Normothermic machine perfusion (NMP) is a technique of kidney preservation designed to restore cellular metabolism after cold ischemia. Kidneys are perfused with an oxygenated banked red blood cell (RBC) based solution for 1h at 36°C. During NMP, RBCs can become damaged, releasing free heme into the perfusate. This can act as a damage-associated molecular pattern (DAMP) activating inflammatory signalling pathways. The aim of this study was to measure the levels of free heme during NMP, assess the effect on kidney function and determine any association with inflammatory and stress related gene expression. Levels of free heme were measured in perfusate samples from a series of donation after circulatory death (DCD) kidneys undergoing NMP as part of a randomised controlled trial (RCT). The age of RBCs and levels of free heme were correlated with perfusion parameters. Changes in gene expression were analysed in a series of kidneys declined for transplantation using the NanoString nCounter Organ Transplant Panel and qRT-PCR. Older units of RBCs were associated with higher levels of free heme and levels increased significantly during NMP . There was no association with levels of free heme and perfusion parameters during NMP (P > 0.05). Transcriptional and qPCR analysis demonstrated the upregulation of differentially expressed genes associated with apoptosis (FOS and JUN), inflammatory cytokines , chemokines  and oxidative stress (KLF4) after NMP. However, these did not correlate with levels of free heme (P >0.05). A significant amount of free heme can be detected in the perfusate before and after NMP particularly when older units of red cells are used. Although transcriptional analysis demonstrated significant upregulation of genes involved with apoptotic, inflammatory and oxidative pathways these were not associated with high levels of free heme. Normothermic machine perfusion (NMP) technologies are being trialled in clinical practice to improve early graft function and assess the quality of kidneys for transplantation –3. An oxNMP can be carried out using a number of different strategies. Experimentally, prolonged durations of NMP have shown benefit in improving early graft function . HoweverCompatible packed red blood cells (pRBCs) from a local blood bank are used in most NMP circuits as an oxygen carrier. RBCs are also important regulators of vascular tone and nitric oxide bioavailability . AlthougThe aim of this study was to measure the levels of free heme during NMP and assess the effect on kidney function and to determine any association with inflammatory and stress related gene expression.Kidneys undergoing 1h NMP from a RCT (ISRCTN15821205) were used to determine the effect of free heme on perfusion parameters (n = 42). The study protocol and trial documents were approved by the NHS Health Research Authority East of England, Cambridge Central Research Committee (15/EE/0356).Human kidneys rejected for transplantation and offered for research were used to investigate the transcriptional effects of free heme on gene expression and cellular damage (n = 15). Consent for research was obtained by specialist nurses in organ donation (SNODs). The study was approved by the National Research Ethics committee and Research and Development office at the University of Cambridge (NRES: 15/NE/0408).NMP was carried out for 1 hour after a period of static cold storage as previously described (The circuit was primed with one unit of compatible packed red blood cells (RBCs) mixed with a priming solution , arterial pressure, temperature and urine output were monitored throughout.During NMP the quality of each transplanted kidney was graded. The NMP quality assessment score was derived from three factors: macroscopic appearance, mean RBF and total urine output. Kidneys with a quality assessment score of 4 or less were considered suitable for transplantation provided there were no other preclusions to transplant .Perfusate samples were collected before and after NMP. Samples were centrifuged at 1600 RPM for 10 minutes at 4°C. Supernatant was removed, flash frozen in liquid nitrogen and stored at –80°C. Urine samples were collected after 1h NMP. Samples were centrifuged at 1600 RPM for 10 minutes at 4°C. Supernatant was removed, flash frozen in liquid nitrogen and stored at –80°C.Free heme was measured in the perfusate samples before and after NMP using the Heme Assay Kit  following manufacturer’s instructions.In the research kidneys, wedge biopsies of cortex were taken before and after NMP. Biopsies were divided into three for fixation in 10% formalin  for paraffin wax embedding, flash frozen in liquid nitrogen and fixed in RNALater Stabilisation Solution .Apoptosis was measured using TUNEL staining on 4µm sections of paraffin fixed tissue. TUNEL staining was carried out following manufacturer’s instructions . Sections were viewed at ×200 magnification and ten fields of view were imaged. TUNEL positive cells were counted in each field of view and an average number of positive cells was calculated for each sample. Apoptotic cells were identified by dark brown staining.Protein lysates were prepared from tissue samples collected before and after NMP. Tissue (5mg) was homogenised in RIPA lysis buffer  with protease inhibitor cocktail  using mechanical homogeniser. Samples were left on ice for 30 mins and then centrifuged for 20 minutes at 4°C 12000 RPM.The protein lysates were used to measure oxidative stress in the research kidneys using the Protein Carbonyl content assay kit  following manufacturer’s instructions.Human neutrophil gelatinase-associated lipocalin (NGAL) was measured in the urine at the end of NMP following manufacturer’s instructions .Gene expression profile was performed using the Human Organ Transplant panel from NanoString technologies. Total RNA was isolated from kidney biopsies and 770 genes related to organ transplant pathways were screened according to manufacturer’s instructions. Normalization, differential of expression and pathways analysis were performed within the nSolver Software Advanced Analysis. Normalisation was based on the selection of a panel of housekeeping genes through geNorm pairwise variation statistic. Differential of expression was displayed in a volcano plot with p-value adjusted by the Benjamini–Hochberg false discovery rate correction method. Further analysis was performed within the R software: the heatmap and principal component analysis were plotted with the top 25 and 100 differentially expressed genes respectively.Gene expression of IL-6, TLR4, HO-1, HMGB1, FOS and JUN  (Reverse transcription (RT) was carried out on RNA samples using a High Capacity cDNA Reverse Transcription Kit  following manufacturer’s instructions. Briefly, each sample was mixed with the master mix provided and RT was carried out in the thermal cycler (BioRad T100) at 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes and 4°C until used in qPCR.qPCR was carried out on the BioRad CFX RealTIme System. Samples were analysed in triplicate and each experimental well contained SsoAdvanced Universal Probes Supermix , primer  and DCD kidneys were compared.Values are presented as the mean ± SD for parametric data and median and inter-quartile range (IQR) for non-parametric distributed data. P values of >0.05 were considered significant. Statistical analysis was carried out using Prism statistical software version 9 .A total of 42 kidneys underwent NMP with pRBCs. Donor demographics and ischemic times are listed in The majority of kidneys were of good quality and had a quality assessment score of 1 and 2 . Baseline levels of free heme were associated with significantly higher levels of baseline potassium (P = 0.044), baseline lactate (P = 0.001), 1h lactate (P = 0.001) and 1h free heme (P <0.0001).There was no association with the age of pRBCs or levels of free heme and functional parameters  (The donor characteristics and ischemic times for the research kidneys (10 DBD and 5 DCD) are detailed in NanoString nCounter Organ Transplant Panel was used to examine the transcriptional changes in gene expression in four human DBD research kidneys that underwent 1h NMP.Seventeen differentially expressed genes were significantly upregulated after NMP. These included genes associated with apoptosis (FOS and JUN), inflammatory cytokines , chemokines  and oxidative stress (KLF4). Several genes associated with anti-inflammatory properties (IL-10) and endothelial cell recovery (VEGF) were also upregulated though did not reach statistical significance , median fold increase 59 (3-364), IL-6 (P = 0.0001), median fold increase 11 (3-81), JUN (P = 0.0001), median fold increase 11 (1-146) and TLR4 (P = 0.0353), median fold increase 1.4 (0.3-16.2). There was no significant fold change in HO-1 (P = 0.2293) and HMGB1  (The number of apoptotic cells significantly increased during NMP compared to baseline samples (P = 0.027). The majority of positive staining was found in the tubular epithelial cells  and the cold ischemic time was matched between groups .Baseline and 1h NMP levels of free heme were similar between DCD and DBD kidneys .DBD kidneys had a significantly greater fold change in IL-6 gene expression compared to the DCD kidneys after 1h NMP (DCD 6.08 (4.09-14.83), DBD 29.51 (8.41-52.25); P = 0.029). There were no significant differences between groups in the five other target genes .The NMP system uses adapted clinical grade pediatric cardiopulmonary bypass technology designed to minimize hemolysis. However, factors such as the heat generated by the centrifugal pump, shear stress, negative pressures, turbulent flow and gravity assisted venous drainage contribute to hemolysis and release of free heme. In cardiac bypass procedures excess levels of heme are associated with organ damage and increased mortality , 14. TheTo determine the molecular effects of heme, changes in transcriptional gene expression in a number of the discarded kidneys were assessed. There was significant upregulation of cellular stress related genes that are consistent with ischemic injury. Genes upregulated after NMP included heat shock proteins, oxygen free radicals (KLF4), hypoxia and inflammatory genes  and chemokines . FOS and JUN gene transcription were amongst the top five significantly upregulated differentially expressed genes. They regulate activator protein 1, a prominent transcription factor involved with regulating cellular fate involving the mitogen-activated protein kinases (MAPKs) pathways . Together with the NLR pathway, the MAPK pathway is involved in the downstream signalling of the innate immune response and pro-inflammatory cytokine production with activation of NF-κB/NLRP3 inflammasome .To quantify the findings from the transcriptional data, a number of genes were investigated using qRT-PCR. In agreement with the NanoString transcriptomic data, there was a significant upregulation of IL-6, FOS and JUN. We also measured HMGB1, a nuclear protein that in situations of stress and ischemia, translocates from the nucleus to the cytoplasm and is excreted into the extracellular space to act as a DAMP , 22. HMGThere was no association in levels of free heme and gene expression in the series of discarded kidneys. However, based on the known effects of free heme we cannot dismiss the possibility of its contribution to the stimulation of these inflammatory and oxidative stress pathways. With such a short perfusion time, we were unable to quantify the protein expression of these pathways. As a DAMP, free heme influences the expression of many genes and, in the NMP environment in the absence of plasma proteins, free heme can interact with TLR4 to upregulate inflammatory mediators , 25, 26.The upregulation of inflammatory mediators during NMP has been associated with prolonged early graft dysfunction  and therThe addition of an oxygen carrier during NMP is considered essential for adequate oxygen delivery to the tissues. Venema L et al, found that the addition of RBCs during more prolonged periods of NMP of porcine kidneys resulted in higher levels of oxygen consumption and function compared to conditions without RBCs . ArtificAnother strategy is to use an oxygenated acellular solution to rewarm kidneys in a controlled manner. Zlatev H et al. recently published the results of six extended criteria donor kidneys demonstrating the safety and feasible of the technique . NonetheThis is the first study to examine the effects of free heme during kidney NMP. It involved two populations of kidneys: to determine the effects of free heme on kidney function during NMP a series of DCD kidneys included in a RCT were used; to determine the effect of free heme at a molecular level a series of DBD and DCD kidneys rejected for transplantation and offered for research were used. Although the research kidneys had longer cold ischemic times and were from DBD and DCD donors, levels of free heme and functional parameters during NMP were similar to the RCT kidneys. The population of DCD research kidneys were further disadvantaged with more of the DCD kidneys being perfused with older units of pRBCs. The small sample size limits any conclusion on the effect of free heme on DCD compared to DBD kidneys in this study but previous analysis of transcriptional gene expression after NMP has found results to be similar between DBD and DCD kidneys .In conclusion, high levels of free heme are found in units of stored RBCs and increase significantly during 1h of NMP. There was no direct association between levels of free heme, kidney function or the upregulation of inflammatory and stress related genes during perfusion. However, this may have consequences for longer periods of perfusion and levels of hemolysis should be minimized during NMP.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE197304)The data presented in the study are deposited in the GEO repository, accession number (GSE197304) The studies involving human participants were reviewed and approved by East of England, Cambridge Central Research Committee (15/EE/0356), National Research Ethics committee and Research and Development office at the University of Cambridge (NRES: 15/NE/0408). The patients/participants provided their written informed consent to participate in this study.SH conceived the research idea, planned and conducted the experiments, analysed the results and co-wrote the manuscript; TE conducted the experiments, analysed the results and co-wrote the manuscript; NJ conducted the experiments, analysed the results and revised the manuscript; MN conceived the research idea and revised the manuscript. All authors contributed to the article and approved the submitted version.The research was funded Kidney Research UK and by the National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Organ Donation and Transplantation at the University of Cambridge in collaboration with Newcastle University and in partnership with NHS Blood and Transplant (NHSBT) Grant RG75628.The views expressed are those of the authors and not necessarily those of the NIHR, the Department of Health and Social Care or NHSBT.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To compare the performance of abbreviated breast magnetic resonance imaging (AB-MRI)-based transfer learning (TL) algorithm and radionics analysis for lymphovascular invasion (LVI) prediction in patients with clinically node-negative invasive breast cancer (IBC).Between November 2017 and October 2020, 233 clinically node-negative IBCs detected by AB-MRI were retrospectively enrolled. One hundred thirty IBCs from center 1 (37 LVI-positive and 93 LVI-negative) were assigned as the training cohort and 103 from center 2 (25 LVI-positive and 78 LVI-negative) as the validation cohort. Based on AB-MRI, a TL signature (TLS) and a radiomics signature (RS) were built with the least absolute shrinkage and selection operator (LASSO) logistic regression. Their diagnostic performances were validated and compared using areas under the receiver operating curve (AUCs), net reclassification improvement (NRI), integrated discrimination improvement (IDI), decision curve analysis (DCA), and stratification analysis. A convolutional filter visualization technique was used to map the response areas of LVI on the AB-MRI.In the validation cohort, compared with RS, the TLS showed better capability in discriminating LVI-positive from LVI-negative lesions . The diagnostic performance of TLS was not affected by the menstrual state, molecular subtype, or contrast agent type . Moreover, DCA showed that the TLS added more net benefit than RS for clinical utility.An AB-MRI-based TLS was superior to RS for preoperative LVI prediction in patients with clinically node-negative IBC. Lymphovascular invasion (LVI) is a well-recognized risk factor for disease recurrence and shorter survival in patients with invasive breast cancer (IBC), especially those with negative lymph nodes –3. FurthMagnetic resonance imaging (MRI), which can characterize the entire lesion with high spatial resolution, is increasingly studied with LVI assessment in IBC –9. HowevRadiomics is a promising tool for the characterization of breast cancer by extracting quantitative features, but the main drawbacks of using traditional radiomics analysis are time-consuming lesion segmentation and hard-coded feature extraction . CompareHence, the purpose of this study was to evaluate the performance of an AB-MRI-based TL algorithm and compare it with that of radiomics for LVI prediction in patients with clinically node-negative IBC.A schematic illustration of the study design is presented in The ethics committee approved this retrospective study of two participating centers with a waiver for informed consent.in situ or special types of invasive breast carcinoma at final pathologic evaluation (n = 69). Finally, a total of 233 patients  were included. Patients from center 1 were assigned as the training cohort (37 LVI-positive and 93 LVI-negative), and patients from center 2 were the external validation cohort (25 LVI-positive and 78 LVI-negative).Between November 2017 and October 2020, the study enrolled consecutive women with new IBC detected by AB-MRI and clinically lymph node-negative in the study. These patients underwent AB-MRI for breast cancer screening or problem resolving. The inclusion criteria were as follows: (a) underwent AB-MRI and have enhanced lesions on MR image; (b) lesions diagnosed as invasive ductal carcinoma based on pathologic evaluation of surgical specimens; (c) time interval between surgery (mastectomy or lumpectomy) and MRI examination: <2 weeks. The exclusion criteria were as follows: (a) biopsy performed before AB-MRI (n = 58); (b) received neoadjuvant chemotherapy (n = 30) or radiotherapy (n = 55) before MRI examination; (c) node-positive diagnosed on preoperative ultrasonography (n = 37): rounded hypoechoic node, complete or partial effacement of the fatty hilum, focal or diffuse cortical thickening (≥5 mm), complete or partial replacement of the node with an ill-defined or irregular mass, extra-hilar blood vessel flow on color Doppler images, or microcalcifications in the node; (d) MR image with obvious artifacts (n = 15); (e) diagnosed as benign tumors, high-risk lesions, pure ductal carcinoma The AB-MRI protocol included a pre- and early postcontrast phase using a 3D T1 gradient echo with a fat saturation sequence. Detailed AB-MRI parameters are listed in All of the surgical specimens were examined by two senior pathologists with 16 and 13 years of experience in breast pathology, and the pathologic evaluations