Conclusion: Maternal age, folic acid supplementation, and previous history of miscarriages is involved in the etiology of CHD in DS fetus in Indian population. Maternal MTHFR and MTRR polymorphisms are also involved in the occurrence of CHD and DS in Indian population when controlling for periconceptional folic acid supplementation. Limitations: Single-Centered StudyObjectives: Maternal MTHFR and MTRR polymorphisms as a risk of CHD in DS fetus were studied along with maternal folic acid supplementation, which could influence the folate metabolism along with other risk factors. Material and Methods: A case-control study comprising of mothers of DS with and without CHD along with controls were recruited from a tertiary care center since 2018–2019. Genomic DNA was isolated followed by PCR-RFLP. Results: Mothers with age ≥35 years and having history of miscarriages have a higher risk of giving birth to DS with CHD . Mothers who carried the MTHFR 677CT/TT and MTRR 524CT/TT genotypes combination in the folic acid nonusers group during pregnancies had six-fold (OR = 6.909,  Down syndrome (DS), also called as trisomy 21, is caused due to maternal non-disjunction of chromosome 21. The prevalence of DS is approximately 1–2 per 1000 live births, and it is characterized by the presence of mental retardation along with congenital anomalies, including congenital heart defects (CHD), Hirsprung’s disease, Alzheimer’s disease, leukemia, etc. CHD in DS account for 40 to 63.5% increased incidence of atrioventricular septal defects and ventricular defects in these population [The maternal age and previous history of miscarriages are well-defined risk factors for DS. Additionally, few reports have showed the association of maternal age and previous history of miscarriages with CHD as well, respectively ,7,8,9,10MTHFR) and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) enzymes encoded by MTHFR and MTRR gene. The polymorphism of MTRR and MTHFR in mothers have been previously linked to incidence of DS globally. Moreover, owing to the role of folate metabolism in cardiovascular development, the MTRR and MTHFR maternal polymorphisms may also pose a risk for CHD in DS offspring [Studies have investigated that folate, an important vitamin, plays essential role in reducing the risk of developing of cardiovascular diseases in during fetal life . Folate ffspring . Howeverffspring ,12,13. TEpidemiological studies have shown that folic acid supplementation can influence the occurrence of CHD and DS independently ,12,13,14In the present study, we investigated the effect of genetic variants in maternal folate-metabolizing genes, such as MTHFR and MTRR, as a risk of CHD in DS fetus. In addition, the effect of maternal folic acid supplementation was inspected, which could influence folate metabolism. Additionally, we also examined other maternal risk factors, such as maternal age and previous history of miscarriages, for the occurrence of CHD in DS fetus.This case-control study comprised of two groups of cases, including mothers of DS with CHD (Group I) and mothers of DS without CHD (Group II), along with age matched maternal controls who had previously delivered healthy offspring (Group III), and it was designed for analyzing the maternal risk factors for the occurrence of CHD in DS.Settings: The study was conducted in a tertiary care and advanced research center in India from September 2018 till December 2019.2PQ/d2, where z = statistic for the level of confidence, P = reported prevalence, Q = 100-P, and d = 5%–precision/error). The sample collection was obtained from those referred to the Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, and controls were healthy volunteers visiting general hospital, SGPGIMS, Lucknow, India. Inclusion criteria for cases were recruitment of those mothers who had delivered DS babies, and the study was further classified by the presence and absence of CHD along with DS in those offspring. All the clinical details of the study, including previous history of miscarriages, age, and folic acid supplementation, were recorded while interviewing and preparing the study questionnaire from the participant. Informed consent from participants was also collected at the same time. Other details, including mean maternal age and weight, total number of pregnancies, history and number of previous miscarriages, tobacco use, and educational level, were also recorded at the same time.Group I, Group II, and Group III comprised of forty, sixty, and fifty mothers, respectively. Cases and control numbers were calculated using a formula on the basis of prevalence of DS , and the DNA was stored 4 °C for further use. Polymerase chain reaction was used to amplify MTHFR and MTRR gene for both the variants . The PCRp-values were considered to be significant at 0.05, and 95% confidence intervals (95%CI) were reported.The chi-square test was used to evaluate the genotypic frequencies and also to test for deviance from Hardy–Weinberg Equilibrium. The odds ratio (OR) was calculated to estimate the odds of CHD in DS individuals in maternal cases for various combinations of MTHFR and MTRR genotypes and maternal risk factors during pregnancy. The OR was also used determine the odds of having an offspring with CHD in DS in maternal MTHFR 677CT + TT versus the wild-type CC genotype and maternal MTRR 1298AC + CC versus the wild-type AA genotypes stratified by folic acid supplementation. For maternal age as a risk factor, subjects with age <35 or ≥35 years were recruited since increased risk of DS offspring among women over the age of 35 was previously reported. All analyses were performed using SPSS for Windows, version 16.0. The The frequencies of maternal age ≥ 35 years in Group I, Group II, and Group III are 65%, 42%, and 40%, respectively. The frequency of maternal age < 35 years in Group I, Group II, and Group III are 35%, 58%, and 60%, respectively. The The frequency of folic acid supplementation was found to be highest in Group III (N = 90%), followed by Group II (N = 65%) and Group I (52%). In Group III, highest frequency was observed in participants taking folic acid supplementation  compared to Group I (52%) and II (65%), which suggests that folic acid is associated with the occurrence of CHD in DS fetuses. The frequency of mothers having previous history of any miscarriages shows that the highest risk of miscarriages influences the occurrence of CHD in DS fetuses in maternal subjects in Group I (N = 52%), and the least frequency was seen in Group III (N = 2%), respectively .p-value = 0.253; 95% CI = 0.628 ± 5.79), and the mothers who did not take folic acid during pregnancies had an increased odds ratio of 6.909  of having a DS child with CHD , and the those mothers who did not take folic acid during pregnancies had an increased odds ratio of 4.75  of having a DS child with CHD.  and the occurrence of CHD in DS fetuses ,12,13. TMaternal age is a well-established risk factor for DS, and previous studies have concluded that women over 35 years of age have a higher probability of having an affected child with DS ,15,16. Ap-value = 0.027; 95% CI—1.23 ± 38.51) and four-fold  increased odds of having a DS child with CHD, respectively, as compared to mothers who had taken the folic acid supplementation , whereas some studies have reported inconclusive results due to limited availability of data ,21,22,23Maternal risk factors, such as age, folic acid supplementation, and previous history of miscarriages, are involved as the potent maternal risk factors in the development of CHD in DS fetuses in the Indian population. Additionally, maternal MTHFR and MTRR genetic polymorphisms are also involved in the occurrence of CHD and DS in the Indian population along with folic acid supplementation during pregnancy. These findings can in turn be used as a maternal biomarker for the prevention of CHD in DS fetuses, leading to better diagnosis treatment and management of disease, which could open an window for reproductive counseling both before and during pregnancy.


