measurementswere performed using electrochemical impedance spectroscopy (EIS)employing a dip probe cell with two platinum foils in a glass casing,with a cell constant of 1.04 cm+ laser as an excitationsource with a resolution of 2 cm–1 by three accumulationsof 30 s of integration. The beam was focused on a circular spot throughthe optics of a microscope (Olympus), a long working distance objectivewith 50× magnification, and a numerical aperture of 0.60. Measurementsin the temperature range from −150 to 20 °C were carriedout by means of a cryostat working with liquid nitrogen flux and aprogrammable heater, with a final thermal stabilization within ±2°C.Raman spectra werecollected in backscattering configuration using a Labram Dilor spectrometer(JobinYvon) and the 488 nm line of an Ar–1.The ESW was estimatedby linear sweep voltammetry (LSV) in a flooded three-electrode cellequipped with glassy carbon as working, platinum mesh as counter,and double junction SCE as reference electrodes (REs), respectively.A volume of 10 mL of electrolyte was used in each LSV measurement.The working electrode (WE) was a glassy carbon pin (3 mm diameter)well polished with alumina paste and sonicated prior to measurement.The counter electrode (CE) was a flame-cleaned platinum mesh at adistance of about 2 cm from the WE, while the tip of the working electrodeis 0.5 cm from the WE at the side of the WE–CE current lines.For each electrolyte, the scan was first performed toward cathodicand then anodic potentials. The WE was cleaned between the two scans.The open-circuit potentials were independent from the compositionand were approximately −0.10 ± 0.05 V vs RE. The initialpotential of LSV was set to zero vs reference for scans on both cathodicand anodic sides. All of the LSV measurements were performed at 25°C under nitrogen flux using a scan rate of 0.5 mV STg, in the rangefrom −90 to −70 °C depending on the salt concentration, the DSC traces showed cold crystallization, startingat Tc, followed by melting endotherms,starting at Tm. The onsets of the Tc were at −46 and −40 °Cfor the 5N20K and 7N20K samples, while the corresponding Tm were at −28 and −20 °C, respectively. Hm, and coldcrystallization enthalpy, ΔHc, forthe two samples 5N20K and 7N20K. In the case of the sample 5N20K,the two enthalpies are nearly equal, which means that this sampleis fully amorphous at low temperatures, i.e., below the glass transition.In the case of the sample 7N20K, in contrast, ΔHm is ∼15% greater than ΔHc, which points to a semicrystalline nature of this sampleat low temperatures. This was confirmed by X-ray measurements performedvs. temperature  that showedthe growth of a crystalline pattern below −35 °C togetherwith a broad feature centered at ∼30°, which is attributedto the amorphous phase. The semicrystalline nature of the sample 7N20Kwas further confirmed by the DSC thermogram obtained during coolingand reported in Figure S2. While the othersamples did not show any reversible crystallization, the sample 7N20Kdisplayed a clear exotherm around −35 °C with reversibleenthalpy ΔHc,r ≈5 J g–1. As expected, the sum of this value and the coldcrystallization enthalpy reported in Figure S1 to known anycrystal phase(s). Further work is needed to solve this problem. Reberet al.30 reported liquidus temperaturesfor several WISEs, such as 35 mol kg–1 NaFSI, 30mol kg–1 NaFSI with 5 mol kg–1 NaFTFSI, 25 mol kg–1 NaFSI with 10 mol kg–1 NaFTFSI, and 19.5 mol kg–1 LiTFSIwith 8.3 mol kg–1 LiBETI, at 50, 44, 38, and 28°C, respectively. They also reported liquidus temperatures of28 and 22 °C for 35 mol kg–1 LiFSI and for25 mol kg–1 LiFSI with 10 mol kg–1 LiFTFSI, respectively. In this scenario, the proposed electrolytesin the current work provide a significant advantage by assuring thermodynamicallystable liquid phases well below 0 °C.tionsee 2. In parB is the pseudo-activationenergy for iontransport , T is the absolute temperature,and T0 is an empirical parameter thatcan be interpreted as the ideal glass-transition temperature. In organicliquids and gel polymer electrolytes, the T0 value obtained by fitting with Tg value measured by DSC, which may be explainedin terms of decoupling between the ion motion and lattice structuralrelaxation.31Here, T0 ≈Tg + 30 °C  withthe cations without causing any significant reassociation resultingin the modification of the local hydrogen bonding.35 The molar conductivities (Λm), activationenergies (Ea), ionic conductivities (σ),and T0 and Tg values are reported in The ionic conductivity decreases with increasing the electrolyteconcentration 2b, as ex5 °C see 2. The beions see 2.34 The Ea is theactivation energy that is related to ion–ion and ion–solventinteractions, while kB is the Boltzmannconstant. Ea values of the electrolytes calculatedfrom the viscosity data. The activation energy increases with increasingthe concentration and the values are in good agreement with thoseobtained from conductivity measurements. This confirms that the mechanismsat the base of bulk properties such as ionic transport and viscosityare well correlated, in agreement with the thermal results.The resultson the charge transport are also supported by the viscositymeasurements vs the temperature, which follow an Arrhenius behavior3a for alFigures S3–S6). As expected,the viscosityincreased as a function of concentration. However, it did not varylinearly as it is in the case of diluted solutions due to complexion interactions at elevated concentrations.36 In the case of salts having a similar anion (as in our case), thechange in viscosity is predominantly caused by the cations.37 Moreover, the behavior of concentrated aqueousacetates rendered them as the so-called “structure-making salts’’because none of the samples showed any decrease in viscosity withincreasing salt concentration. Furthermore, this behavior signifiesthe reduced activity of water molecules since the cations and acetateanions being strongly hydrated are contributing to some fashion ofmolecular ordering.38All of the electrolyte samples behaved like Newtonianfluids . At 25 °C,Λ0 has a value of 52.90 S cm2 mol–1, while the constant A is 15.50.Thus, the Kohlrausch behavior calls for complete salt dissociationin the whole compositional range.42Classical Waldenplot can be used to probe the ionicity of theacetate electrolytes. –2 wastaken as a limit on the glassy carbon electrode. The present proceduremay lead to overestimated decomposition limits because it involvesa large amount of electrolyte and a quasi-ideal