are provided in U test was used to compare the between-group differences of each radiomics feature in the LVI-positive and LVI-negative groups, and intra-class correlation coefficients (ICCs) were used to evaluate the reproducibility and stability of the radiomics features. The specific process is presented in An RS was developed with the following steps: region of interest (ROI) acquisition, feature extraction, feature selection, and model construction. The ROI was manually segmented by a professional radiologist . Reader 2  randomly chose 30 patients from the training cohort and performed tumor segmentation for inter-reader agreement analysis. Based on the ROI, 10,402 radiomics features were extracted using the in-house software developed with MATLAB 2016 , including first-order, shape-based, and texture features. Then, the Mann–Whitney A least absolute shrinkage and selection operator (LASSO) logistic regression was used to build an RS using a linear combination of features based on the selected features. The features with nonzero coefficients were considered valuable predictors for predicting the LVI status, and the tuning parameter was selected by 10-fold cross-validation for the radiomics method. Finally, the output of the RS was labeled as the radiomics score (R-score).The development of a TLS consisted of two steps: TL features extraction and classification layer training. The first step is the training of feature extraction network. In order to avoid an overfitting of the model, the TL strategy  was usedU test. The second step is the classification layer training based on the LASSO logistic regression; the training process was similar to that of RS.Based on the TL feature, the differences of the transfer learning signature (TLS) between the LVI-positive and LVI-negative groups were assessed using the Mann–Whitney For investigating the interpretability of the TLS, the convolutional filter was visualized with gradient-weighted class activation mapping (Grad-CAM) , which cGiven an ROI image, each convolutional filter generated a response map showing all the corresponding feature patterns extracted from the lesion. A valuable convolutional filter should have different responses to different types of lesions. Thus, the visualization of the response map for convolutional filters in different lesion groups was helpful to understand the TLS.We compared the TLS with the RS to comprehensively evaluate the performances of the models.ROC analysis was performed for the training cohort and external validation cohort to evaluate the diagnostic performance of the TLS and RS. The following parameters were calculated: the area under the curve (AUC), sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV). The AUCs of the TLS and RS were compared using the DeLong test. In addition, to compare the classification ability of the DLS and RS, the net reclassification index (NRI) and integrated discrimination improvement (IDI) were calculated. Decision curve analysis (DCA) was used to estimate the clinical utility of the TLS and RS. Moreover, a stratified analysis was performed on the menstrual state, molecular subtype, and contrast agent type.http://www.rproject.org). All radiomics features were extracted with Matlab 2016, and the TL features were extracted with Python 3.6. LASSO was performed using the “glmnet” package, and the ROC curve analysis was performed using the “pROC”. Clinicopathologic characteristics between the LVI-positive and LVI-negative groups were compared using chi-squared test or Mann–Whitney U test. p Values <0.05 were considered indicative of a statistically significant difference.All statistical tests were performed using R3.0.1  and intraclass correlation coefficient (ICC) values greater than 0.75 were used in the LASSO logistic regression. In the LASSO logistic regression, 14 features with nonzero coefficients  was poor in the validation cohort Table 2,There were several limitations in our study. First, the sample size was relatively small, especially the training cohort, which did not meet the traditional CNN modeling requirement. To overcome this shortage, we pretrained the network with TL. The model based on a transfer learning strategy can avoid overfitting in a training dataset, which reduces the amount of data required for modeling. Nevertheless, the generalization of a TLS still needs to be validated in other centers. Second, multiparametric MRI-based TLS performance was not investigated as the purpose of the study is to investigate whether LVI status can be assessed simultaneously when IBC is detected on AB-MRI. The included patients only underwent AB-MRI for breast cancer screening or lesion diagnosis. However, full diagnostic protocol should be compared with an AB-MRI for LVI prediction in prospective studies. Finally, our study was based on the construction of a two-dimensional slice feature model, and the performance of the three-dimensional features remains to be further studied.In summary, the TLS was superior to the RS for LVI prediction in IBC patients with clinically negative nodes. The proposed AB-MRI-based TLS could potentially serve as an easy-to-access and easy-to-use approach to assist individual breast cancer treatments.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Jiangmen Central Hospital and Nanfang Hospital. The patients/participants provided their written informed consent to participate in this study.WL and LZ designed the research. XL, XC, and RL collected the data. BF, YL, YC, HZ, and TY contributed data analysis tools and performed the analysis; BF also acquired the funding. ZL and BF wrote the paper. WL, EC, and LZ supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , the National Natural Science Foundation of Guangxi (2021GXNSFAA075037), the Guilin University of Aerospace Technology Foundation (XJ21KT17), the Guangdong Basic and Applied Basic Research Foundation (2019A1515011773), the Pearl River S&T Nova Program of Guangzhou under Grant (201906010043), Elite Young Scholars Program of Jiangmen Central Hospital (J201904), and Medical Scientific Research Foundation of Guangdong Province of China (A2020622).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer WL declared a shared affiliation with several of the authors, TY and LZ, to the handling editor at time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Molecular Plant Pathology 22, 683–693, Figure 6c (p. 689) contained an inadvertent image duplication in the water control panel. This did not affect the experimental data or conclusions.In Tian et al. , publishThe correct version is provided here:FIGURE 6 Mgx1LysM undergoes chitin‐induced polymerization. (a) Amino acid sequence alignment of Mgx1LysM and Mg1LysM. The LysM is indicated with black underlining. (b) I‐TASSER software‐based in silico prediction of the three‐dimensional structure of Mgx1LysM (left) based on the recently generated crystal structure of Mg1LysM (right) . The N‐terminal 15 amino acids of both proteins are depicted in orange. Structures are visualized using the PyMOL molecular graphics system . (c) The LysM effector Mgx1LysM, together with RiSLM and Mg1LysM as positive controls, and Ecp6 as negative control, were incubated with chitohexaose (chitin) or water. After overnight incubation, methylene blue was added and protein solutions were centrifuged, resulting in protein pellets (red arrowheads) as a consequence of polymerization for Mgx1LysM, Mg1LysM, and RiSLM, but not for Ecp6


The goal of this study was to analyze the content availability and accessibility of preventive medicine residency program websites. In COVID-related travel restrictions, the information provided on program websites has become increasingly crucial for residency applicants.A cross-sectional study was conducted by extracting the list of preventive medicine residency programs on the Fellowship and Residency Electronic and Interactive Database (FRIEDA). A 40-point criterion was used for the quality evaluation of residency programs. The study was conducted and analyzed in 2021.82 preventive medicine residency programs were identified, and listed on FRIEDA, out of which 65 program websites were accessible. The median number of 40-point criteria met by the preventive medicine residency website was 25. The criteria fulfilled by the greatest number of program websites was research opportunity/facilities (94%).The majority of the preventive medicine residency program websites were not up to the mark regarding accessibility and quality. An updated preventive medicine residency program website is essential for the applicants. Programs with relevant and precise information on their websites have higher chances of attracting potential candidates and better chances of finding the match between applicants and programs. •Preventive medicine residency programs train physicians competent in public health, epidemiology, population health, and clinical medicine.•The majority of the preventive medicine residency program websites were not up to the mark regarding accessibility and quality.