Following target prediction, we show that 71.6% of miRNA predicted targets were altered as a consequence of SNPs located within the seed region of the mature miRNAs. Next, we identified variations in the Minimum Free Energy (MFE), which represents the capacity to alter molecule stability and, consequently, miRNA maturation. A total of 48.6% of the sequences analyzed showed values within those previously reported as sufficient to alter miRNA maturation. We have also found 131 SNPs in 46 miRNAs, with altered target prediction, occurring in QTL regions. Lastly, analysis of evolutionary conservation scores for each SNP locus suggested that they have a conserved biological function through the evolutionary process. Our results suggest that SNPs in microRNAs have the potential to affect bovine phenotypes and could be of great value for genetic improvement studies, as well as production.Single nucleotide polymorphisms (SNPs) can have significant effects on phenotypic characteristics in cattle. MicroRNAs (miRNAs) are small, non-coding RNAs that act as post-transcriptional regulators by binding them to target mRNAs. In the present study, we scanned ~56 million SNPs against 1,064 bovine miRNA sequences and analyzed,  Single nucleotide polymorphisms (SNPs), the most common mutation in mammalian genomes, are single-base exchanges that naturally occur in the genome and have long been studied as valuable genetic markers, as they can affect and/or aid the selection of phenotypic traits ,2. CurreIn humans, approximately 70% of the genome is translated into RNA, but 98.5% are non-coding RNAs (ncRNA), i.e. not translated into protein ,7. FolloIn humans, the occurrence of SNPs at target sites of miRNAs on messenger RNAs (mRNAs) has been reported to alter mRNA:miRNA binding, creating or destroying targets, and potentially regulating a wide variety of diseases, including cancer (reviewed by Moszyńska ). Notwitin silico predictions on the effects of these SNPs on target binding, primary miRNA formation, occurrence of miRNAs with altered target prediction in QTL regions, and the evolutionary conservation for each SNP locus.Through the use of bioinformatic tools and the largest bovine SNP database currently available (1000 Bulls Genome Project), we sought to identify SNPs that occur in miRNA sequences and to perform Bos taurus UMD 3.1.1 [http://www.1000bullgenomes.com/) INDELS were not used in this study. Primary and mature miRNA location, positioning, and sequences were obtained from miRbase (www.mirbase.org), release 21 [http://128.206.116.18:8080/faangmine/begin.do) [In this study, we used the MD 3.1.1  as the rlease 21 –21. Posilease 21 . Data miegin.do) .An in-house Python script (2.7 version) was used to identify all SNPs (Run5 of 1000BGP) that are located within all bovine miRNA sequences available on miRBase (release 21).The miRmap  package To calculate variations in Minimum Free Energy (MFE) between reference and altered sequences of primary miRNAs, the program RNAFold—ViennaRNA Package 2.0  was utilAn in-house Python script (version 2.7) was used to identify which SNPs, with altered target prediction, were located in QTL mapped regions available in Animal QTLdb .−3) [PhyloP scores are used to measure non-neutral nucleotide substitution rates, indicating conservation and acceleration independently at each nucleotide . To calc−3)  of all p−3) ,33. Once−3) , followeWe performed a scan in 1,064 miRNA primary sequences and found 452 miRNA sequences with SNPs. Out of 452 miRNAs with SNPs, 193 of those had SNP(s) located within miRNA´s mature sequences, with 91 of them within seed regions. Some miRNAs harbored more than one SNP per sequence. Nucleotide changes were comprised of 47% transitions and 53% transversions .Out of 193 miRNA mature sequences with SNPs, 91 had SNPs occurring within their seed region, and 70 of these 91 (77%) had changes in target prediction as a consequence of the nucleotide present. In regards to target prediction alteration observed for the 70 miRNAs, 48% of all targets were newly created, and 35.1% were lost , as a coIn order to investigate the influence of a SNP in the hairpin structure of the miRNAs, we calculated the energy change (ΔΔG) between reference and altered sequences of 917 miRNA sequences  using thFocusing on the 70 mature miRNAs with altered target prediction, we crossed their positions with that of known QTL regions available on Animal QTLdb . We founTo investigate the conservation level of each altered base occurring in miRNA primary sequences, we obtained the PhyloP score based on the alignment of 100 vertebrates  relativeWe were able to contrast the largest SNP database available in the bovine species to date, with all available bovine miRNA sequences. The publicly available SNP databases, Bovine Genome Database  and SNPcIn our study, SNPs within seed sequences greatly affected target prediction, with less than 20% of similar targets between miRNAs containing the reference or altered alleles . Our preCyprinus carpio) [We found a significant increase in the variation of MFE in the transversions when compared to transitions. This variation was also reported by Guo et al. , however carpio) . Positiv carpio) . In our  carpio) .LCORL for downregulation. The LCORL gene encodes the PALI1 and PALI2 proteins, essential for the activation of the PRC2.1 and PRC2.2 complexes and regulation of the suppressive histone mark H3K27me3, essential for the normal development, as demonstrated in mice [LCORL gene is highly conserved among mammals and is related to skeletal height and size in humans [LCORL gene with body conformation indexes in Nordic red cattle [LCORL, caused by a SNP in bta-miR-1256, may affect overall size and therefore calving ease.In our study, we have identified 125 miRNAs with SNPs in previously identified QTL regions. These loci are large, spanning several Kbs within chromosomes, therefore caution should be taken when discussing point variations in such large regions. Notwithstanding, 47 of these miRNAs associated with QTLs presented altered target prediction as a consequence of a SNP within their seed region, which could translate directly in significant protein differences in individuals harboring different bases in non-coding regions of the genome. The influence of SNPs on the divergence of traits between cattle breeds, or within breed, are well reported in several studies, as revised by Seidel , however in mice . The LCOn humans –53, bodyn humans –57, dogsn humans  and pigsn humans , body wen humans ,61 and sn humans . Previoud cattle , carcassd cattle  and orgad cattle  in Simmed cattle  and statd cattle , thus, sIGFBP-2 gene in a QTL related to calving difficulty. Human growth hormone (GH) regulates the expression of IGF (insulin-like growth factor) and the deficiency of GH / IGF1 results in short stature in children. IGFBP (Insulin-like growth factor-binding protein) belong to a group of 6 proteins that bind to IGF and have as main function to regulate the access of IGF1 and 2 to the IGF1 receptor [In addition, Purfield et al.  detectedreceptor ,69.Evolutionary conservation of miRNAs, as well the conservation of their target genes, have already been evidenced in previous studies ,71. In tCD163 gene and was recently identified by Ju et al., where increased expression levels of these miRNAs in the udder of Holsteins were associated with mastitis [CD163, which could influence immune response to infection. In 2017, Guan et al. identified the ZNF423 gene as a target for miR-23a, affecting cellular differentiation in bovine fetal muscle. Authors reported that the level of expression of miR-23a is low during cell differentiation, while the forced overexpression of a miR-mimic 23a leads to a reduction in the accumulation of lipids [ZNF423 acts as a positive regulator of PPARγ expression, promoting the differentiation of undifferentiated cells into adipocytes. They have also demonstrated that the reduction of ZNF423 expression, through small RNAs, negatively regulates the rate of adipocyte differentiation [ZNF423 as a target, and in the gain of ZNF521 which, interestingly, was previously reported as a negative regulator of ZNF423 in mice [ZNF423 as a target for miR-23a, in addition to downregulation of ZNF521, caused by the presence of a SNP in the miR-23a seed region, can result in increased adipogenesis and could impact meat marbling.Among the miRNAs with altered target-binding prediction, we highlight miR-15a and 23a, which have their binding to target genes previously validated in cattle. MiR-15a targets the mastitis . Interesf lipids . Gupta entiation . In the  in mice . Thus, tStreptococcus uberis in bovine mammary epithelial cells. A pathway analysis highlighted MAPK signalling, Cytokine-Cytokine Receptor Signalling and the JAK-STAT Signalling Pathway as important pathways for mastitis response in these cells. Of the 2,491 target genes predicted by the authors, 93 genes were predicted as targets for mir-29e, which was upregulated at 4 hours of infection and downregulated at 6 hours. When we compare these targets with the prediction performed in our study, the presence of a SNP in the miR-29e seed region results in the loss of 52 of these 93 target genes and a gain of 4 target genes, suggesting that a SNP in the seed region of this miR-29e miRNA can alter more than 50% of the target genes for this miRNA, as predicted by Lawless et al., potentially affecting important pathways related to innate immunity in response to gram-positive bacterial infection.A phenome search carried out through the FAANG  Consortium , using oFinally, the present study points to an approach focused on differences in genomic sequences that are not present in coding genes, but can indirectly generate differences in the phenotype of individuals, through epigenetically regulated processes. In fact, our group has previously identified SNPs that, through the creation or destruction of CpG sites, may mechanistically induce a quantitative difference in methylation, which in turn modulates gene expression and consequently phenotypes . Out of In the present study we demonstrated that SNPs in miRNA sequences can alter their target prediction, as well as pre-miRNA processing, possibly increasing or decreasing mature miRNA production, which in turn could affect production traits in cattle.S1 File(XLSX)Click here for additional data file.