electrocatalytic electrodewith a small surface area. However, it has the advantages of beinghighly reproducible and it is useful to compare different electrolytes.22 This is due to the combination of two effects:the increase in both pH (see Table S3)and concentration of the acetate ion. In the first case, the concentrationof OH– ions increases from 8.7 × 10–5 to 1.7 × 10–4 M, however, it is reasonablethat in the concentrated solution, their activity is lower and inverselyproportional to the salt concentration. Then, acetate ions shouldbe involved in the anodic decomposition process and it is reasonablethat the onset of the decomposition potential decreases as the concentrationincreases.The cathodic and anodic decomposition potentialsfor all of thesamples obtained in these conditions are summarized in –1) as reported in –1, whichincludes all of the main vibrational modes of the acetate group, didnot vary significantly with the cation composition and will be deeplydiscussed in the section devoted to the temperature evolution of thespectra. However, for the sake of completeness, the entire Raman spectraare reported in Figure S7 for all of theinvestigated compositions. In the high wavenumber region, based onthe behavior of a specific water molecule as either a proton donor(D) or acceptor (A) in a hydrogen bond, the characteristic OH stretchingband can be deconvoluted into five sub-bands, namely, DAA, DDAA, DDA,DA, and nonhydrogen bonding motifs located at ∼3000, ∼3300,∼3450, ∼3600, and ∼3650 cm–1, respectively44 . In the same spectral region, some acetatevibrations can be assigned, namely, the CH3 symmetric stretching(CH3:SS) at ∼2845 cm–1, the CH3 Fermi resonance (CH3:FR) at ∼2935 cm–1, and the CH3 asymmetric stretching (CH3:AS) at ∼2981.47 The spectra were fitted as asum of CH3- and OH-related peaks for a total of eight Gaussianbands as described above (the fitting results are reported in Tables S1 and S2). It is important to underlinethat all five OH stretching vibrations are present even at very highconcentrations, including contributions from both water–waterand water–solute interactions. Minor changes in the positionand in the full width at half-maximum (FWHM) of the bands were registered.However, they are within experimental and fitting errors and, apparently,they do not follow a clear trend as a function of salt concentration.In contrast, band intensities were strongly influenced by acetateconcentration. Considering the relative intensities of the differentOH bands, a large part of the signal came from the DDAA and the DAbands which, together, were responsible for 95% of the total peakarea. Focusing attention on these two peaks, as shown in 48 In fact, the hydrogen bond strength is inverselyrelated to the frequency; therefore, the bands at lower or higherfrequencies are attributed to stronger or weaker hydrogen bonding,respectively.49 It can be assumed thatat higher concentrations, the interaction of water with the acetateanion is more favored than the water–water or water–cationinteractions. Hence, water molecules are effectively seized by theacetate anions present in such a high population, where the carboxylicgroup is accepting the hydrogen bonds in a bidentate-like structure.These findings may explain the electrochemical behavior of the differentsolutions. In fact, there is a direct correlation between the DDAApopulation and the ESW, in particular with the cathodic decompositionpotential. An increase in the overpotential for water degradationis due to the reduced activity of water because of strong hydrogenbonding with the carboxylate group of the acetate anion.49To better understand the interactions between thecomponents ofthe solution and to correlate the structure with the functional properties,Raman analysis vs. both sodium acetate concentration and temperaturewas performed. Raman study vs electrolyte concentration was carriedout at room temperature to understand the evolution of hydrogen bondingin these highly concentrated electrolytes. A deeper insight into thelocal hydrogen-bonding network is key to understanding the peculiarproperties of water in such solutions. Thus, we focused our analysison the high wavenumber region of the Raman spectrum . Similar resultswere obtained for the sample 1N20K . All of the spectra presented the expected features ofaqueous solution of acetate salts even though clear changes couldbe appreciated as a function of temperature.To further characterize the solution structureand to better explainthe thermal behavior of the system (DSC curves in –1), the C–C stretching (926cm–1), the CH3 deformation (1345 cm–1), and C–O stretching (1413 cm–1), while water- and CH3-related stretching bands occurabove 3000 cm–1 as discussed previously. The thermalevolution of the Raman spectra could be grouped into three major families,respectively, amorphous glass-like state at T <−50 °C, crystalline state at −50 °C < T < 10 °C, and liquid state at T > 10 °C, in good accordance with the DSC signals. The spectralfeatures most affected by the change pertained both to the acetateand water groups. In particular, passing from the liquid to crystalphase, we observed the sharpening of the main C–C stretchingaccompanied by a shift from 926 to 931 cm–1. Atthe same time, the C–O stretching mode also sharpened withthe emergence of a second peak at 1443 cm–1. Thesmall peak due to the methyl deformation also became sharper and registereda small shift passing from 1345 to 1348 cm–1. Theoverall peak sharpening points toward the formation of a crystal structure.Thus, we compared our data with the Raman spectra of prototypicalcrystals of acetate salts to figure out the arrangement of the metastablecrystal phase . Indeed, this can be due to the differentexperimental protocols of DSC and Raman measurements, reflecting thekinetic nature of cold crystallization.The main vibrational modes of the acetate groupare the COO rocking. As summarized in Finally, the crystallizationkinetics of the sample 7N20K was isothermallymonitored in the 1200–1600 cm–1 CH3COOK, with different amounts of CH3COONa, ranging from 1 to 7 mol kg–1. DSCanalysis carried out in a temperature range of −120 to 90 °Crevealed that the glass-transition temperature Tg increases with increasing the salt concentration. Moreover,the DSC also confirmed the low liquidus temperature for the most concentrated7N20K sample. Viscosity measurements carried out vs. temperature classifiedthe acetate salts to belong to the so-called “‘structure-makingsalts”’. Ionic