•There are no previous study on the accessibility and content of preventive medicine residency program websites. The study was conducted and analyzed in 2021.Based on the type of program and geographic location, subgroup analysis was performed. Program types were defined as university-based, community-based, and military-based, as stated on FRIEDA. Four geographic regions were used to divide the program: Northeast , South , Midwest , West  . Data fo2.3Based on the type of program and geographic location, we evaluated the mean number of criteria fulfilled by each program. An unpaired T-test was used to calculate the statistical significance of the mean number of criteria fulfilled by each program. Furthermore, Kruskal-Wallis H tests were performed to compare the variables based on the number of groups being compared. A p-value of <0.05 was considered significant for all cases. Statistical Product and Service Solution Software  was used to analyze all study data.3●Numerical Distribution of Residency Programs●Analysis of Residency Programs Website 40-Point CriteriaOut of 82 preventive medicine residency programs listed on FRIEDA, 65 programs fulfilled the inclusion criteria. These programs included 46 (71%) university, 15 (23%) community, and 4 (6%) military programs. Programs excluded from the analysis included 5 programs due to inaccessible links and 12 programs that had no information on their website, as shown in The median number of 40-point criteria met by the preventive medicine residency website was 25 ± 7.74 (range = 7–35). The top 5 prevalent domains to be fulfilled by each website included Research (94%), program overview (88%), faculty (72%), current residents (65%), and residential/housing information (64%), as shown in i.Program Overview: There were 3 criteria in this domain. It included Overview Mission Statement/Introductory Notes/Message from Program Director , Residency Program Director Credentials , Residency Program Director Contact Information .ii.Application: Seven criteria were evaluated in this domain. The top three criteria that were fulfilled in this domain were residency application requirements , USMLE step scores , interview dates information .iii.Curriculum: This domain had 6 criteria that were evaluated for each website. The top three criteria included were didactics , clinical rotation sites , rotation schedule .iv.Current Residents: In this domain, 4 criteria were assessed. The top three criteria included in this domain were the number of residents , name of residents , picture of residents .v.Alumni: Alumni is the lowest (50%) fulfilled domain. Therefore, 3 criteria were assessed in this domain. The criteria with the highest percentage of the information available included alumni names , followed by alumni job location , that was followed by alumni pictures .vi.Faculty: This domain contained 3 out 40 criteria assessed. It included faculty name , faculty picture , faculty background information, credentials .vii.Research: The Research was the highest (94%) fulfilled domain, and it contained 1 criteria research Opportunities/Facilities .viii.Benefits and Incentives: Out of the 40-point criteria, this domain contained 7 criteria. The three highest criteria present in this domain were educational fund , insurance , salary .ix.Residential/Housing Information: There were 4 criteria evaluated in this domain. The top three criteria in this domain included social media , diversity , surrounding area/neighborhood/city Information .●Analysis of 40-Point Criteria with Study CharacteristicsThe percentage of highest cited criteria fulfilled by each website were research opportunities/facilities (94%), overview mission statement/introductory notes/message from Program Director (P.D.) (89%), didactics (83%), residency application requirement (82%), clinical rotation sites (75%), and faculty name (75%) as shown on 4The study acts as a snapshot of the 2021 status of preventive medicine residency programs' websites, which can subsequently provide insights into why preventive medicine programs are minimal in number compared to other residency programs and face decreasing attention from prospective applicants . ForemosAll website domains reported 50% or greater mean percentage fulfillment as shown in The second most fulfilled domain was "Program Overview," approaching near to 90% fulfillment; however, "Residency Program Director Contact Information" was provided by 78% of the programs, which means around less than a quarter of the programs did not provide any information to get in touch with the program director. This may be due to a reluctance to share contact information on a public website but contacting program directors through formal channels is a potential source of in-depth information for applicants. The least fulfilled domain was "Alumni Information," which was only reported by 50% of the websites that will result in applicants connecting with the graduates of only half of the programs. Alumni, alongside faculty members, can serve as important mentors for current and prospective residents regarding work-life balance, program characteristics, and future career paths ,24.Alarmingly, most of the programs (12%) lack information regarding board pass rates of their trainees, a finding shared by Silvestre et al. and Khan et al. as they reviewed plastic surgery residency and cardiology fellowship websites, respectively ,24. BoarInternational medical graduates (IMGs) are an essential addition to the U.S. health care system. Every year IMGs from around the world apply for residencies in the U.S., including 7000 non-U.S. visas requiring IMGs, according to the data provided by National Residency Match Program (NRMP) . HoweverWe evaluated 65 programs, and none of them completely fulfilled the criteria checklist. The information provided on program websites was found inadequate for the applicants. It is recommended that the website content should be frequently monitored and updated annually at least 3–4 months before the match application process begins. Joint efforts should be initiated between the American Board of Preventive Medicine  and the There were certain limitations to our study. First, the department or person responsible for the maintenance of the website was not contacted directly. Second, websites were solely assessed for the presence and absence of content; the quality and accuracy of the content were not analyzed. Third, we studied the website for a restricted timeframe, rather than a complete analysis that may not include changes made afterward. Fourth, some websites were inaccessible through their links, and they were not contacted any further to assess their content. Fifth, some of the programs may please provide necessary information to candidates via electronic or postal mailing methods, or maybe through online events were therefore unreachable. In the last, information gathered by candidates via any other resource was not analyzed as we included variables gleaned from Accreditation Council for Graduate Medical Education (ACGME) and some other published studies on the same topic.5Most U.S. Preventive Medicine residency websites lack valuable and updated information. This lack in quality indicates many opportunities are vulnerable to being missed by potential candidates. Future directions and more research work/educational awareness regarding these websites and information are necessary among medical community. Efforts to constantly improve website content and add relevant information regarding the program such as selection criteria, visa problems, incentives and benefits, alumni, and board pass rates can ensure better recruitment of applicants. Furthermore, it helps candidates make an informed decision and impact applicants’ decision to choose the program for applying, interviewing, and ranking during the match process.The study was reviewed and approved by the Institute of Medical Sciences, GAMBAT, Pakistan's institutional review board, and was registered under the reference number IRB/21/13.The study was reviewed and approved by the Institute of Medical Sciences, GAMBAT, Pakistan's institutional review board, and was registered under the reference number IRB/21/13.The study was self funded.Maaz Hasan Khan: conduct, data acquisition, data analysis, statistical analysis, manuscript preparation, manuscript editing and manuscript, final approval, and agreeing to the accuracy of the work.Ariba Salman: planning, conduct, data acquisition, data analysis, statistical analysis, manuscript editing, and manuscript review, final approval, and agreeing to the accuracy of the work.Alina Sehar: planning, conduct, data acquisition, data analysis, statistical analysis, manuscript editing, and manuscript review, final approval, and agreeing to the accuracy of the work.Farah Yasmin: planning, conduct, data acquisition, data analysis, statistical analysis, manuscript editing, and manuscript review, final approval, and agreeing to the accuracy of the work editing and manuscript review, final approval, and agreeing to the accuracy of the work.Shehzeen Fatima Memon: planning, conduct, data acquisition, data analysis, statistical analysis, manuscript editing, and manuscript review, final approval, and agreeing to the accuracy of the work editing and manuscript review, final approval, and agreeing to the accuracy of the work.Maham Abbasi: planning, conduct, data acquisition, data analysis, statistical analysis, manuscript editing, and manuscript review, final approval, and agreeing to the accuracy of the work editing and manuscript review, final approval, and agreeing to the accuracy of the work.Muhammad Sohaib Asghar: planning, conduct, manuscript preparation, manuscript editing and manuscript review, final approval, and agreeing to the accuracy of the work.Mohammed Mahmmoud Fadelallah Eljack: planning, conduct, manuscript preparation, manuscript editing and manuscript review, final approval, and agreeing to the accuracy of the work.Kaleem Ullah: manuscript editing and manuscript review, final approval, and agreeing to the accuracy of the work.Muhammad Junaid Tahir: manuscript editing and manuscript review, final approval, and agreeing to the accuracy of the work.1Name of the registry: Institute of Medical Sciences, GAMBAT, Pakistan's institutional review board2Unique Identifying number or registration ID: IRB/21/13.3Hyperlink to your specific registration (must be publicly accessible and will be checked):Muhammad Sohaib Asghar.N/A.Authors reports no conflict of interest.