Mild neurocognitive disorder (MND) is an intermediate state that can progress to dementia, and the cognitive reserve of MND is an important task in preventing dementia. Acupuncture and neurofeedback (NF) training have been used to improve cognitive function and treat MND or dementia, but their effectiveness remains controversial. In this trial, we will evaluate the efficacy and safety of combined NF-acupuncture treatment in comparison with single acupuncture treatment.This study is a randomized, assessor-blind, pilot trial. It is designed in accordance with the Standards for Reporting Interventions in Controlled Trials of Acupuncture. A total of 44 MND participants who meet the inclusion and exclusion criteria will be enrolled, and each will be randomly assigned to 1 of 2 groups of 22 subjects. Each subject will visit 24 times over 12 weeks and receive either acupuncture or NF-acupuncture combined treatment. At visit 25 (week 13), a follow-up evaluation will be performed, and then the investigator will analyze the results. The primary outcome is defined by the Korean version of the Montreal Cognitive Assessment score from screening to visit 25. The secondary outcome includes the following: change in Alzheimer Disease Assessment Scale–Cognitive, the Korean version of the Beck Depression Inventory, Body Awareness Questionnaire, delayed matching to sample task scores, and functional near-infrared spectroscopy values, from visit 1 to visit 25; heart rate variability values from visit 1 to visit 5, visit 9, visit 13, visit 21, visit 25; breath per minute values from visit 1 to visit 1 to 25.We will evaluate the effectiveness and safety of combined NF-acupuncture therapy, and expect that it will serve as the basis for the use of NF together with acupuncture in the clinical setting.https://cris.nih.go.kr/cris/search/detailSearch.do/16239)KCT0004972 (registered in Clinical Research Information Service of the Republic of Korea,  In South Korea, the prevalence rate of MND is 23.7% in people older than 65 years. To date, there have been no drug interventions that delay conversion to dementia. To prevent progression to dementia in MND, various interventions are needed that include decreasing neuropathological damage and maximizing function.Mild neurocognitive disorder (MND) is considered an intermediate state between healthy aging and early dementia, or as a potential state that represents a higher risk of dementia. An increasing number of studies that present complementary and alternative approaches of acupuncture intervention for cognitive reserves have also been reported.,7 The afferent neural circuit of somatosensory induced by acupuncture specifically increase cognitive-related functions. Altered brain function and functional networks of cognitive-related brain regions as well as sensorimotor areas following acupuncture in patients with cognitive impairment support the central nervous system mechanism of acupuncture.–11To increase and maintain the cognitive reserve, several cognitive training programs have been increasingly used in MND. Several studies have reported the effectiveness of NF training in improving cognitive function in dementia,14 and MND–17 patients.Neurofeedback (NF) training is a noninvasive brain stimulation technique with operant conditioning closed-loop feedback control mechanism, used in the treatment of a range of neurological and psychiatric conditions.Based on these neural-neurofunctional groundworks, we believe that combined NF-acupuncture treatment could be a meaningful approach to effectively improve cognitive function. However, due to the lack of clinical research, we plan to conduct a randomized, assessor-blind pilot study.22.1The main objective of this pilot study is to evaluate the efficacy and safety of NF-acupuncture combined therapy compared to acupuncture single treatment in MND patients.2.2 score. The secondary outcomes include Alzheimer Disease Assessment Scale–Cognitive (ADAS-Cog) subscale scores, the Korean version of Beck Depression Inventory (K-BDI) score, Body Awareness Questionnaire (BAQ) score, breath per minute (BPM), heart rate variability (HRV) values measured during NF training, functional near-infrared spectroscopy (fNIRS) value, and delayed matching to sample task (DMTS) score. This protocol  follows the Recommendations for Standard Protocol Items: Recommendations for Interventional Trial guidelines. The final approval date of clinical research information service registration is April 28, 2020.This study is designed as a randomized, assessor-blind, parallel trial. The clinical trial site will be set as Daejeon Korean Medicine Hospital of Daejeon University in the Republic of Korea. The number of eligible participants will be set as 44. Subjects will be divided into 2 equal groups: the combined treatment group  and the single treatment group (control group). Each group will be treated twice a week for 12 weeks (visits 1–24). After visit 24, during the follow-up period, participants will be guided to visit for follow-up assessment, which will be visit 25. The primary outcome is the Korean version of Montreal Cognitive Assessment (MoCA-K)2.32.3.1Recruitment will be started on April 22, 2020 . The investigator will elevate eligibility by applying the inclusion and exclusion criteria, conducting vital sign checks and laboratory tests, taking demographic information and medical history of the subject. The investigator will open the envelope that contains the randomization code and give the subject an identification code containing the stratification number .2.3.2.11.Males and females aged between 50 and 75 years.2.Participants diagnosed with MND based on the diagnostic and statistical manual of mental disorders-5 diagnostic criteria.3.Participants with MoCA-K score of 22 or less.4.Participants who have a level of education of more than 6 years (more than elementary education level).5.Participants or authorized surrogates who voluntarily sign the clinical trial consent form.2.3.2.21.Alzheimer disease, vascular dementia, Parkinson disease, Huntington disease, hydrocephalus, etc.2.Patients who have had major psychiatric disorders such as schizophrenia, delusional disorder, bipolar disorder, alcohol, or substance abuse disorder, were diagnosed according to diagnostic and statistical manual of mental disorders-5 diagnostic criteria.3.Patients who have had other neurologic diseases such as epilepsy, brain injury, stroke, etc.4.Patients who have participated in other interventional clinical trials for 1 month before the start of the trial.5.Patients who have performed the diagnostic test of cognitive impairment 3 months before the start of the trial.6.Patients who are pregnant, lactating, or not using appropriate methods of contraception.7.Patients with unstable medical conditions , and chest radiography, etc).8.Patients with hypertension, as defined by a systolic blood pressure above 160 mm Hg, or diastolic blood pressure above 95 mm Hg.9.Patients who had clinically significant liver disease or had serum aspartate aminotransferase, alanine aminotransferase, and/or gamma-glutamyl transpeptidase values more than twice the laboratory test upper limit reference.10.Patients who cannot be measured by fNIRS.11.Patients who disagree with acupuncture treatment.12.Patients assessed as ineligible to participate in the trial by the principal investigator for any other reason.2.4All consenting participants will be subjected to the measurement of vital signs , demographic information survey, medical history , physical examination, laboratory tests, and electrocardiogram tests. Eligible participants who meet the inclusion and exclusion criteria will receive a schedule of visits (screening).At visit 1, the investigator will check the subject's vital signs and test ADAS-cog, K-BDI, BAQ, DMTS (response and accuracy), and fNIRS . The control group will receive acupuncture treatment for 30 minutes, while measuring a simple EEG with NF, and the intervention group will receive acupuncture for a minute while performing the NF training protocol . In the meantime, the investigator will check the subject's BPM and HRV. The investigator will check the subject's adverse reactions and concomitant medications and inform them that cigarettes and caffeine must be avoided 2 hours before the test, as they affect the HRV. From visits 2 to 24, both groups will be treated in the same manner as visit 1, and BPM HRV will be checked in the meantime. The investigator will check the subject's adverse reactions and concomitant medications and inform them in the same manner as on visit 1. At visit 24, the investigator will check the subject's vital signs and test ADAS-cog, K-BDI, BAQ, DMTS (response and accuracy), and fNIRS . Additional visits may be performed at any time if deemed necessary by the investigator's judgment, or following the subject's request. In the event of an unexpected visit, the following tests will be performed: vital signs, checking adverse reactions or concomitant drugs, and laboratory tests. In principle, administration of concomitant drugs will be prohibited during the trial, and when combination therapy may occur, it will be decided whether or not to drop out or exclude at the discretion of the principle investigator (PI). A detailed outline of the schedules is shown in Table 2.5Participants in the control group will visit twice a week for 12 weeks, for a total of 24 visits, to receive acupuncture treatment. Participants in the intervention group will also visit twice a week for 12 weeks, for a total of 24 visits, and receive combined NF-acupuncture treatment. Acupuncture treatment will be performed while abiding by the Standards for Reporting Interventions in Controlled Trials of Acupuncture.2.5.1GV 20, EX-HN1, LI4, and ST36  will be selected as intervention points according to their internal agreement with the acupuncture rationale. The specific operation method will be as follows: the subject sits with their back on the bed to conduct NF training or simple EEG together; the needle is inserted perpendicular to the skin at a depth of 5 to 30 mm; Korean Medical Doctor will leave the intervention room after acupuncture in order to blind NF intervention and return to the room 30 minutes later to remove needles.2.5.2The intervention group will perform the NF sensorimotor rhythm (SMR) training protocol, whereas the control group will have EEG measured for the 30 minutes of acupuncture treatment.The ProComp 2 Neurofeedback system  will be used in this trial. After cleaning the electrode location with an abrasive conductive gel  and filling the electrode cups with conductive gel , an electrode will be placed at the subject's central scalp area according to the international 10/20 system. A reference and ground electrode will be placed on the left and right earlobes, respectively. A 3-electrode location input impedance will be maintained under 5 kΩ.The training session will consist of a 6-minute baseline trial and a 30-minute NF training trial, divided into 6 segments. To determine threshold values, each subject will be measured at baseline SMR wave  for 3 minutes with eyes closed and 3 minutes with eyes open. NF training, which is designed to increase threshold SMR values, will be performed 6 times for 5 minutes with 20 to 30 seconds of rest between each trial. BPM and HRV values will also be collected in each trial. The threshold value at each trial is newly set according to the subject's feedback.2.6 If the dropout rate is higher than expected, even after deliberation, the number of participants can be adjusted.Considering a 10% dropout rate, we conclude that a total of 44 patients is needed to measure the feasibility of combined NF-acupuncture treatment .2.7The investigator in charge of randomization, who will not be involved in the trial, will generate the random sequence list using SAS Version 9.4  program, and each randomized code will be kept in sealed envelopes, to be managed by the PI. For randomized allocation, the stratified block randomization method will be used, for which the stratification factors will be gender and age. This method involves stratification into 4 subgroups, by age and gender: 1 group of 50 to 60 years old individuals and 1 of 61 to 75 years old individuals for each gender. The investigator assigning the randomization code will open the sealed envelope in front of the participants to whom the identification code is assigned. This investigator will record the date of opening, sign the envelope, and store it separately.In this trial, blinding of the subjects is not possible; assessor blinding will therefore be performed to control for bias. An investigator doctor not involved in randomization will perform acupuncture on subjects before NF training or simple EEG, will be blinded to the type of treatment to be administered, and will subsequently perform an evaluation of the effectiveness and safety.Blinding breaking should be determined on a case-by-case basis and considered only in serious medical emergencies. If the investigator determines that blinding breaking is necessary, they can contact the PI or sponsor to obtain consent, and subsequently document the breaking procedure.2.8t test or Wilcoxon rank-sum test to determine normality. Efficacy analysis will be conducted according to the primary full analysis set principle and secondarily by the per protocol principle. All statistical analyses will be based on a two-tailed test and statistically, the significance is set at P < .05. The analysis tool will be SAS Version 