conductivity decreased by increasingsalt concentration. However, the remarkable value of 21.2 mS cm–1 at 25 °C was obtained for the 7N20K most concentratedsample. Electrochemical measurements showed that the electrochemicalstability window increased by increasing the CH3COONa concentration.Such an increase is due to the shift of cathodic decomposition tomore negative potentials, while the anodic decomposition limit isless affected by the salt concentration. The peculiar interactionamong phase components and the emerging structural features were deeplyinvestigated by Raman spectroscopy, which shed light on the evolutionof hydrogen-bonding motifs in these concentrated solutions. A carefulRaman analysis conducted vs. temperature helped in tracing out a possiblestructure of the crystal formation during the crystallization process.Due to the good ionic conductivity, large ESW, and low liquidus point,these concentrated systems are promising as WISEs for batteries andsupercapacitors.In this work, the physicochemical propertiesof a series of binaryaqueous acetate salt solutions were thoroughly investigated via thermal,rheological, electrical, electrochemical, and spectroscopy measurements.Four different solutions were prepared by dissolving 20 mol kg


Studies have indicated that higher numbers of nurses regarding staffing ensure patient safety and a better practice environment. Using citation analysis, this study visualizes the landscape of nurse staffing research over the last two decades to show the overall publication trends, major contributors, and main research topics. We extracted bibliometric information from PubMed from January 2000 to September 2022. After clustering the network, we analyzed each cluster’s characteristics by keyword. A total of 2167 papers were considered for analysis, and 14 clusters were created. The analysis showed that the number of papers published per year has been increasing. Researchers from the US, the UK, Canada, Australia, and Belgium have led this field. As the main clusters in nurse staffing research during the past two decades, the following five research settings were identified: nurse outcome and patient outcome research in acute care hospitals, nurse staffing mandate evaluation research, nursing home research, and school nurse research. The first three clusters accounted for more than 80% of the total number of published papers, and this ratio has not changed in the past 20 years. To further develop nurse staffing research globally, evidence from other geographic areas, such as African and Asian countries, and from long-term care or community settings is necessary. Worldwide, approximately 20 million nursing staff, accounting for more than 70% of healthcare professionals, provide patient care on the front lines 24 h a day, 365 days a year . The effConcurrently, to ensure safe nurse staffing, several countries and states have introduced legislation or financial incentives related to nurse staffing ratios in hospitals and other facilities. California was the first US state to enact a minimum nurse-to-patient staffing ratio law in 2004 ,13. The To improve nurse staffing policies, it is important to conduct policy evaluations of these regulations in various countries and/or states. In countries without legislation, it is necessary to undertake research on safe nursing placement that captures the characteristics of the country’s healthcare system. However, a geographic bias has been indicated in previous research, which has mostly presented evidence from high-income countries ,22. In aRecently, with the advancement of language processing technology, “bibliometric (or citation network) analysis”, which analyzes bibliographic information, has become increasingly useful in showing the landscape and identifying the major topics of a health service research ,28. In nConsequently, this study aimed to visualize the landscape of nurse staffing research over the past two decades. It used citation analysis to show the overall trends in the number of publications and major contributors and reveal the main research topics and the trends in these topics based on similarities in citation relationships.A citation network analysis was conducted. We extracted bibliometric information from PubMed. The search strategy was “nurse staffing” [Title/Abstract] OR “nurse workload” [Title/Abstract] OR “nurse workforce” [Title/Abstract] OR “nurse to patient” [Title/Abstract] OR “patient to nurse” [Title/Abstract] OR “nurse to bed” [Title/Abstract]. We set the publication limit from 1 January 2000 to 30 September 2022  Table A1In the search process, unlike traditional literature review methods, all the literature that met the above search strategy was included in the citation analysis. No limitation was imposed on the study setting. Nursing positions or titles were included, ranging from general nursing staff to advanced practice nurses. We also included a broad range of the literature with titles or abstracts in English, even if the main text was in a different language.https://academic-landscape.com/page/about?next=/ accessed on 1 October 2022)  for the analysis. This system was developed to visualize citation network analyses through the following procedures: (1) retrieving data, (2) determining the maximum connected component and network clustering, and (3) visualization The TF-ICF scores were higher for words that occurred more frequently only in the cluster. A higher TF-ICF score indicates more importance regarding the word that characterizes the cluster.We also carried out a trend analysis of the number of publications, authors, and journals for the articles included in our analysis. For the trend analysis of the number of publications, we visualised each cluster’s annual trends and showed each cluster’s rise and fall over the past 20 years. The affiliations and countries of the top 20 authors, in terms of the number of publications, were listed to reveal the geographical distribution of the nurse staffing studies. The top 20 journals in terms of the number of publications were listed to identify which areas of medicine, nursing, and public health more likely covered the nurse staffing issue.Of the 2308 articles returned by the search strategy, 14 clusters were formed from 2167 papers based on citation relationships. In total, 141 articles were automatically dropped during the clustering process because they had no citation relationship with other articles. The overall number of papers published per year has been