Ganoderma lucidum. Firstly, the enzymatic properties of purified LAC-4 laccase, and the degradation of three chlorophenol pollutants 2,6-dichlorophenol , 2,3,6-trichlorophenol  and 3-chlorophenol (3-CP) by LAC-4 were systematically studied. LAC-4 had a strong ability for 2,6-DCP and 2,3,6-TCP degradation. The degradation ability of LAC-4 to 3-CP was significantly lower than that of 2,6-DCP and 2,3,6-TCP. LAC-4 also had a good degradation effect on the chlorophenol mixture . The results of kinetics of degradation of chlorophenols by LAC-4 suggested that the affinity of LAC-4 for 2,6-DCP was higher than 2,3,6-TCP. The catalytic efficiency and the catalytic rate of LAC-4 on 2,6-DCP were also significantly higher than 2,3,6-TCP. During degradation of 2,6-DCP and 2,3,6-TCP, LAC-4 had a strong tolerance for high concentrations of different metal salts  and organic solvents (such as ethylene glycol and glycerol). Next, detoxification of chlorophenols by LAC-4 was also systematically explored. LAC-4 treatment had a strong detoxification ability and a good detoxification effect on the phytotoxicity of individual chlorophenols  and chlorophenol mixtures . The phytotoxicities of 2,6-DCP, 2,3,6-TCP and chlorophenol mixtures  treated with LAC-4 were considerably reduced or eliminated. Finally, we focused on the degradation mechanisms and pathways of 2,6-DCP and 2,3,6-TCP degradation by LAC-4. The putative transformation pathway of 2,6-DCP and 2,3,6-TCP catalyzed by laccase was revealed for the first time. The free radicals formed by LAC-4 oxidation of 2,6-DCP and 2,3,6-TCP produced dimers through polymerization. LAC-4 catalyzed the polymerization of 2,6-DCP and 2,3,6-TCP, forming dimer products. LAC-4 catalyzed 2,6-DCP into two main products: 2,6-dichloro-4- phenol and 3,3′,5,5′-tetrachloro-4,4′-dihydroxybiphenyl. LAC-4 catalyzed 2,3,6-TCP into two main products: 2,3,6-trichloro-4- phenol and 2,2′,3,3′,5,5′-hexachloro--4,4′-diol.A laccase named LAC-4 was purified from  Laccase is a multi-copper oxidase that has an active center containing four copper atoms. Laccase can oxidize a substrate into free radicals by extracting electrons from it. Additional non-enzymatic secondary reactions could occur due to the instability of free radicals. Meanwhile, the oxygen molecule is reduced to water as the electron acceptor ,2,3. BecChlorophenol is a type of phenol organic compound containing one or more covalently bound chlorine atoms. Chlorophenol compounds are widely used in chemical production and other fields, including wood preservatives, fungicides and herbicides. 2,6-dichlorophenol is an important chemical raw material widely used in chemicals, medicine, and pesticides ,11. Alth2O2 to participate in the catalytic process, has attracted much attention in the treatment of chlorophenol pollutants -4,4′-diol.Thus, conversion reactions of the two chlorophenols 2,6-DCP and 2,3,6-TCP by LAC-4 laccase shared a common attribute; specifically, the free radicals that formed from the oxidization of 2,6-DCP and 2,3,6-TCP by LAC-4 laccase produced dimers as a result of the polymerization reactions. In summary, LAC-4 catalyzed the polymerization of 2,6-DCP and 2,3,6-TCP, forming dimer products.We have also performed an experiment to look at degradation of an equivalent mixture of 2,6-DCP and 2,3,6-TCP catalyzed by LAC-4 laccase. We detected the products of LAC-4 laccase degrading a mixture of chlorophenols  by GC-MS. The result was shown in Homodimer metabolites include the following substances: homopolymeric product of 2,6-DCP  phenol, 3,3′,5,5′-tetrachloro-4,4′-dihydroxybiphenyl); homopolymeric product of 2,3,6-TCP  phenol, 2,2′,3,3′,5,5′-hexachloro--4,4′-diol).Heterodimer metabolites include the following substances: heteropolymeric products of 2,6-DCP and 2,3,6-TCP  phenol, 2,6-dichloro-4- phenol).In summary, the degradation products of an equivalent mixture of 2,6-DCP and 2,3,6-TCP catalyzed by LAC-4 laccase contain four homodimers and three heterodimers.The laccase activity decreased when the temperature exceeded 70 °C. The reasons for the decrease in laccase activity when the temperature exceeds 70 °C are as follows: laccase molecules will unfold with the gradual increase in ambient temperature, and the specific spatial arrangement of peptide chains will also change. In addition, hydrophobic amino acid residues inside laccase molecules will also be exposed. These factors will affect the activity of laccase. When the temperature exceeds 70 °C, the temperature will directly destroy the conformation of laccase and reduce its activity.The stability of laccase greatly reduced over 30 °C. The reasons that the stability of laccase greatly reduced over 30 °C are as follows: Previous studies have shown that when the temperature exceeds 30 °C, although the secondary structure has not been destroyed, the peptide chain of laccase is induced to extend. Laccase changes from tightly ordered structure to loose conformation. Therefore, the stability of laccase significantly decreased over 30 °C.− can enter the active site of laccase and inhibit the transfer of electrons from the T1 site to the T2/T3 site (the center of the trinuclear copper cluster), thereby inhibiting the activity of laccase. An alkaline environment can also deprotonate amino acids in the O2 pocket of laccase -4,4′-diol. Taken together, LAC-4 laccase has great practical value and application potential in the treatment of chlorophenol wastewater or the remediation of an environment contaminated by chlorophenol.The enzymatic properties of the laccase LAC-4 purified from


Toxoplasma gondii serology can yield false-positive IgM results due to aspecific reactions. On the other hand, specific therapy can delay IgG production and, therefore, the diagnosis of seroconversion. There is a need for confirmation tests to early detect seroconversions during pregnancy. We conducted a multicentre study to evaluate the diagnostic accuracy of the Toxo II IgG and a new, not yet commercialised Toxo II IgM western blot (WB) (LDBio diagnostics Lyon France) on 229 sera corresponding to 93 patients with seroconversions and 158 sera corresponding to 68 patients with nonspecific IgM. Sensitivity was 97.8% for IgM WB and 98.9% for IgG WB. Specificity was 89.7% and 100%, respectively. The concordance between IgM and IgG Toxo WB with the final diagnosis was very good, K = 0.89 and K = 0.99, respectively. In 5 cases (5.4%), the appearance of IgM, and in 55 cases (59.1%), the appearance of IgG was recorded by WB earlier than by traditional tests. In 10 cases (10.8%), IgM was detected after the traditional tests and in 2 cases (2.2%) for IgG. The association of IgG and IgM WB on the same sample not only detected all seroconversions but also correctly identified most of the false-positive results.The high sensitivity of the automated tests used for  Toxoplasma gondii. Infection is usually asymptomatic or mildly symptomatic in immunocompetent patients, with spontaneous recovery, but can be life-threatening in immunocompromised patients  and cause severe disorders  in congenitally infected foetuses from mothers with acute infection during pregnancy [Toxoplasma specific IgG and IgM antibodies in serum, on monthly follow-up visits and counselling for hygienic–prophylactic measures in seronegative women, and if necessary, treatment of women who seroconverted [Toxoplasmosis is a ubiquitous protozoan infection due to the Apicomplexa regnancy ,2. Theraregnancy ,4. To thregnancy . Routineonverted ,5,6.Toxoplasma gondii infection (IgG negative and IgM positive), the differential diagnosis between seroconversion and false IgM positivity is often difficult, as the infection is in most cases asymptomatic, and it is necessary to wait until specific anti-Toxoplasma IgG antibodies are measurable [Automated tests are usually employed for the determination of specific IgG and IgM antibodies, but despite vast improvements in test performance in recent years, equivocal results with low levels of IgM without IgG in previously seronegative women are still frequently observed in clinical practice due to a lack of standardisation and high sensitivity of most of the marketed tests. They are thus sometimes difficult to interpret, even for medical parasitologists. In a pregnant woman with a suspected recent asurable ,8,9,10. asurable .A diagnosis of toxoplasmosis in pregnancy requires treatment until delivery, amniocentesis to diagnose infection in the foetus and serological and clinical follow-up of the newborn.This indicates the need for an early and precise diagnosis during pregnancy in order to treat only true seroconversions as soon as possible.