9.4 . Missing values will be imputed by the last-observation-carried-forward method.All categorical data will be presented as frequencies or percentiles, and will be analyzed by the Pearson chi-squared test or Fisher exact test. All numeric data will be presented as mean, standard deviation, and will be analyzed by Student independent sample 2.92.9.1MoCA-K will be the primary outcome. The endpoint will be the change in the MoCA-K score from screening to visit 25. We will use the covariance analysis model that includes each group as a fixed factor, MoCA-K score as a dependent variable, and the difference in demographic characteristics as a covariate.2.9.2t test or Wilcoxon signed-rank test will be performed on the same participant on the secondary outcome before and after treatment in each group.ADAS-cog, K-BDI, BAQ, fNIRS, DMTS, BPM, and HRV will be the secondary outcomes. Endpoints will include the change in ADAS-cog, K-BDI, BAQ, DMTS scores, and fNIRS values from visit 1 to visit 25; HRV values from visit 1 to visit 5, 9, 13, 21, and 25; and BMP values from visit 1 to visit 25. As primary outcome, we will use the covariance analysis model that includes each group as a fixed factor, ADAS-cog, K-BDI, BAQ, DMTS scores and HRV, BPM, and fNIRS values as dependent variables, and differences in demographic characteristics as covariates. A paired 2.9.3Evaluating changes in laboratory test results from screening to visit 25, checking of vital signs, and interviews about adverse events at each visit will be performed for safety assessment and adverse event reports.2.9.4Assessment subjects will be primarily assigned to intention-to-treat and secondary per protocol groups. Adverse events will be reported as follows: symptoms, onset, duration, severity , and intervention causality .2.9.5PI will educate the investigator, subjects, or authorized surrogate to inform of any reaction that may occur during or after intervention. If adverse events occur, the involved parties will take appropriate action to minimize adverse reactions. For subjects with adverse reactions, follow-up should be performed until symptoms disappear and the condition is stabilized and a progress report should subsequently be submitted. When a serious adverse event occurs, the investigator will immediately report it to the PI. The PI will thereafter suspend all or part of the clinical trial and report it to the Institutional Review Board (IRB) within 24 hours.2.10The identification information of all documents related to the trial, including the case report form, will be recorded according to the identification code, and the information will be kept confidential in a locked place even after the results of the clinical research are published. For monitoring and trial process management, sponsors or monitoring staff can access and duplicate the document. A separate center's document storage will be prepared for document security. For 3 years after the completion of the result report, a staff in charge of security will be allocated in the storage.2.11An independent staff member on the sponsor side can conduct monitoring by calling and visiting. There are three regular visits, but one or two additional visits can be made depending on the speed of recruitment. The specific visit frequency depends on the monitoring Statement of Purpose. When visiting, the staff will check adherence to the registration schedule and compliance with Korea Good Clinical Practice guidelines. The potential presence of omissions will be checked in records by checking all documents and consulting with the investigator if there is any problem.2.12This trial will be conducted under the principles of the Declaration of Helsinki, and guidelines of the International Council of Harmonization-Good Clinical Practice guidelines. This study has been approved by the IRB of Daejeon University Daejeon Medical Center (No. DJNP-20–01) on April 22, 2020. Whenever there are important protocol modifications, the revision date, reason, content will be recorded and kept. Moreover, protocol revision should be approved by the IRB, and relevant parties, including investigators and participants, should be informed. Consent documents that include participants or authorized surrogates’ voluntary signatures and signature dates should be obtained by the investigator with sufficient explanation of this trial, the appropriate time to determine consent or assent. The trial result dataset cannot be published or used without the consent of the sponsor.3NF training is used as a treatment for memory deficit worldwide; however, despite several trials indicating its effectiveness, it remains controversial. Acupuncture has also long been used as a treatment for memory deficits; although significant evidence has been accumulated, controversy remains regarding the level of evidence, and its effectiveness is also controversial. This randomized, assessor-blind, pilot trial protocol aims to evaluate the efficacy and safety of NF-acupuncture combined therapy compared to acupuncture single therapy for MND patients. We consider the outcome of this test as the primary outcome in this trial, because it appears useful in evaluating the effect of combined NF-acupuncture treatment.MoCa-K was developed for the purpose of screening patients with mild cognitive impairment. Compared to other tests, it is more useful for screening patients with mild cognitive impairment as it includes more prefrontal executive function evaluation items.Although this study is limited in the number of subjects, it is the first randomized controlled trial conducted with NF-acupuncture combined therapy. It will clinically support the wide use of NF training with acupuncture for the treatment of MND. Moreover, we expect that this trial will lead to further study of NF-acupuncture combined therapy or NF training for MND patients.All of the authors have read and approved the final manuscript.Conceptualization: Jun-Hwan Lee, In Chul Jung.Methodology: Jieun Kim, Ju-Yeon Kim, Hae Sook Kim, Yang-Chun Park.Supervision: Jun-Hwan Lee, In Chul Jung.Writing – original draft: Jin Hyung Jeong, Changjin Jung.Writing – review & editing: Jin Hyung Jeong, Ju-Yeon Kim.


Background and objectiveThe coronavirus disease 2019 (COVID-19) pandemic has affected the health, social, and economic sectors all over the world. With a view to assessing the impact of COVID-19 on the mental health of healthcare workers (HCWs), we conducted a study to find out the incidence and severity of depression, anxiety, and post-traumatic stress disorder (PTSD) among HCWs.Material and methodsThis was an observational cross-sectional study conducted in the Department of Obstetrics and Gynaecology in collaboration with the Department of Psychiatry at AIIMS, New Delhi from March 2021 to June 2021. One hundred HCWs working in the Department of Obstetrics and Gynaecology were surveyed using a set of semi-structured interview schedules and structured questionnaires distributed via email or manually. The structured questionnaire included the demographic profile; other baseline information; the 42-item Depression, Anxiety, and Stress Scale (DASS-42); and the Impact of Event Scale-Revised (IES-R) instrument. Data analysis was carried out using the statistical package STATA version 14.0 .ResultsA total of 100 HCWs participated in the study, out of which 39 (39%), 45 (45%), and 16 (16%) were doctors, nursing staff, and supporting staff, respectively. Overall, 92 (92%) of the participants were women, and the mean age of the participants was 29.87 ±4.85 years. Out of the 100 participants, 17 (17%), 25 (25%), 13 (13%), and two (2%) participants had depression, anxiety, stress, and PTSD, respectively. Occupation-wise, among the nursing staff, doctors, and supporting staff, the incidence of depression was 24.4%, 15.4%, 0.0%, respectively; the anxiety rate was 33.3%, 25.6%, and 0.0%, respectively; and the rate of stress was 17.8%, 12.8%, and 0.0%, respectively. The IES-R score was significantly higher among unmarried as compared to married participants . Participants living in joint families had a higher DASS-42 score  and lower IES-R score  as compared to those living in nuclear families. Depression , anxiety , stress  and PTSD  were all higher among HCWs having exposure to COVID-19 more than 10 hours per week compared to participants with an exposure of less than 10 hours per week. The participants having psychiatric illness in the family showed significantly higher mean values for DASS-42  as compared to those without any psychiatric illness in the family.ConclusionBased on our findings, occupational and environmental factors at the workplace play a key role in mental health outcomes, and COVID-19 has had a significant impact on the mental health of HCWs. Furthermore, we have also observed that effective planning can significantly reduce mental stress. In December 2019, the world witnessed the emergence of several cases of pneumonia of unknown cause in China, which eventually led to a pandemic of global proportions called coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The WHO declared COVID-19 a global pandemic in March 2020 . Since tAt our institute also, following a concerted effort by the department in collaboration with hospital administration, we prepared ourselves to face the challenges posed by COVID-19. A road map for the new normal was developed and implemented to smoothen the obstetric care to be provided during COVID-19 as early as possible . The worVarious studies have shown that this pandemic has profoundly affected the mental health of HCWs, and many doctors and nurses have been suffering from depression, anxiety, and increased levels of stress -11. HighThis study aimed to assess the incidence and severity of depression, anxiety, and post-traumatic stress disorder (PTSD) among HCWs and also to explore the association of these with background variables including gender, marital status, occupation, medical history, and level of involvement with COVID-19 patients. Additionally, we aimed to identify the more vulnerable subgroups and the potential risk factors so that early intervention can be planned and continuity of care maintained.This was an observational cross-sectional study conducted in the Department of Obstetrics and Gynaecology in collaboration with the Department of Psychiatry at AIIMS, New Delhi from March 2021 to June 2021 after obtaining ethical approval from the ethics committee of the institute. We enrolled 100 HCWs working in the Department of Obstetrics and Gynaecology, including doctors, nursing staff, and supporting staff members. HCWs of both genders who were willing to participate were included in the study. Those having any prior history of depression, anxiety, psychosis, or other psychological problems were excluded from the study.Data collectionThe survey was conducted using a set of semi-structured interview schedules, and structured questionnaires were distributed via email or manually as per the convenience of the participants. The semi-structured questionnaire schedule sought information regarding the demographic profile of the participants, including age, gender, religion, educational level, relationship status, occupational history, medical history, level of direct involvement with COVID-19 patients, the source of information regarding COVID-19, and whether the training provided by the institute had helped them or not. The structured questionnaire included the Depression, Anxiety, and Stress Scale (DASS-42) , which cStatistical analysisData analysis was carried out using the statistical package STATA version 14.0 . All questions were given numerical codes and the answers were categorized into mild, moderate, and severe according to the cut-off value for each domain. Descriptive measures such as mean, standard deviation (SD), and range values were calculated for each domain and compared across the categories using the analysis of variance (ANOVA) test. The