on the rise, starting with 26 per year in 2000 and gradually increasing to more than 100 per year in 2011 and more than 160 per year after 2020 a.Out of the 14 clusters, the top five clusters comprised 97.5%. The 6th to 14th clusters, thus, only consisted of a total of 54 papers (2.5%). Therefore, in this paper, we discuss only the top five clusters, which included a level of papers that could be interpreted . ClusterCluster #1, “nurse outcome research in acute care hospitals”, was the largest, with 771 articles. “ICU” (0.00171), “workload” (0.00075), “environment” (0.00061), “quality” (0.00050), and “outcome” (0.00045) were identified as representative key terms with high TF-ICFs  Table A2Cluster #2 was titled “patient outcome research in acute care hospitals” and comprised 614 articles. “Mortality” (0.00127), “patient outcome” (0.00104), “hospital” (0.00103), “fall” (0.00078), “surgical” (0.00066), and “readmission” (0.00062) were identified as key terms with high TF-ICFs  Table A2Cluster #3, “nurse staffing mandate evaluation research”, comprised 537 articles. “Staffing level” (0.00225), “patient outcome” (0.00084), “registered nurse” (0.00064), “California” (0.00063), “policy” (0.00063), and “cost” (0.00058) were identified as key terms with high TF-ICFs  Table A2Cluster #4, “nursing home research”, comprised 138 articles. “Nursing home” (0.00728), “resident” (0.00433), “facility” (0.00154), “quality” (0.00127), “medicare” (0.00093), “rating” (0.00076), and “star” (0.00072) were identified as key terms with high TF-ICFs  Table A2Cluster #5, the “school nurse research” cluster, comprised 53 articles. “School nurse” (0.01518), “school” (0.00933), “student” (0.00395), “mental” (0.00238), “school nurse workload” (0.00231), “asthma” (0.00130), and “epinephrine” (0.00111) were identified as key terms with high IFICFs  Table A2In terms of spatial location, Cluster #1 and Cluster #2, as well as Cluster #1 and Cluster #3, were found to be in close spatial distance and highly similar. Conversely, Clusters #4 and #5 were far from the other clusters and, thus, represented independent research topics .To the best of our knowledge, this study is the first to capture the landscape of articles on nurse staffing from 2000 to 2022 using a citation network analysis. In terms of the overall publication trends, we found that nurse staffing research has expanded, led by US and UK researchers. We also found that nurse staffing research can be divided into five main clusters according to the co-citation relationship: #1 “nurse outcome research in acute care hospitals”, #2 “patient outcomes research in acute care hospitals”, #3 “nurse staffing mandate evaluation research”, #4 “nursing home research”, and #5 “school nurse research”.The overall trend of annual publications has been rising, indicating that the nurse staffing research field has been growing. However, it is suggested that more robust evidence on new adequate nurse staffing methods is needed, for instance, flexible nurse staffing methods beyond the use of ratios , shift wResearchers from the US, the UK, Canada, Australia, and Belgium have led this field. This finding is consistent with the findings of a previous study focusing on the overall trend of high-impact nursing research papers, which are defined as papers ranked in the top 10% of citation frequency . Among tAs for journals in which nurse staffing research is published, nursing administration journals are ranked highly. The International Journal of Nursing Studies, which has a broad scope across the nursing field, and other high-impact journals within the broader medical and health service research field also ranked highly, including Medical Care, Health Services Research, Health Affairs, BMJ Open, and the Journal of the American Geriatrics Society. Higher attention to nurse staffing in nursing administration, health policy, and health service research is indicated. However, the publication trends and leading authors and journals over the past two decades demonstrate that there is still limited knowledge and evidence on adequate nurse staffing from other countries, such as low-middle income counties and/or Asian regions with different nurse staffing regulation systems within their healthcare systems.Five main clusters in nurse staffing research over the past two decades were identified in this study: #1 “nurse outcome research in acute care hospitals”, #2 “patient outcome research in acute care hospitals”, #3 “nurse staffing mandate evaluation research”, #4 “nursing home research”, and #5 “school nurse research”. The first three clusters accounted for more than 80% of the total, and this share has not changed in the past 20 years.The largest clusters were #1, “nurse outcome research in acute care hospitals”, and #2, “patient outcome research in acute care hospitals”. Although there was no small number of articles that examined both patient and nurse outcomes, those in Cluster #1, “nurse outcome research in acute care hospital”, were relatively more focused on the working environment and nurses’ turnover or retention in relation to the shortage in the nursing workforce.Clusters #1, “nurse outcome research in acute care hospitals”, and #2, “patient outcome research in acute care hospitals”, were found to be spatially close, indicating the similarity in their context, with the only difference being whether they both focused on nurse outcomes or patient outcomes in nurse staffing research in acute care hospitals. Both clusters examine acute care hospital settings and reflect the trend of quality assurance in healthcare systems, mainly in the US, since the Institute of Medicine’s “To Err is Human” report. To assess the extent to which nursing personnel in acute care hospitals contribute to healthcare quality, patient safety, and a professional and safe work environment, the National Quality Forum developed 15 performance measures for nursing-sensitive care in 2004 . Our finCluster #3 was titled “nurse staffing mandate evaluation research”. The most famous minimum nurse staffing ratio mandate is that of California, US. Since the minimum nurse staffing mandate was introduced in 2004 and became active in 2005, it has been empirically examined as a natural experiment ,42. ReseIn Cluster #4, “nursing home research”, most articles were related to policy changes in Medicare Medicaid certification made by the Centers for Medicare Medicaid Service (CMS) in the US . The NurCluster #5, “school nurse research”, was a relatively newly