® western blot (Toxo II IgG) as a second-line, confirmatory test is already established. The test has received the CE mark, and it is recognised as a very good alternative to the Sabin–Feldman dye test, which is still considered the gold standard [The diagnostic accuracy of LDBio Toxo II IgGstandard ,17,18,19Toxoplasma gondii IgG detection during infection in pregnant women was significantly shortened by the use of Toxo II IgG WB [The time to anti-I IgG WB ,13. ToxoWe performed a retrospective multicentre study involving four referral centres for the diagnosis of toxoplasmosis  on the leftovers of laboratory samples.For seroconversion patients, at least two samples were collected. The first sample had to be either the last sample with negative results, whenever available, or the first sample with IgM (IgG negative or positive). In all the first samples, the IgG titer with traditional tests was very low, often negative, or in the grey zone, according to the threshold recommended by each manufacturer.For false-positive IgM, at least one sample was kept for the study, but whenever available, further samples without the appearance of IgG were also included. The clinical status of the patients from whom each sample was drawn was determined by each centre according to the combination of tests used in each laboratory ; the preInclusion criteria for the samples were full anonymisation, samples stored frozen at no more than −15 °C and available volume of at least 400 μL (200 μL for testing and 200 μL in storage for retesting in case of discrepancies). Patient samples were selected from those already in local registries at participating centres with a signed informed consent to the use of the residual samples. Centres had to ensure that all the clinical and laboratory information needed for diagnosis was available. All samples present in the existing database of each centre analysed in the previous 6 years [Toxoplasma lysate obtained in vivo. The bands have been defined experimentally as being found in patients. Their identification was made by comparison with molecular weight markers and, therefore, corresponds to an approximate molecular weight. The P30 protein seems to be SAG1, the major T. gondii surface protein and the most immunogenic constituent of tachyzoïtes. The cutoff of Toxo II IgM WB was defined by the presence of two bands in the specific area (between 30 and 40 kDa) and including the P30 . The presence of a single P30 or very pale bands was also recorded and interpreted as equivocal. The criteria of the IFU requiring three bands for IgG did not lower sensitivity compared with using two bands [LDBio performed two tests on all the samples: TOXO II IgG was run following the corresponding instructions for the use (IFU) of the commercial kit, and Toxo II IgM, for which no IFU is available, as the test is not commercialised, was performed under the same conditions as Toxo II IgG  using the commercial LDBio anti-IgM conjugate. The cutoff for IgG WB was set following the criteria of the IFU by the presence of three of five specific bands , including the 30 kDa band. The antigen is a wo bands . This alToxo II IgG and Toxo II IgM were performed with the help of a Dynablot plus instrument  by an operator blinded to reference test results within LDBio facilities.To validate the test, two different analyses were performed: concordance (Cohen’s kappa) with final diagnosis  and diagnostic accuracy . With a sample size of 100 patients with proven seroconversion and 100 with nonspecific IgM, we estimated >95% power to identify both sensitivity and specificity of 90% . The unit of analysis was the patient (not the samples) to avoid over/under estimation of performances due to repeated samples of the same patient. At the same time, an analysis at the sample level was performed to determine which technique detected IgG and IgM first.We performed two sensitivity analyses for equivocal IgM WB results, considering them once as negative and once as positive, even if, for clinical purposes, it might be better to consider the equivocal results as positive, as they could be an earlier marker for seroconversion. Finally, the frequency of antigenic bands for positive samples was calculated. Stata computer software version 16.0  was used for the statistical analyses.Three hundred and eighty-seven sera were collected: 229 corresponding to 93 toxoplasmic seroconversions (2 to 4 sera/patient) and 158 sera corresponding to 68 patients with cross-reactions and/or nonspecific IgM obtained with the automated tests and/or IgM ISAGA Tests (1 to 7 sera/patient) . In the The interpretation of the results was easy and reproducible. Some examples of WB strip results are shown in Toxoplasma IgM and negative for anti-Toxoplasma IgG. Therefore, the performances of the tests in this study do not reflect their actual performances in the field, but only in this specific population, in which diagnosis is difficult due to the puzzling serology results. The aim here was to answer the question: is it real seroconversion or false IgM positivity?In this multicentre study, we evaluated for the first time the diagnostic efficacy of Toxo II IgM WB in a selected population of patients positive for anti-Toxoplasma IgG and positive anti-Toxoplasma IgM antibodies on the first samples, thus avoiding the wait until the IgG seroconversion.The Toxo II WB IgM not only detected almost all seroconversions with a sensitivity of 97.8% but also well discriminated the false-positive results  with a sThe high number (27.9%) of equivocal Toxo II WB IgM results in the false IgM group, 19 of 68 patients , shows tTreatment (spiramycin) of the 56 patients from Pavia was started immediately after the detection of IgM in the absence of IgG, but no difference in the time to appearance of IgG was recorded compared with the other patients in centres where therapy was started only after documented seroconversion. LDBio Toxo II IgG and IgM WB allow an early and precise diagnosis of seroconversion, even in pregnant women who had been treated with spiramycin. All pregnant women who seroconverted can be treated, counselled and, if necessary, addressed to prenatal therapy and diagnosis earlier. Moreover, most women with a nonspecific response can be reassured, have their treatment stopped if it had been started and after another sampling 1 and 2 months after therapy interruption, they can be considered negative. With this study, we also confirmed the good sensitivity and specificity of LDBio Toxo II WB IgG, as well as its superiority in detecting seroconversion faster than Elisa, as reported in the literature. In about half of the patients (59.1%), samples tested positive earlier with WB compared with conventional techniques, as already reported in previous studies ,13,16,19


Healthcare systems worldwide are challenged by the increasingly aging populations, characterized by multimorbidity and declining physical and mental function . The capAs an approach developed with a global perspective, the 19 implementation actions of ICOPE were developed to fit all countries. Still, its current implementation faces several barriers of heterogeneous magnitude overall, particularly in LMIC. For example, the cultural systems modulate access to health services in different ways, such as negative  or positive (strong support networks) . So whenThe approach of the ToC can articulate the rationale of the intervention from the expected or desired outcome back to the resources needed to accomplish it, including the identification of the steps (pre-conditions) required, key players, and assumptions involved. The evaluation of the proposed model requires an understanding of the context, answering questions such as if the goal and objectives of the ICOPE program are logical and plausible. These goals and objectives are feasible? Are the procedures for identifying members of the target population, delivering service to them, and sustaining that service through competition well-defined and sufficient? The process to answer these questions may use a consensus approach with relevant stakeholders. The ToC is graphically represented in a flowchart that integrates all the elements from the program's theory and makes them explicit. It contains the outcomes, indicators, assumptions, rationales, and linkages between the previous factors with a proper methodology described elsewhere . ExampleOnce a context-specific ToC has been developed, a formal evaluation of the ICOPE design may improve the likelihood of success. While from the development of the ToC, it is expected that there is a strong base on the rationale of the ICOPE as the model is based on available evidence, discussing its assumptions and its implementation feasibility is given existing constraints, both in resources and in a managerial will for transformation. The ToC framework to define a plausible plan to evaluate the theory and design for ICOPE in LMIC has the advantage of addressing the public problem. It also requires a description of the expected changes in the participants' behaviors, attitudes, and skills. To define it, we need to review the ICOPE documents, interview the program stakeholders, and conduct site visits and observations of the program functions and circumstances.Developing health interventions based on the best available evidence is a high resource-demanding process that multilateral and international organizations may facilitate . To bettAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intervention programs for children with developmental