association of the severity of the anxiety, depression, and stress level with background variables was compared using Pearson's chi-squared test. A two-sided probability of p<0.005 was considered statistically significant for all statistical tests.A total of 100 HCWs participated in the study, out of which 39 (39%) were doctors, 45 (45%) were nursing staff, and 16 (16%) were supporting staff, including health assistants, technicians, guards, and sanitary assistants. Overall, 92 (92%) of the participants were women, and the mean age of the participants was 29.87 ±4.85 years. Among the cohort, 70 (70%) were graduates, 46 (46%) were married and living with their families, and 74 (74%) had nuclear families  participants had less than 10 hours/week of exposure, and 64 (64%) participants had more than 10 hours/week of exposure to COVID-19-infected patients , 25 (25%), 13 (13%), and two (2%) participants had depression, anxiety, stress, and PTSD respectively. Occupation-wise, among the nursing staff, doctors, and supporting staff respectively, the incidence of depression , anxiety , and stress  were higher among the nursing staff compared to doctors and supporting staff, but this difference was not statistically significant  and IES-R score  than males; but due to a minimal proportion of males in the study, the difference was not statistically significant. The IES-R score was significantly higher in unmarried as compared to married participants . Participants living in joint families had a higher DASS-42 score  and lower IES-R score  as compared to those with nuclear families. The mean value of the DASS-42 score  and IES-R score  was higher among doctors than nursing staff and supporting staff respectively, but the difference was not statistically significant. Depression , anxiety , stress , and PTSD  were all higher among HCWs having exposure to COVID-19 for more than 10 hours per week compared to participants having exposure for less than 10 hours per week. The participants having psychiatric illness in the family showed significantly higher mean values for DASS-42  as compared to those without any psychiatric illness in the family , four (23.5%), three (17.6%), and two (11.8%) participants respectively. When the severity of anxiety was observed, mild, moderate, severe, and extremely severe anxiety was found in 12 (48%), six (24%), three (12%), and four (16%) participants out of 25. With regard to stress, out of 13, six (46.2%), four (30.8%), and three (23.1%) were having mild, moderate, and severe stress. None of them had extremely severe stress. Two participants were having extremely severe PTSD.The COVID-19 pandemic is an ongoing global challenge, and as the disease spreads rapidly, the most affected countries are facing huge challenges to fulfill the demands for PPE kits and infrastructure. However, India witnessed the emergence of the COVID-19 pandemic slightly later than the rest of the world, and the strict initial strategies implemented by the Government of India, like complete lockdown, had delayed the spread of the disease and provided sufficient time to our healthcare system to be prepared for the pandemic.In our department, our proactive team took timely decisions and helped us prepare ourselves for handling this pandemic with the administration's help. Measures such as the sufficient supply of PPE kits, appropriate segregation of patients and treating HCWs in COVID-19-positive, suspect, and regular areas, continued provision of information regarding COVID-19, the training regarding the proper use of PPE kits, and other safety measures enabled our team to be prepared effectively and efficiently during COVID-19. This adequate level of preparation and the lower number of participants must be the reason that our results are not in concordance with the results of most of the studies conducted outside or inside India. In our study, the incidence of depression, anxiety, stress, and PTSD was 17%, 25%, 13%, and 2%, respectively, which was much lower than many international and national studies, as shown in one latest study where out of 1,563 HCWs, 50.7%, 44.7%, and 36.1% showed symptoms of depression, anxiety, and sleep deprivation, respectively . A studyZhang et al.  and Lu eBeing a frontline worker and the duration of exposure also affected the outcomes. At our institute, we had posted the staff on a rotation basis, and every participant was directly exposed to the patients; however, there was a difference in exposure time depending upon their ward postings. The participants with more than 10 hours per week of exposure to COVID-19 patients were affected more than the participants with less than 10 hours per week of exposure, and this difference was statistically significant. This finding is in line with the results shown in many other studies ,23,24. AThe female gender has been considered a risk factor for developing depression, anxiety, and insomnia by Lai et al. . AnotherWhen we considered the protective factors, almost all HCWs agreed that the provision of adequate PPE kits, good hospital guidance, and the facilities provided to them were very satisfactory and helped them maintain their mental stability during this pandemic. In addition, measures such as continuous training in the form of mock drills, segregation of all the wards and the pathways heading to the COVID-19 areas, the sterilization system, contact-tracing of affected persons, and provision of appropriate isolation and quarantine facilities for HCWs and their families ensured the HCWs that the institute is keenly concerned regarding their safety. Various studies have also shown that good hospital guidance, increased knowledge about the disease, and proper training reduce the stress experienced by the HCWs ,26,27. SThe major strength of our study is that we had included participants from all segments of HCWs, including doctors, nursing staff, health attendants, sanitary attendants, and technicians, and conducted a subgroup analysis to find out the most vulnerable group in terms of psychological impact so that appropriate counseling and management can be provided. In addition, many previous studies did not address the medical history of psychiatric disorders in the family or the participant itself; we have included both and excluded participants who had any form of psychiatric illness.This study has some limitations. Firstly, it was a self-reported survey, and this may have led to recall bias. Our sample size was very small and represented only a small group of our team, especially the supporting staff, which may have affected the results. The study was conducted at an apex institute, and due to the lack of generalized SOPs, the management system can be different at different institutes; hence the results do not apply to the whole population of HCWs working in the obstetrics and gynaecology departments in the country. The study was conducted during the end of the first wave of COVID-19 when the HCWs had become more prepared mentally and socially. The first wave did not have so much impact and this factor can impair the generalizability of results. Finally, we did not have any other data to compare our results against, such as those collected during pre-COVID-19 or post-COVID-19 periods; hence, it is not easy to ascertain if the psychological impact is associated with COVID-19 alone or if any other factors also played a role.Our study clearly showed that even after providing adequate facilities to the HCWs, their mental stress levels were very high, and we need to take more action to make the environment more comfortable for our HCWs. We can be exposed to this type of situation again; hence, we should be prepared to overcome the stress related to such circumstances in the near future and should put in place proper facilities and resources for our HCWs so that they can deal with the challenges effectively.Our study showed that occupational and environmental factors at the workplace play a key role in mental health outcomes, and COVID-19 has significantly impacted the mental health of HCWs. Adequate planning, complete and timely provision of information, proper provision of PPE kits, along with regular training of the staff significantly contributed to reducing their mental stress at our institute. We may encounter similar outbreaks and pandemics in the future as well, and hence to protect and improve the psychological well-being of the HCWs who are often at the forefront of fighting these events, we can initiate screening programs and can explore various psychotherapeutic interventions and support mechanisms for HCWs.


The purpose of this study is to develop metrics for stigma-producing factors by conducting a survey among stigma inflictors, that is, people who participate in the stigmatization of others. This study attempted to develop a stigma measurement scale for service industry workers. This study focused airline cabin crew members in accordance with a seven-step scale development procedure to derive stigma measurement variables. As a result, the stigma scale developed in this study consists of 6 factors  containing 28 measurement items. This study indicates the need to find countermeasures that can reduce stigmatization of employees within organizations. At a personal level, the practical implication is to prevent stigmatization from occurring within the organization by improving the perception of stigma by cabin crew within the airline organization. At the organizational level, the practical implication is to analyze and reduce the factors of social stigma that negatively affect organizational performance. Human prejudices greatly influence decision making ,2. FirstIn most social environments, we interact with various people. When we perceive negative attributes in strangers, we often treat them with disdain and value them less than other ordinary people who are aligned with social norms. The phenomenon where a person is perceived as less worthy of honor, due to a certain attribute, by almost the entire society is defined by scholars as stigma .Social stigma is the damaged social identity of a person . It refeMost research on stigma consists of medical and psychosocial studies on the effects of stigmas caused by acquired immune deficiency syndrome (AIDS), mental illness, alcoholism, physical disability, and smoking, etc. The areas of interest were mainly mental and physical damages caused by stigma, and education, treatment, and countermeasures to solve them. Recently, the research field has been expanding to include stigmas on brands and corporate bankruptcy, the impact of stigma on organizational members, and stigmas that occur within an organization. However, there has been little research on stigmas on workers in the hospitality industry. This study attempts to develop a stigma scale by identifying factors that cause airline cabin crew workers to be stigmatized by their co-workers.Considering the hierarchical team-based structure of cabin crews, when stigma occurs in the organization, the victims are mostly juniors, not seniors. This study analyzed the stigma phenomenon in airline organizations from the perspective of the inflictors (seniors who impose stigmas on juniors) and developed a measure of social stigma. The development of a stigma scale for airline cabin crew may contribute to preventing unnecessary conflicts within the organization by helping us to understand the stigma phenomenon in the service industry and identifying factors that cause stigma within cabin crews.This study highlights the need to form countermeasures for reducing stigma within the organization. At a personal level, the practical implication is to prevent stigma from occurring within the organization by improving the perception of stigma by a cabin crew within an airline organization. At the organizational level, the practical implication is to analyze and reduce the factors of social stigma that negatively affect organizational performance.Stigma