developed area of research. The annual number of papers was over five for the first time in 2018, and the total amount of research in this cluster is somewhat limited compared to research in acute care hospital settings. School nurses take on the role of case managers, bringing providers, families, and schools together to support students’ health; as a result, better attendance and academic success are gained ,65. SomeThis study has some limitations. The first relates to the search database. The WOS database is widely used in bibliometric analysis; however, we used PubMed because we could only access the current trends via WOS from 2000 to 2011 due to funding limitations. However, using PubMed has some benefits. For example, PubMed has the advantages of being the best database in the medical field, having an optimal update frequency, and including early online articles . Future Using citation analysis, nurse staffing research over the past two decades formed five major clusters, depending on the study setting and outcomes focused on. This landscape of over 2000 nurse staffing articles revealed that evidence regarding long-term care settings and/or those from other geographic areas is still small compared to those in acute care settings from the US or UK. To ensure the safety of patients and nurses in all practice settings and locations, diverse geographic and setting knowledge is essential.


A systematic PubMed search identified 35 studies. All in vivo MRS studies (N = 13) reported good to excellent reproducibility of GSH measures. In brain, 3 out of 4 MRS studies reported decreased GSH with age, measured in precuneus, cingulate, and occipital regions, while 1 study reported increased GSH with age in frontal and sensorimotor regions. In post-mortem brain, out of 3 studies, 2 reported decreased GSH with age in hippocampal and frontal regions, while 1 study reported increased GSH with age in a frontal region. Oxidized glutathione disulfide (GSSG) was reported to be increased in caudate with age in 1 study, suggesting OS. Although findings in the brain lacked a clear consensus, the majority of studies suggested a decline of GSH with age. The low number of studies (particularly ex vivo) and potential regional differences may have contributed to variability in the findings in brain. In blood, in contrast, GSH levels predominately were reported to decrease with advancing age , while GSSG findings lacked consensus. The larger number of studies assessing age-specific GSH level changes in blood (N = 16) allowed for more robust consensus across studies than in brain. Overall, the literature suggests that aging is associated with increased OS in brain and body, but the timing and regional distribution of changes in the brain require further study. The contribution of brain OS to brain aging, and the effect of interventions to raise brain GSH levels on decline of brain function, remain understudied. Given that reliable tools to measure brain GSH exist, we hope this paper will serve as a catalyst to stimulate more work in this field.Aging is characterized by a gradual decline of the body’s biological functions, which can lead to increased production of reactive oxygen species (ROS). Antioxidants neutralize ROS and maintain balance between oxidation and reduction. If ROS production exceeds the ability of antioxidant systems to neutralize, a damaging state of oxidative stress (OS) may exist. The reduced form of glutathione (GSH) is the most abundant antioxidant, and decline of GSH is considered a marker of OS. Our review summarizes the literature on GSH variations with age in healthy adults in brain ( Examples of ROS include superoxide anion radical (O₂.-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•)  will be 60 years (yrs) or older by the year 2050 . Aging ial (HO•) . As demoal (HO•) . Howeveral (HO•) , which cal (HO•) A  .Fig. 1MiThe free radical theory of aging posits that excess ROS-induced damage significantly contributes to aging and age-associated diseases . With thAs illustrated in in vivo using magnetic resonance spectroscopy (MRS), a powerful non-invasive technique for cerebral metabolite quantification, as well as in autopsy brain tissue and peripheral blood using biochemical assays, such as high-performance liquid chromatography (HPLC)  . While l22.1in vivo, using proton MRS (1H-MRS) of healthy adults, or ex vivo biochemical analyses of autopsy tissue from people without neurological or psychiatric conditions in life, and/or (ii) blood from healthy adults – using biochemical analyses of the fluid component . Using the operators “AND” and “OR”, and the filters “Humans” and “English”, we conducted searches using the following seven expressions: Search 1: (“MRS” OR “magnetic resonance spectroscopy”) AND (“GSH” OR “glutathione”) AND “reproducibility”; Search 2: (“MRS” OR “magnetic resonance spectroscopy”) AND (“GSH” OR “glutathione”) AND “aging”; Search 3: (“brain tissue” OR “brain autopsy”) AND (“GSH” OR “glutathione”) AND “aging”; Search 4: “plasma” AND (“GSH” OR “glutathione”) AND “aging”; Search 5: “serum” AND (“GSH” OR “glutathione”) AND “aging”; Search 6: “brain” AND (“GSH” OR “glutathione”) AND “aging”; and Search 7: “blood” AND (“GSH” OR “glutathione”) AND “aging”. Search results were filtered for duplicates, and unique studies investigating age-related changes in GSH levels in adult humans were included. In general, we considered individuals 18 to 39 yrs of age as young adults, 40 to 59 yrs of age as middle-aged adults, and ≥60 yrs of age as older adults ; case control studies; clinical trial or intervention without baseline data; review articles or meta analysis; and studies not published in English. In addition, for the purposes of this review, we focused on GSH level readout in the liquid portion of blood (plasma or serum), since we were interested in the systemic readout of GSH. We, therefore, excluded studies investigating GSH levels in erythrocytes, as this readout would be specific to the blood tissue itself.2.2in vivo and in autopsy tissue) the region of interest (ROI) was noted. For in vivo brain studies using MRS, magnetic field strength and the pulse sequence used were noted. For MRS studies assessing the reproducibility of GSH quantification, we also noted the echo time (TE), metabolite referencing scheme , and reported reproducibility statistics. GSH results from studies using the same cohort were pooled under the PubMed unique identifier or PMID of the earliest publication and treated as results