disabilities increasingly target caregiver training to implement effective strategies for child development. Research conducted in different countries shows that the Caregiver Skills Training Program (CST) developed by the World Health Organization and Autism Speaks could also be a recommended intervention.The pre-pilot phase included seven, and the pilot phase included 29 families of children with developmental disabilities trained to implement the intervention program. The caregivers were asked to complete the Autism Treatment Evaluation Checklist at the beginning and at the end of the program.z = −2.99, p < 0.05) and Health/Physical/Behavior  after caregiver participation in the training program. In the pilot phase, the paired t-test also determined a statistically significant improvement in Speech, Language and Communication between the first  and the second testing , t(28) = −2.29, p < 0.05, as well as a significant improvement between the first  and the second testing , t(28) = 2.11, p < 0.05 in Health/Physical/Behavior. Eta squared values (0.16 and 0.14) indicate that the intervention effect was significant. No differences were determined in Sociability and Sensory/Cognitive Awareness between the first and the second testing.In the pre-pilot phase, the Wilcoxon signed-rank test determined a statistically significant improvement in Speech, Language and Communication (The initial results of the Caregiver Skills Training Program are encouraging. For this program to be recommended as an evidence-based intervention, further research should be conducted on larger samples, controlling possible intervening variables. Children with developmental delays (DD) need continuous lifelong support, from birth to adulthood. In many countries, especially in low and middle-income countries (LMIC), adequate services are poorly accessible or completely inaccessible to this vulnerable group . The chiHowever, sometimes even with appropriate support systems and care providers, there is not a sufficient improvement in child's behavior and development. This can happen in cases where experts are mainly focused on delivering interventions to children, neglecting the role of caregivers in overall treatment , 6. All Serbia is a country of ~7 million people where about 1 million people are aged 0–17 . Even ifOne of the most recognized parent-based education programs to date is Caregivers' Skills Training (CST) developed by Autism Speaks (AS) and the World Health Organization (WHO) . The traTo date, the CST program has been implemented, or is in the process of implementation, in more than 30 countries all over the world, and many of these countries are LIMC . The CSTThe main aim of this research is to evaluate the effects of the CST program on different domains of child's development. In addition, this research aims to evaluate the process of implementation, cultural appropriateness and parental opinions on the CST program in Serbia.The CST program in families of children with developmental disabilities was conducted in a pre-pilot and a pilot phase. The pre-pilot phase included seven and the pilot phase 29 families. There was one child with developmental disabilities in each family. Five boys and two girls, 4–8.5 years of age , participated in the pre-pilot phase, while 20 boys (69%) and nine girls (31%), 2–9 years of age , participated in the pilot phase of the study. Both research phases included a convenience sample of caregivers who requested health service support due to developmental disabilities in children . At the time of research, three children showed significant developmental delay but had not yet been diagnosed.The program was divided into two consecutive phases: pre-pilot and pilot phases. In the first (pre-pilot) phase, the master trainers were trained and delivered the training to the parents. ‘Master trainers' is a term determined by the program which describes experts who are in charge of implementation, supervision and delivery of the program in their country. After their extensive training by the founders of CST, the master trainers are able to deliver the CST to other experts in their country (in later text: facilitators) who will continue their work with the families under supervision. Each country has a certain number of trained master trainers who are usually experts in child mental health and development. In our country, eight master trainers (six women and two men), 28–49 years of age , participated in the training program. All master trainers were experts in mental health or health associate professionals in primary healthcare. Two of them were employed in private healthcare institutions, and six worked in public healthcare institutions. Given the differences in their age, the master trainers were expected to have different work experience ranging from four to 22 years. Also, they spent between two and 22 years working with children. Two master trainers had no previous experience in training parents, while six did.In the second (pilot) phase, each master trainer trained two facilitators to conduct the program with the families. After their training, the facilitators delivered the program to the families under the supervision of a master trainer. Apart from one facilitator who worked in a public education institution, all others were employed in a public healthcare institution.In each phase, the program was comprised of nine parental group sessions with three home sessions.All instruments and measures created for CST and used in this research were recommended and provided by the WHO and AS. The measures were translated, back-translated and adapted to Serbian language.Information on master trainers, caregivers, and facilitators, partially presented in the Participants and Procedures sections, was collected using CST background measures: Demographic and professional background information for facilitators and Information about caregivers: Demographic and service history information.The Post session Feedback Form—Caregivers and the Home Visit Participant Feedback Form were used to assess the implementation of the CST program. After each session, the caregivers completed the Post session Feedback Form stating their observations and impressions. The Form included 14 questions requiring the caregivers to assess the degree of understanding, the relevance of the information received, the usefulness of key messages and tips, the usefulness of advice in achieving their own program goals, learning activities, duration of the session and its parts, etc.In addition to expressing their views about the sessions, caregivers were asked to share their experiences after each home visit with master trainers/facilitators by completing the Home Visit Participant Feedback Form. This Form included only five questions, requiring caregivers to assess the duration and benefits of the home visit, and the benefits of the feedback received during the recording. Open-ended questions were used to collect additional information about whether video-recording was acceptable from the caregivers' perspective and their suggestions for improving home visits.The Autism Treatment Evaluation Checklist (ATEC)  was usedt-test was used to compare the first and the second test results in the pilot phase. Repeated measures two-way ANOVA was used to determine whether the participants' scores on ATEC in the pilot phase differed depending on whether the program was implemented in person or online. The repeated factor tested the difference between the two measurements, and the factor that was not repeated referred to the difference in the way the training was implemented.The statistical analysis was performed in IBM SPSS Statistics version 27. Due to the small sample in the pre-pilot research phase, the difference in the participants' achievements on ATEC between the first and the second testing was determined by the Wilcoxon signed-rank test. The paired samples This research has been approved by the Ethics Committee of the Institute of Mental Health in Serbia. All participants have signed the Informed Consent Form prior to inclusion in the research.The main aim our research was to evaluate the effects of the CST on child's development and to evaluate the appropriateness of the program in our country. To achieve this, we collected data from caregivers in two subsequent phases on their attitudes toward the program as well as their views on how the program effected their child's development. In this section we will present the obtained quantitative and some qualitative data.The data obtained using the Post session Feedback Form—Caregivers, after each session, and Home Visit Participant Feedback Form, after each home visit, could only be processed descriptively. All scores were very high. Thus, due to the small variability of the obtained data, the satisfaction with individual sessions and home visits was not observed as a control variable in further analysis of the training outcomes. The degree of understanding, the relevance of the information received, the usefulness of key messages and tips, and the usefulness