is a phenomenon in which an individual possesses a degraded social identity due to a group’s prejudice towards that individual within a social context . StafforStigma: notes on the management of spoiled identity, basic concepts and approaches were classified from the perspective of social psychology. The three distinctions for stigma are ethnic identity, personal character flaws, and abomination of the body. Link and Phelan (2001) have suggested a multidimensional model in which the concept of stigma is separated into various components, including: discrimination, stereotyping, labelling, separation, and status loss within the context of power differential [The term stigma relates back to the ancient Greeks, who burned or cut marks into the skin of slaves, criminals, and traitors in order to identify them as immoral or tainted people who should be avoided . Today, erential .Newly developed measures or scales should have an empirical connection to “gold standard”-like standards. This is known as criterion validity , and meaJob Competency (JC)Job competency refers to an individual’s characteristics and abilities that are exhibited when performing a job . CompeteWork-related competency of the cabin crew is crucial to an airline company because success in the airline industry depends largely on the quality of service provided by the cabin crew . Also, tConscientiousness (CS)McCrae and Costa (1985) developed NEO , a personality test that measures the reliability and validity of the Big Five . As per Researchers have found that conscientious employees: are not only more punctual ,25, but Honesty (HH)The HEXACOLeader member exchange (LMX) is defined as the exchange relationship with the leader from a subordinate’s point of view. Leaders and subordinates affect each other’s work by forming a social exchange relationship . LMX theThe organization of airline cabin crew, which is a representative service organization, has introduced a team system to improve service quality and efficiency of manpower management through teamwork.Physical Attractiveness (PA)Physical appearance is characterized by a frontline employee’s facial features, physique, and attire ,51,52. SMany studies have found that attractive physical appearance of frontline service providers helps to create positive impressions on customers ,52. PhysMental Health (MH)In response to the economic and social impacts of common mental health disorders such as depression , a combiPositive mental health is expressed as psychological well-being, and negative mental health is expressed as psychological stress such as depression, anxiety, and loss of emotional and behavioral control . The stiPsychological Well-Being (PW)Psychological well-being is similar to the concept of welfare or happiness, but it is hard to define clearly because the meaning varies according to time, people, and place . PsycholPsychological well-being indicates accepting oneself positively, forming relationships with people, performing autonomous actions, and taking independent decisions in accordance with the standards and principles set by oneself . PsycholPerceived Insider Status (PIS)Perceived insider status refers to the degree to which an employee feels like an insider at the workplace . It descEmployees who have a high insider status are more likely to have positive organizational attitudes  was conducted to verify whether the initial items derived through expert surveys were suitable for the cabin crew stigma evaluation scale. The data collection period was for about two weeks from 10 September to 22 September 2020 and the data were collected at Incheon Airport and Gimpo Airport in South Korea. A total of 174 questionnaires were collected from Korean cabin crew members working at domestic airlines, out of which 4 were excluded due to reliability concerns and 170 were used in the analysis. To understand the demographic characteristics of this study, a total of 170 questionnaires were analyzed using the SPSS 24 statistical program.Study 2: The main survey was conducted to verify whether the refined items through the 170 questionnaires were suitable for the cabin crew stigma evaluation scale. A total of 29 questions were derived from the results of the exploratory factor analysis conducted through the first pre-survey, and the questionnaire was modified and distributed. The main survey data collection lasted for 16 days from 25 September to 10 October 2020, and was conducted at Gimpo Airport in South Korea with Korean cabin crews working for domestic airlines. A total of 179 questionnaires were collected and 172 were used for the analysis, excluding seven unreliable ones. The 172 questionnaires were analyzed using the SPSS 24 statistical program to analyze the demographic characteristics of the survey.Study 3: A total of 353 questionnaires were collected based on the pre survey and the main survey, and 342 were used for analysis, excluding 11 unreliable ones. In order to understand the demographic characteristics of this survey, the 342 questionnaires were analyzed using the SPSS/AMOS 24 statistical program. The frequency analysis results consisted of gender, position, marital status, education level, age, annual salary, and job tenure (number of years worked).In this study, we attempted to develop a scale based on previous studies of ,87,88,89First, in the research stage, related literature was investigated for the composition of initial items. This literature review was conducted in order to use in-depth interviews conducted for scale development for reference validation analysis to find out the correlation between refined measurement items and refined measurement variables based on literature research. Second, initial items were derived by conducting in-depth interviews with airline cabin crew members. Based on the extracted contents, the measurement items were refined through the process of correction, deletion, and addition through the opinions of experts. Third, based on the in-depth interviews, key words were selected and the first expert survey was conducted on a total of 10 people, consisting of academia and field experts. Reflecting the opinions of experts, measurement items were refined through deletion, modification, and addition. Fourth, the second expert survey was conducted based on the measured items refined through the first expert survey. The procedure was the same as that of the first item refinement. Fifth, the measurement items were developed through content validity verification. The content validity ratio (CVR) and a 5-point Likert scale average are calculated to verify the validity of the content, and statistically insignificant values were removed from the items. Sixth, a study 1 (pre-test) was conducted to verify the first scale, and validity factor analysis and confirmatory factor analysis were conducted based on the collected questionnaire data to verify validity and reliability.This survey was conducted to perform the 2nd scale verification by supplementing and revising the 1st scale verification step. To evaluate the constructed scale, the dimensionality was re-verified through confirmatory factor analysis and the construction validity was verified. Finally, statistical significance was reviewed through the criterion validity to see if the airline cabin crew stigma factors derived through previous studies and in-depth interviews are suitable and have any correlation. For the criterion validity analysis, Pearson correlation analysis was performed to verify the statistical significance between variables.This study was conducted on airline cabin crew members in accordance with a seven-step scale development procedure to derive stigma measurement variables. Through a literature review, a total of eight stigma factors  and initial items were derived. Based on previous studies, in-depth interviews were conducted with cabin crew members using free association questionnaires and the validity of the content was reviewed by referring to expert opinions. Based on this, the first and second expert surveys were conducted to refine the airline cabin crew stigma factors into seven factors  and 41 initial items.Study 1 was conducted on 170 airline cabin crew members through a questionnaire consisting of the derived measurement items. As a result of conducting exploratory factor analysis (EFA) and confirmatory factor analysis (CFA), 7 factors and 41 items were reduced to 6 factors and 29 items, deleting 12 items. To verify that the items refined in the first pre-test were suitable for the cabin crew stigma evaluation scale, a second survey was conducted on airline cabin crew members. Confirmatory factor analysis was conducted using 172 questionnaires, and one factor was deleted because it did not show statistical significance, and a total of 6 factors  and 28 measurement items were derived. Finally, to verify the theoretical validity of the airline cabin crew stigma, based on the 342 questionnaires collected in the first and second surveys, criterion validity analysis was conducted on 8 leading factors presented through literature study and the 6 factors derived from the in-depth interviews. As a result of Pearson correlation analysis, it was found that the correlations between all variables were statistically significant.In the research analysis, we utilized program of IBM SPSS Statistics 21 and AMOS 21 for all statistical analyses. Descriptive statistics were utilized to identify the sample characteristics. To achieve research objectives, we followed Churchill ; DeVelliStep 1: Based on literature studies, in-depth interviews were conducted with airline cabin crew. The following specific questions were asked.Have you ever had a stigma in your workplace?Have you ever experienced being stigmatized in your workplace while working at an airline?Have you ever seen someone else experience stigma? Step 2: This study refined the measurement items based on literature studies, in-depth interviews, and first and second expert surveys. As shown in Study 1: Among the 170 survey participants, 145 (85.5%) were female and 25 (14.7%) were male. The participants were asked to indicate their annual incomes. Most respondents had an annual income of 30,000,000 to 39,999,999 Korean won (31.8%), followed by 40,000,000 to 49,999,999 won (27.1%), and 50,000,000 to 59,000,000 (23.2%). Regarding education level, 123 (72.4%) had a bachelor’s degree, followed by 26 (15.3%) graduate-degree holders, 20 (11.8%) 2-year college or some college graduates, and 1 (0.6%) high-school graduate or less. The survey also asked participants their work tenure. The majority  replied more than 10 years. As for respondents’ age, the age group with the highest proportion  was 30–39 years, followed by 20–29 years , and 40–49 years . Regarding job positions, most of them, that is, 86 (50.6%), were flight attendants (SS/SD), 40 (23.5%) were assistant pursers (AP), 35 (20.6%) were pursers (PS), 8 (4.7%) were senior pursers (SP), and 1 (0.6%) was chief purser (CP).Study 2: Among the 172 survey participants, 152 (88.4) % were female, and 20 (11.6) % were male. The majority reported an income of 30,000,000 to 39,999,999 Korean won , followed by 40,000,000 to 49,999,999 won , and 50,000,000 to 59,000,000 . Regarding education level, 124 (72.1%) respondents had a bachelor’s degree, followed by 31 (18.0%) college graduates or 2-year college degree holders, and 17 (9.9%) graduate-degree holders. The survey also asked participants their work tenure. The majority  had been working for 5–7 years. The majority age group  was 20–29 years, followed by 30–39 years , and 40–49 years . Regarding job positions, 99 (57.6%) were flight attendants (SS/SD), 45 (26.2%) were assistant pursers (AP), 20 (11.6%) were pursers (PS), 7 (4.1%) were senior pursers (SP), and 1 (0.6%) was chief purser (CP).Study 3: Among the 342 (170 + 172) survey respondents, 297 (86.8%) were female, and 45 (13.2%) were male. The most common annual income was 30,000,000 to 39,999,999 Korean won , followed by 40,000,000 to 49,999,999 Korean won , 50,000,000 to 59,999,999 Korean won , 60,000,000 won or more , and 24,999,999 won or less . Regarding education level, 247 (72.2%) respondents had a bachelor’s degree, followed by 51 (14.9%) 2-year college or some college graduates, 43 (12.6%) graduate-degree holders, and 1 (0.3%) respondent with a high-school graduate degree or less. The majority were of age 20–29 years  followed by 30–39 years  and 40–49 years . Regarding the job positions, 185 (54.1%) were flight attendants (SS/SD), 85 (24.9%) were assistant pursers (AP), 55 (16.1%) were pursers (PS), 15 (4.4%) were senior pursers (SP), and 2 (0.6%) were chief pursers (CP).As a result of conducting exploratory factor analysis in the initial items, a total of 20 items were deleted from the initial items reducing 8 factors (49 items) to 6 factors (29 items). In addition, neuroticism and human relations were grouped into the same factor, forming a single factor.  