from a single study to avoid counting the cohort multiple times. Moreover, the risk of bias for included studies was assessed by two independent reviewers (F.D. and S.S.) using the Joanna Briggs Institute (JBI)’s analytical Cross Sectional studies guidelines (Two reviewers (F.D. and A.B.) conducted the searches and one (F.D.) checked for duplicates. Two reviewers (F.D. and A.B.) independently screened all unique search results for potential inclusion in the review. Data extracted from studies that met inclusion criteria were as follows: number of study participants, age range of participants, sex distribution, method(s) used to assess glutathione (GSH (reduced form), GSSG (oxidized form), tGSH, or GSH/GSSG ratio), and variations in metabolite levels with age. Specific to the brain studies   . Resonanon (ppm) . Each ofon (ppm) . This efon (ppm) . A numbeon (ppm) . Of theson (ppm)  combinedon (ppm) , togetheon (ppm) . At highon (ppm) . These non (ppm) , with imon (ppm) , thereby0) inhomogeneity and radiofrequency excitation (B1) inhomogeneity, and elevated radio frequency power deposition. Therefore, studies comparing results across different field strengths are helpful for evaluating benefits of UHF strengths, given the additional technical challenges.We identified 13 MRS studies reporting on the reproducibility of GSH measurement in young or young to middle-aged healthy adults  Table 1Table 1, r, 15%, 2/13) , the intraclass correlation coefficient  and Pearson’s correlation coefficient  . Reprodu%, 2/13) , one stu%, 2/13)  and one %, 2/13)  reportin%, 2/13)  reportin%, 2/13)  and four%, 2/13)  reportin%, 2/13) , half  , and thr%, 2/13) . Reprodu%, 2/13) , (b) MEG%, 2/13) , and (c)%, 2/13) . While n%, 2/13) . The mea%, 2/13)   .3.2.2Using MRS, four studies investigated age-specific alterations in GSH levels in the brain of healthy, cognitively unimpaired adults . Two of The relationship between GSH levels and cognitive function, using the Montreal Cognitive Assessment (MoCA), was interrogated by half (2/4) the MRS studies, and no significant association was found in the ROIs investigated, namely medial frontal , sensori3.2.3In the human autopsy brain, almost all glutathione (>98.80%) is in its reduced form, with greater GSH concentrations in the WM (∼1.18 mM) compared to the grey matter (GM) (∼0.83 mM) . Three iin vivo MRS studies, the brain autopsy studies lacked clear-cut separation of groups by age  or increases  in old adults. In autopsy brain tissue, only one study measured GSH and reported an increase in levels with age  , medial frontal and prefrontal dorsolateral regions) had been investigated by reproducibility studies with reliable GSH measures demonstrated. The majority of the  adults) . While G adults)  and occi adults)  brain re3.33.3.1Under normal conditions, GSH levels in blood plasma are usually of 1 to 6 μM . To dateThe majority of GSH studies  reported significantly lower plasma levels with increasing age in adults (18–85 yrs) , as wellWhen investigating sex-specific changes in plasma GSH levels, four studies reported a lack of significant differences between age-matched men and women . SpecifiIn addition to GSH, 6 studies also assessed plasma GSSG (N = 3) , GSH/GSS3.3.2In serum, two studies investigated GSH, GSSG, and/or tGSH in serum of healthy adults, using two techniques  Table 3Table 3. 3.3.3In summary, of the 16 cross-sectional studies in blood, 11 (69%) assessed GSH, with fewer studies  investigating GSSG, GSH/GSSG and/or tGSH. Relative to young adults, GSH levels in peripheral blood were decreased in old adults in the majority  of studies, indicating an increase in OS with age . We also4We conducted a comprehensive review of the literature to examine the consistency of age-specific GSH level changes in the brain and peripheral blood across studies.4.14.1.1in vivo is dependent on the reproducibility of MRS measurements. However, studies interrogating the reproducibility of GSH measures is a relatively recent phenomenon, with publication dates of the 13 identified studies ranging from 2014 to 2022. This is not surprising given that MRS has been historically used to study (a) high-concentration and easily detectable metabolites at lower field strengths, such as N-acetylaspartate, choline, creatine and myo-inositol inositol . DemonstThe literature investigating age-related brain GSH level variations using MRS or biochemical assays is in its infancy, with only seven cross-sectional studies meeting our inclusion criteria. Together, these studies investigated GSH variations in 10 ROIs distributed in the frontal (N = 2), limbic (N = 2), occipital (N = 2), parietal (N = 1), caudate (N = 1), sensorimotor (N = 1) or cerebellar (N = 1) regions. With increasing age, GSH levels were observed to significantly decrease in 40% of the ROIs , increase in 30% , and remain stable in 30% . Of the above-mentioned ROIs, GSH levels in the frontal  and occiGss, Gclc and Gclm, three genes encoding enzymes involved in GSH synthesis, at a single time point, as well as (b) change in the expression level of these genes with age, with examples that include age-related increases of Gclc and Gclm in the hypothalamus and medulla, respectively  in the liquid portion of blood can act as a readout of systemic (whole body) OS . In our 4.1.2.1Moreover, in our review, plasma, and not serum, was the liquid blood portion used by the majority (88%) of studies to interrogate age-specific changes in GSH levels. This preference for plasma may be attributed to some of its advantages over serum, such as a larger volume, no clotting time delay and a lower risk of haemolysis or the breakdown of erythrocytes . In fact4.2clinicaltrials.gov , we recorphisms , only onThis work was supported by the Fonds de Recherche Québec - Santé (FRQS)  (F.D.); FRQS , Fonds de soutien à la recherche pour les neurosciences du vieillissement from the Fondation Courtois and the Quebec Bio-Imaging Network [#PP 19.20] (A.B.); and the Canadian Institutes of Health Research grant [#153005] (S.N. (grantee), S.S.).The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Actinomycetota, Pseudomonadota, Acidobacteriota, Chloroflexota, and Cyanobacteria. At the genus level, the analysis revealed a dominance of the genus Crossiella, which is actively involved in biomineralization processes, followed by Pseudomonas, Bacillus, Chujaibacter, and Sphingomonas. The presence of these taxa is associated with the carbon, nitrogen, and ammonia cycles, and some are possibly related to