shows thχ2 = 3568.284, df = 406, p < 0.001). The total cumulative proportion of variance is 71.931%, which satisfies the appropriate level of 70% or more, and all factor loadings are 0.6 or more, suggesting that the convergent validity of the items has been secured.As shown in Cronbach’s alpha(α) reflects the internal consistency reliability among indicators of a construct . With vaCommonality of 0.4 or less consisted of a total of 29 items excluding 12. The 12 items which were deleted included three items of work ability , one selfishness item (selfishness 2), four work ethic items , two appearance items , and two items of human relations .χ2 = 764.059, df = 362, p < 0.001, χ2/df = 2.111, RMSEA = 0.081, CFI = 0.882, IFI = 0.883, TLI = 0.867). Internal consistency for each research construct was evaluated. As shown in We analyzed the measurement model using the CFA. The result from the CFA indicated that the measurement model included an adequate fit to the data , conscientiousness (3), selfishness (6), work ethics (3), appearance (2), neuroticism (8)—were finally derived.χ2 = 897.431, df = 362, p < 0.001, χ2/df = 2.479, RMSEA = 0.093, CFI = 0.901, IFI = 0.902, TLI = 0.889).As shown in In this study, Pearson’s correlation was used to analyze the correlation between variables. The correlation between all variables was found to be significant, and the contents are shown in A total of 13 items that did not satisfy statistical significance were deleted from the initial items. As a result, the stigma scale developed for cabin crew consisted of a total of 6 factors and 28 items, including work ability (6), conscientiousness (3), selfishness (6), work ethics (3), appearance (2), and neuroticism. Over the past few decades, American researchers have studied the stigma phenomenon of various social groups . Many prThe purpose of this study was to develop metrics for stigma-producing factors by conducting a survey among stigma inflictors, that is, people who participate in the stigmatization of others. The stigma scale for cabin crew members was composed of a total of six factors: work ability (6), conscientiousness (3), selfishness (6), work ethics (3), appearance (2), and neuroticism (8).Work ability (WA) showed that cabin crew members often suffered alienation and stigmatization due to negative performance evaluations. Additionally, there were many cases where individuals who were unable to communicate well with other team members or did not understand the work properly, felt stigmatized.Conscientiousness (CS) indicated that stigma occurs when a cabin crew member exhibits inadequacy at work, and neglects briefings or work. Due to the nature of the work that cabin crews do, team members are required to collaborate within a short period of time, and one of the team’s members may experience a setback in their work if they leave due to unfaithful factors.Selfishness (SF) includes being obsessed with appreciation letters, being greedy, flattering and manipulating the leader, excessive devotion to the company to achieve success, and focusing solely on promotion.Work ethics (WE) includes taking in-flight goods for personal use, lying at work, and the tendency to exaggerate . Work ethics as a moral and ethical factor refers to employees’ shared perception of what is right or wrong within the organization and how to deal with related ethical issues .Appearance (AP) deals with violation of company attire regulations or the overall grooming being unsatisfactory. The appearance of the cabin crew represents not only an individual but also a company image, creating an overall image and atmosphere in an environment that provides services .Lastly, neuroticism (NT) includes emotional ups and downs, violence in words and actions, hysteria, hypersensitivity, harassment of juniors, inclusion in the blacklist, causing disruption of teamwork, humiliating others in front of many people, and innocence. Some of these items were originally part of the human relations factor but were later integrated into neuroticism after looking at the statistical results of exploratory factor analysis.The purpose of the study was as follows. First, this study attempted to develop a measurement scale for stigma factors by investigating the areas from which stigmas arise by looking through the perspective of stigma inflictors (senior cabin crew). These areas include work ability, conscientiousness, selfishness, appearance, work ethics, and neuroticism.Second, this study attempted to develop a stigma scale for service industry workers, particularly airline cabin crew members. This study revealed how stigmatization occurs in the service industry and developed a scale for assessing stigma in the specific context of airline cabin crew members.Third, by developing a stigma measurement scale, this study seeks to understand how stigma occurs among airline cabin crews in order to find solutions to minimize its negative effects. Stigma depends on social, economic, and political power, and has negative consequences for individuals and organizations, including loss of status, discrimination, stereotyping, alienation, and rejection . By helpThe practical implications of this study are as follows. First, this study is meaningful as it analyses the causal factors of stigmas occurring within airline cabin crews. Second, by identifying the direct causes of workplace stigmas, they can be better prevented and self-stigmatization of victims can be minimized, thereby strengthening competency. Third, this study shows that all cabin crew members need to be faithful to their assigned responsibilities and roles in order to avoid being stigmatized. Fourth, for efficient performance at the organizational level, strategic measures should be taken to create a positive organizational culture such that stigmatization does not occur. Finally, victims who have suffered stigmatization must undergo psychological treatment to overcome the trauma of their stigma and improve their quality of life. At the organizational level, it should be emphasized that prevention is a more efficient way to manage airline personnel performance than post-trauma intervention.First, stigma is a concept established by Goffman (1963), and various studies have been attempted, centering on the socially vulnerable classes. However, there is a relative lack of domestic and foreign literature on workplace stigmas. Notwithstanding the significance of developing a scale specifically for airline cabin crew, the present study is somewhat lacking in the literature review on the relationships within cabin crews and how stigmas affect them. In the future, we intend to conduct stigma research in various fields to create a foundation of research literature on workplace stigmas that could guide future researchers.Second, this study conducted qualitative interviews and surveys only for cabin crews working for domestic South Korean airlines. Domestic cabin crews differ from international cabin crews in terms of working conditions and work environment. In future research, we will conduct a comparative analysis study on the causes of stigma not only in domestic cabin crews but also in the cabin crews of airline companies in other countries.Third, the demographic analysis of this study showed the proportion of women to be significantly higher than that of men. Even though this reflects the general trend of more women being employed in airline cabin crews, it is somewhat regrettable that the gender ratio was not evenly distributed. In future studies, a survey will be conducted by subdividing the sample with a longer survey period. Finally, when a factor is defined by 4–5 items with loadings above 0.50, it is a solid factor with practical relevance but the factors conscientiousness (3), work ethics (3) and appearance (2) are defined by fewer items. The sample was not entirely representative of airline cabin crews. In future research, more participants will be interviewed to construct more items for each factor.


SCFA-WA) on growth performance and microbiota of weaned piglets. In total, 192 male piglets, approximately 4 wk of age, were allocated to 24 pens (12 per treatment) with 8 piglets per pen. The piglets received either regular drinking water (negative control) or drinking water with the acidifier supplied at 2 L/1,000 L. Body weight and feed intake were measured weekly on pen level. During the first 2 wk, daily visual assessment and scoring of the feces was conducted. Fecal samples of three piglets per pen were collected on days 14 and 42 for high-throughput sequencing analysis of the microbiota. Piglets offered SCFA-WA had significantly improved feed efficiency in the third week (P = 0.025) and over the whole study period  compared with piglets in the negative control group, with a strong tendency observed during the first feeding phase . Furthermore, the water acidifier group had a higher water intake than piglets provided with control water during the second feeding phase  and over the whole study period . There was no significant difference in body weight, average daily gain, or average daily feed intake . Furthermore, there was no overall significant difference in fecal scoring between the treatments. In terms of the fecal microbiota response, piglets offered the water acidifier showed a significantly higher relative abundance (RA) of genus Clostridium sensu stricto 1 and a lower RA of genus Streptococcus compared to the control. Furthermore, the redundancy analysis showed a positive association between improved feed efficiency and daily weight gain and RA of Butyricicoccus and Faecalibacterium. In conclusion, consumption of the water acidifier containing free and buffered SCFA modulated the microbiota and improved feed efficiency in piglets.This study examined the effect of a water acidifier containing free and buffered short-chain fatty acids ( The weaning process is a stressful time for piglets, as they adapt to new diets and transition to new physical and social environments. During the weaning phase, piglets often have reduced feed intake, subsequently leading to inadequate utilization of nutrients in terms of both digestion and absorption, and an increased incidence of diarrhea . Recent SCFA), which are known to have beneficial effects for the host  prior to animals being sourced and was carried out under Home Office licence. The study was discussed and approved by the AWERB prior to the start of the in-life phase.The farm housing and husbandry were representative of EU farming conditions and met relevant ethical, hygienic, and animal welfare requirements.SCFA-WA containing formic acid, acetic acid, propionic acid, copper, zinc acetate, ammonium formate, and a pH of 2.7 to 3.3; Selko B.V., Tilburg, the Netherlands), with 12 pens per treatment. Each room was equipped with two water lines, each connected to a water tank containing the respective treatment. The control group received regular drinking water without any additions. The SCFA-WA group received drinking water with SCFA-WA treatment added to the water tank, where the inclusion rate for the first 3 d was 1 L/1,000L , from days 4 to 6 the inclusion rate was 1.5 L/1,000 L, and from day 7 to the end of the experiment (day 42), the inclusion rate was 2 L/1,000 L. The dosing was based on titration of the drinking water with SCFA-WA until a pH of 3.8 was reached, as per manufacturer recommendations. Once weekly, samples of water were obtained from the drinkers of two pens per treatment per room to monitor pH. One common wheat-based diet was offered to both treatment groups over the two phases, phase 1 being from day 0 to 21 and phase 2 being from days 21 to 42  at an average live weight of 8.73 kg (between 6.6 and 11.6 kg) and approximately 4 wk of age were housed at Drayton Animal Health Ltd. . The newly weaned piglets were randomly allocated to 24 pens, in groups of 8 piglets, blocked by weight, equally divided over 3 rooms. The pens were randomly allocated to two treatments, 1) negative control (Control) or 2) water acidifier containing free and buffered SCFA (21 to 42 . The feeBW) and feed intake were measured on pen level at days 0, 7, 14, 21, 28, 35, and 42. Based on this data, the average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (FE) were calculated. The water intake was calculated per water line by measuring differences in the level of water in the water tank. To measure the difference in water level, dipsticks were used with 10-L increments. Each morning, the water left in the tank was measured with these dipsticks and rounded up or down to the nearest 10 L. The tanks were subsequently emptied and refilled with fresh water mixed with or without the SCFA-WA. In the afternoon, the water level was checked to make sure the piglets had enough water overnight. Water intake was averaged between the three rooms. Mortality was monitored daily.Body weight  feces, score 3 = thick or thin, liquid feces). The staff members did the scoring sessions together to standardize their interpretations. On days 14 and 42, three average sized piglets per pen, which were representative for the pen, were selected and each used to collect approximately 10 g of feces via rectal palpation. The same piglets were used at both sampling days. Of the three individual samples per pen, two subsamples of at least 1 g each were taken in a cryovial and stored at −80 °C for microbiota analysis. The residual part of the sample was stored at the animal facility as retention sample.qPCR) with 926F  following the manufacturer’s instructions with some modifications to extract DNA instead of RNA, such as omitting the β-mercaptoethanol and DNase I steps. The fecal samples were weighted and approximately 70 mg of sample was mixed with 650-µL PM1 solution including 100-µg PureLink RNaseA . Bacteria in the samples were lysed with MagNA Lyser  for 2× 40 s at 5,500 rpm prior to DNA extraction. The DNA was extracted from this cell lysate on the spin filter columns following the manufacturer’s instruction manual, the genomic DNA was eluted from the spin columns in 100 µL 10 mM Tris–HCl buffer (pH 8.0). The concentration of the extracted prokaryotic DNA in each sample was calculated by quantitative polymerase chain reaction (ith 926F  and 1027ith 926F  primers RA) at each taxonomic level was determined.Sequencing reads for each time point were analyzed with NG-Tax pipeline  trimmingt-test to compare treatments. Body weight, ADG, ADFI, and FE data were analyzed by week, by feeding phase and overall study period. Fecal consistency scores were analyzed by Kruskal–Wallis test per day. Where a significant difference was observed in body weight, ADG, ADFI, FE, or fecal consistency scores, Duncan’s multiple range test was carried out. No outliers were removed for analysis.Data analysis of the growth performance and fecal scoring was carried out by the statistician of DAH  using GenStat 12.2  software, accepting a level of probability of less than or equal to 0.05 as indicating significance and a level of probability between 0.05 and 0.10 as indicating tendency. Body weight, ADG, ADFI, and FE were analyzed by T-test: P > 0.05). For the beta diversity analyses, the Bray–Curtis index and Unifrac were used with ANOSIM to assess statistical differences between groups using OUT-level data. The Bray–Curtis index is based on microbial OTU counts and indicates a difference in OTU abundance between treatments  to determine significance.Alpha and beta diversity microbiota analyses were performed on rarefied data using MicrobiomeAnalyst. Alpha diversity measures included Chao1 index, Observed Species, Shannon index, and Simpson index and were performed at each taxonomic level data , multivariate analysis  with forward selection of the effect of experimental variables , and the partial RDA to evaluate the effect of experimental variables separately were performed in Canoco5 ( Canoco5 . In the P = 0.055). FE was significantly improved in SCFA-WA (0.696) compared with the control  in the third week of the study, which likely contributed to the strong tendency observed in the first feeding phase. Over the entire study (days 0 to 42), FE was significantly improved in SCFA-WA piglets (P = 0.042). During the second feeding phase (days 21 to 42) and over the whole study period (days 0 to 42), the SCFA-WA group consumed significantly more water ; however, the overall differences were not significant (data not shown). In general, the diarrhea incidence was relatively low throughout this study.The sum of total incidence of feces with score 1 (no diarrhea) was numerically higher in the control group , whereas feces with the score 3 (diarrhea) was numerically higher in the treatment with SCFA-WA . Whether piglets in the treatment with SCFA-WA had more diarrhea cannot be concluded as statistical analysis was not performed over the total sum per treatment. On day 13, there was a significant difference in fecal scoring , but there was no difference in the unweighted and weighted Unifrac. On day 42, there was a significant difference in the Bray–Curtis index and weighted Unifrac between the two treatments (P = 0.002 and P = 0.003). On day 14, T-test statistics showed that none of the alpha diversity metrices differed between the treatment groups. On day 42, Shannon and Simpson indexes at Class level were significantly lower in the SCFA-WA treatment (P = 0.047 and P = 0.018) and Shannon and Simpson indexes at Order level were significantly higher in the SCFA-WA treatment (P = 0.029 and P = 0.024).On day 14, ANOSIM analysis showed a significant difference between control and SCFA-WA treatment in beta diversity Bray–Curtis index , and a trend in Bacteroides pectinophilus group (P = 0.054) and Eubacterium nodatum group (P = 0.059). At Family level, a higher RA was seen in Streptococcaceae and Clostridiaceae and a lower RA of unassigned family  showed a significantly higher RA of Clostridium sensu stricto 1 and lower RA of Streptococcus at Genus level for the treatment for the treatment with SCFA-WA , FE 0 to 14 , and several pens. The taxa most related to the average daily weight gain and FE were Lactobacillus and Butyricicoccus, unknown genus in Butyricicoccaceae family, Prevotella 9, and Faecalibacterium . On day 42, Butyricicoccus and Faecalibacterium seemed to be associated with greater FE, similar to the observation made on day 14.RDA showed that control and SCFA-WA explained 1.82% (The SCFA-WA used in the current study reduced the pH of drinking water from 6.89–8.03 to 2.90–3.79 and improved FE in piglets at weaning. During weaning, the stomach pH in piglets increases, resulting in less activity of the enzyme pepsin as well as less stimulation of the secretion of pancreatic enzymes . This coClostridium sensu stricto 1 and lower RA of Streptococcus at Genus level when piglets were supplemented with SCFA-WA. Clostridium sensu stricto 1 belong to the Clostridiaceae. Clostridiaceae was more abundant in pigs with a higher FE in a feed intake model in which piglets with the same ADG were divided in two groups based on feed intake. In the current study, the piglets that received SCFA-WA had a higher FE along with a higher RA of members of the Clostridiaceae taxa. Furthermore, some members of Clostridium can produce SCFAs by consuming mucus-derived saccharides as energy source. This in turn has a beneficial effect on intestinal mucosa barrier, which leads to the possible inhibition of pathogen adherence (There was a significantly higher RA of dherence . In the Streptococcus belong to the lactic acid bacteria. Some species have probiotic characteristics (Streptococcus suis, which is the cause of various diseases such as meningitis, septicemia, and endocarditis (S. suis mainly infect piglets through the upper respiratory tract, studies have shown that the gastrointestinal tract of weaned piglets is rapidly colonized by S. suis (Streptococcus species could cause disease, a lower abundance of Streptococcus could be considered beneficial for the animal. In the current study, the SCFA-WA showed a lower abundance in Streptococcus. As no species identification was possible with the current method, we cannot conclude whether the lower abundance of Streptococcus observed here were commensal or pathogenic species.eristics ; howevereristics . The moscarditis . Althoug S. suis . The gas S. suis . As multButyricicoccus and Faecalibacterium. Although no species could be assigned in this study, the aforementioned genera are known to include species that could impart performance and health benefits, particularly via the production of butyrate.The RDA of the fecal samples showed a positive correlation between improved FE and daily weight gain and higher RA of genera Faecalibacterium is F. prausnitzii, known as a beneficial bacterium which shows anti-inflammatory effects due to the secretion of metabolites blocking NF-κB activation and IL-8 secretion as well as decreasing proinflammatory cytokine synthesis and increasing anti-inflammatory cytokine secretion (Faecalibacterium, like Butyricicoccus, belong to the butyrate producers (Faecalibacterium is expected to be beneficial for the animal, as in humans, a low abundance of Faecalibacterium is often associated with inflammatory bowel disease (One of the most known ecretion . Faecaliroducers . A highe disease .Butyricicoccus pullicaecorum has previously been used as probiotic in broilers (Butyricicoccus pullicaecorum seems to be enhanced in response to a corn-soybean diet, probably due to its starch-degrading capacities (Butyricicoccus is not known, it is possible that the genus members included butyric acid producing organisms that had an effect via volatile fatty acid production.The butyrate-producing broilers . The autpacities . This laFaecalibacterium, associated with higher feed intake. However, in this study, fecal SCFA including butyrate, were not measured; thus, this hypothesis needs to be further investigated.Most of the bacterial genera found to be associated with higher FE are described as butyrate producers. Butyric acid functions as the main source of energy for colonocytes . It furtClostridium sensu stricto 1 and lower RA of Streptococcus. Furthermore, the RDA showed a positive association between improved FE and RA of Butyricicoccus and Faecalibacterium known to include butyrate-producing species considered to be beneficial for the health of the animals. The results from this study demonstrate that providing water acidifier containing free and buffered SCFA to newly weaned piglets can modulate the microbiota and improve FE in piglets.In conclusion, piglets provided with SCFA-WA had a significantly improved FE and higher water intake compared with controls. There was no significant difference in body weight, ADG, or ADFI. Furthermore, there was no overall significant difference in fecal scoring between the treatments. Piglets provided with SCFA-WA showed a significantly higher RA of