the anthropic disturbance of these caves. This study provides the first insight into the microbial diversity of the Etna volcano lava tubes, and expands on previous research on microbiology of volcanic caves across the world.While microbial communities in limestone caves across the world are relatively understood, knowledge of the microbial composition in lava tubes is lagging behind. These caves are found in volcanic regions worldwide and are typically lined with multicolored microbial mats on their walls and ceilings. The Mount Etna  represents one of the most active volcanos in the world. Due to its outstanding biodiversity and geological features, it was declared Natural Heritage of Humanity by the UNESCO in 2013. Despite the presence of more than 200 basaltic lava tubes, the microbial diversity of these hypogean systems has never been investigated so far. Here, we investigated bacterial communities in four lava tubes of Mount Etna volcano. Field emission scanning electron microscopy (FESEM) was carried out for the morphological characterization and detection of microbial features. We documented an abundant presence of microbial cells with different morphotypes including rod-shaped, filamentous, and coccoidal cells with surface appendages, resembling actinobacteria reported in other lava tubes across the world. Based on 16S rRNA gene analysis, the colored microbial mats collected were mostly composed of bacteria belonging to the phyla The online version contains supplementary material available at 10.1007/s00248-023-02181-2. In the last 20 years, considerable effort has been made to shed light on the microbial communities of karst caves, particularly speleothems and walls hosting wall paintings , 2. In cMicrobial communities in lava tubes grow forming extensive colored biofilms on speleothems and walls , similarActinomycetota, as revealed by cDNA analysis of yellow colonies from a lava tube in La Palma, Canary Island, Spain . Re. Re20]. “Grotta di Monte Corruccio” (SICT1056) is an outflow tunnel partially contained in the effusive eruptive apparatus of the homonymous mount formed in a time interval of 15,000–3930 ± 60 years . It has Despite no information is available on the precise number of tourists entering each cave, based on an estimate of the tourist frequentation and the presence of waste due to visits, we can sort the four caves in two categories: those that experience low tourist use (“Grotta Catanese I” and “Grotta di Monte Corruccio”) and the ones experiencing higher tourist use (“Grotta del Santo” and “Grotta Lunga”).Small fragments of each sample collected in the four lava tubes were observed using a Zeiss Discovery V8 stereomicroscope (200 × maximum magnification) coupled to a MOTICAM 10.0 system to perform a detailed macroscopic examination of the sample surface.Subsequently, samples were examined by field emission scanning electron microscopy (FESEM) using a high-resolution FEI Teneo SEM  equipped with an Oxford X-ray energy dispersive spectroscopy (EDS) detector for characterizing the microtopography of the samples and detect microbial cells.Air-dried bulk samples with microbial mats were directly mounted on a sample stub and sputter coated with a thin platinum film, with an acceleration voltage of 5 kV, using the SE detector.The statistical analyses were performed in R . The taxMolecular biology techniques based on 16S rRNA gene analysis were conducted for the identification of the bacterial communities present in the Etna lava tube samples.Genomic DNA was extracted from 12 samples collected in the Etna lava tubes using the DNeasy PowerLyzer PowerSoil Kit according to the manufacturer’s protocol (Qiagen), and quantified using a Qubit 4.0 fluorometer (Invitrogen). Sequencing libraries of the V3-V4 hypervariable region of the 16S rRNA gene were prepared according to the Illumina 16S Metagenomic Sequencing Library protocols. The gDNA input (2 ng) was amplified by PCR using the universal primer pair 314F (5′- Illumina overhang- CCTACGGGNGGCWGCAG -3′) and 805R (5′- Illumina overhang- GACTACHVGGGTATCTAATCC -3′) with Illumina adapter overhang sequences. The thermocycling conditions were as follows: 3 min at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by a 5-min final extension at 72 °C. The PCR products were then purified with AMPure beads . Two microliters of each purified product was PCR amplified for final library construction containing the index using NexteraXT Indexed Primer, under the same thermocycling conditions as mentioned before, except for 10 cycles. After purification with AMPure beads, the final purified products were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape . The purified amplicons were then sequenced using the MiSeq™ platform  by Macrogen Sequencing Services (Korea).The raw data obtained from Illumina platform MiSeq, for producing 300 PE reads, was initially quality checked, trimmed, and clustered in ASVs using QIIME2  with DADUnder the stereomicroscope, samples showed colored stains coating the rock substrate or associated with mineral grains Table . There wMicrobial cells and structures were imaged by high-resolution FESEM, as an effective and fast method for microbial life detection in complex samples. FESEM images showed abundant microbial cells with different morphotypes in all the samples Figs. , and 5. 2FESEM observations showed an abundant presence of actinobacteria-like cells for the four samples collected in “Grotta del Santo,” mainly comprising spores with a hairy surface Fig.  or chainOrthoseira roeseana  revealed the presence of several actinobacteria-like coccoid (1-µm diameter) and rod-shaped cells Fig.  with spiana Fig.  and Humiila Fig. , which iSamples collected in “Grotta di Monte Corruccio” showed greater variety in microbial structures and cell morphologies Fig. . In the 3 microspheres found in Kipuka Kanohina lava cave in Hawaii, USA [3 formation [Observations conducted on sample GMC_5 revealed the presence of Ca-rich spheroids closely associated with filamentous cells Fig. , H, reseaii, USA . Precipiaii, USA  or throuormation . Numerouormation . Calciumormation .Interestingly, sample from “Grotta Lunga” (GL_1) showed the presence of reticulated filaments embedded in a matrix of EPS Fig. , B. The We identified a total of 6310 unique ASVs for the 12 samples, with the samples collected in “Grotta Lunga” being richer than the others . These samples also showed the highest values in terms of alpha diversity indices, i.e., Shannon index  and PielP < 0.001). By comparing the differences among caves with the Tukey’s post hoc test, we could highlight that most of this effect is due to the significant difference between the “Grotta Lunga” and the others, with the former being significantly richer than the others . Regarding the other pairwise comparisons, we could record a significant difference between “Grotta Catanese I” and “Grotta del Santo” , while no other significant differences were identified .When considering the taxonomic richness measured at the order level, the “Grotta Lunga” resulted the richest cave with 174 orders, followed by “Grotta Catanese I” with 150 orders, and then by “Grotta del Santo” and “Grotta di Monte Corruccio” caves with 148 and 147 orders, respectively  and high temperature (18.7 °C). On the contrary, lower values of richness were observed in “Grotta di Monte Corruccio,” which is located at 1365 m, with a temperature of 13.9 °C. Tentatively, differences among caves in terms of richness could also be explained by the different environmental conditions and biogeochemical processes operating in caves  as well Independent libraries of DNA sequences from each sample were built targeting the V3-V4 hypervariable regions of the 16S rRNA gene, with the objective of detecting the total bacteria present in these samples.Actinomycetota, Pseudomonadota, Acidobacteriota, Chloroflexota, and Cyanobacteria, followed by other phyla with lower representativeness. Taxonomic identification resulted in a prominent importance of the phylum Actinomycetota in almost all samples, ranging from 78.4% in GS_2B to 14.3% in GC1_2, corroborating the observations by FESEM.The relative abundances of the dominant phyla in each cave sample are shown in Fig. Actinomycetota has been widely found in caves where are actively involved in the biomineralization processes . Moreovelopment , 61.Pseudomonadota was also present in each cave, being the most representative group in “Grotta del Santo” and “Grotta Lunga” . The Pseudomonadota are well represented in lava caves across the world [The phylum he world , includihe world , 15.Actinomycetota and Pseudomonadota were among the most representative groups in lava tubes from La Palma (Canary Islands) and Galápagos (Equador), respectively.A similar trend was observed by Gonzalez-Pimentel et al.  and MillAcidobacteriota was equally distributed in all caves, ranging from 3.02% in “Grotta di Monte Corruccio” (sample GMC_5) and 12.39% in “Grotta del Santo” (sample GS_2A). Several studies have detected the presence of acidobacterial 16S rRNA gene sequences in caves . Howeves et al. .Cyanobacteria was abundant in Grotta Catanese I but poorly represented in the other caves. The presence of this taxon in sample GC1_2 is due to its proximity to the cave entrance, which receives sunlight allowing the development of photosynthetic-based biofilms on the cave wall. Accordingly, Cyanobacteria are mainly favored by the presence of light and thereby generally frequent near the cave entrance [Chloroflexota, Planctomycetota, Bacillota, Nitrospirota, Bacteroidota, Patescibacteria, and Verrucomicrobiota. They are also common at lower rates in other lava tubes [The phylum entrance , 69, altentrance ), which va tubes , 6, 9. OActinomycetes, Gammaproteobacteria, Alphaproteobacteria, Oxyphotobacteria, followed by the less abundant Bacilli, Bacteroidia, and Nitrospira .The bacterial communities identified at the class level showed differences between caves and sectors. Most bacteria belonged to the following class: Actinomycetes was the most abundant class in “Grotta del Santo” (GS_2B: 78.21%) and “Grotta di Monte Corruccio”  sample, while Gammaproteobacteria was predominant in “Grotta Lunga” (GL_1: 28.60%) and “Grotta del Santo” (GS_4: 25.99%). Oxyphotobacteria were instead the most abundant class in “Grotta Catanese I” (GC1_2). The class Alphaproteobacteria was also well represented in all samples, ranging from 22.37% in “Grotta del Santo” GS_4 and 4.66% in “Grotta Catanese I” (GC1_2). The class Actinomycetes is common in caves and its studies in different locations have revealed the presence of several novels and rare taxon [The relative abundance of the different classes varied in each sample, suggesting differences in the local environment and element composition of the volcanic substrate . Actinom GS_2B: 7.21% and re taxon . Investire taxon  as well re taxon ; Gonzale GS_2B: 7.21% and Gammaproteobacteria is among the most dominant group in habitats with either natural or anthropogenic organic inputs [Bacillus spp., Escherichia coli, and Staphylococcus aureus [The class c inputs . Accordic inputs –80 or cac inputs . This tac inputs ). Among s aureus , 83, 84.Crossiella is well present in all samples, being the most abundant group in 8 out of 12 analyzed samples  and rec Northup , 92. Othronments .This study provides the first insight into the taxonomic groups constituting the microbial communities of Mount Etna lava tubes. Although the limited number of samples did not allow us to properly correlate these data with any environmental variables, we could detect the higher richness in “Grotta Lunga,” which is located at a lower elevation (850 m a.s.l.), whereas we observed a general lower richness at higher elevation (1365 m a.s.l.) in “Grotta di Monte Corruccio.” Accordingly, differences among caves in terms of richness could be explained by the different environmental conditions and biogeochemical processes operating in caves . FurtherOur results revealed the abundant presence of phylotypes previously detected in other lava tubes worldwide. The presence of a large number of unclassified bacteria in the 12 sampling sites suggests that these lava tubes have a great potential for the isolation of novel species. However, the land use overlying the cave, as well as the uncontrolled presence of visitors into these caves, may pose major threats to these ecosystems, especially those located at lower altitudes beyond the strictly protected areas of the volcano, generally exposed to a higher tourist frequentation. Further research is needed to ascertain the possible presence of novel bacteria in caves from the Etna lava caves, as well as the presence of contaminants and their potential risk associated with human health. Data of pathogens should be therefore considered not only for the conservation of these unique geosites, but also for the risk associated with human health.Supplementary file1 (DOCX 1180 KB)Below is the link to the electronic supplementary material.