of advice in achieving program goals were assessed by a five-point Likert scale in the Post session Feedback Form. The average scores in both research phases ranged between 4.29 and 5.00. Participants listed different key messages and tips they considered the most or the least significant in each session, but the general tendency was to choose the first ones in a line. With regard to learning activities, group discussion and demonstration were most often mentioned as the favorite ones. More than 80% of the participants positively evaluated the duration of individual sessions and activities. Over 90% of the caregivers felt very or somewhat willing to apply the advice received during sessions at home. Depending on the session, between 80 and 95% of the participants stated that the messages they received during sessions did not contradict what they or their family members considered good or significant.In addition to expressing their views on the sessions, the caregivers were asked to share their experiences with master trainers/facilitators after every home visit. Almost all participants stated that the home visit lasted as long as necessary, that it was useful, and that the feedback obtained by video recording was valuable. In both research phases, the caregivers were positive about the fact that video recording was an integral part of the program.N = 7), while the N = 29).The Z = −2,232, p < 0.05) and Health/Physical/Behavior , with a large effect size by Cohen's criteria .The Wilcoxon signed-rank test determined significant improvements in Speech/Language/Communication  and Health/Physical/Behavior , while there were no significant differences between the first and the second testing on the remaining two subscales. Eta squared values indicate that the intervention effect was significant.Similar results were obtained in the pilot phase on a somewhat larger sample. Significant improvements were found in Speech/Language/Communication , and in Health/Physical/Behavior . This suggests that the effects of the program did not depend on whether the training was conducted in person or online.Two-way ANOVA did not indicate the interaction between the two factors (repeated assessment and the way the program was implemented), both in Speech/Language/Communication  were trained by the representative of AS. All MTs are experts in the field of mental health or health associates in primary health care. The training was supervised by the representatives of AS, who had many years of experience in the CST program implementation. The training of master trainers (TOMT) lasted for 5 days. It was conducted in person and included explaining procedures, key recommendations and advice, role-playing, modeling expected behavior, and direct work with patients. The other part of the TOMT was designed to deliver the whole CST program to the parents. In other words, the trained MTs delivered the whole program to the families of children with DD. Their activities were recorded, thus allowing the supervision of experts from AS.The CST training of parents was provided in nine parental group sessions and three home visits. The entire training and research material  was translated into Serbian. Then, a material adaptation strategy was developed in direct meetings with the representatives of AS. The Adaptation Team and Adaptation Advisory Group were formed to work together on linguistic and cultural material adaptation. The Adaptation Advisory Group included experts in working with children with DD and neurodevelopmental disorders and their families.In the pilot phase, the certified MTs trained the facilitators to implement training. The final goal was that the facilitators could then work directly with families of children with DD under the supervision of the MTs. The facilitators had different professions: nurses, educators, health associate professionals in primary healthcare, community nurses, mental health experts, etc.Most of the respondents from both phases of the research were mothers. This was not surprising, as in Serbia childcare responsibilities are traditionally the job of mothers. Contrary to expectations, two participants in each research stage were fathers. The study that pre-piloted the CST in Ethiopia had a similar result with a low number of fathers . The litThe majority of families who participated in the study were married parents. We do assume that married parents are more willing to participate in training and education processes as sharing caring responsibilities allows greater time for participation. However, we also need to highlight the importance of single-parent and divorced families that already have a significant childcare burden, and even more so if they are a single parent to a child with DD. This is especially the case with single mothers, who face additional burdens associated with gender inequalities typical in the region  . A UniteThis research was conducted in the centers that implemented the CST, all of which are located in urban areas . The resulting sample hence only included participants from urban surroundings. However, 46% of families in Serbia live in rural surroundings with very limited resources and access to health and social care . BecauseThe outbreak of the COVID-19 pandemic during the training program affected the implementation of group sessions and home visits. During the pandemic, the CST program was adapted in order to help in switching the training to online setting . Some MTMost of the families included in the sample received some kind support, commonly in the form of special education, speech therapy and medical attention. In our previous research, we have shown that over ninety per cent of families of children with DD reported that additional support from all systems is important and necessary . Other sThe majority of the participants evaluated the program positively, as very successful and important for them; the degree of understanding, the relevance of the information received, the usefulness of key messages and tips, and the usefulness of advice in achieving program goals were evaluated between 4.29 and 5.00. This feedback can be interpreted as an endorsement of the CST program, as very well-organized and informative and useful for parents. Our results indicate that there is now an extremely practical, step by step program in Serbia which allows parents to feel understood, appreciated, and respected. This program allows parents to feel as the most valuable actor in their child's treatment and not only a passive observer in the process. It provides parents with very high professional support, as well as with much needed emotional support. The research from other countries that implemented the CST reports similar results, especially in the LIMC , 13, 14.Group discussions and practical exercises were most often voted as favorite parts of the training. Previous literature reviews show that group-based parenting programs show positive and promising results in improving emotional and behavioral functioning of young children as well as parental confidence, knowledge and wellbeing , 21. A nEven though the groups were small to be analyzed in more detail, the parents believe that CST helped them in improving Speech/Language/Communication and Health/Physical/Behavior of their children. This is not surprising, as the program is specifically designed to improve the above-mentioned domains of functioning; out of nine sessions, two sessions are dedicated to understanding and improvement of the child's communication, and two sessions for understanding and controlling the child's behavior. Research conducted on the CST program in other countries reports similar results , 13, 14.This research had several limitations. The first is a limited sample size. This was partly due to fact that this was an implementation (pilot) phase of the program which had a limited number of participants. In addition, due to the COVID pandemic outburst, the research suffered a certain dropout of families before the ending of the program. Namely, some families had technical difficulties, some had health-related issues, and some did not want to continue the program due to the shift of priorities in the time of crisis. All of this also resulted in a limited amount of collected data delivered to the research team.The adaptation of the program in Serbia took place mainly in urban areas, in clinical settings and delivered mainly by professionals with long years of experience in working with families of children with DD. We are aware that delivering the program in rural settings by non-specialists would possibly have different benefits and recommendations, but also possible additional challenges that were not discovered in this round of research.Caregiver's education is a process of systematic provision of necessary information to caregivers which can provide them with the specific knowledge and skills for encouraging their child's development and competence. In the past decade, some systematic reviews, mostly from high-income countries, suggest that caregivers are able to learn and acquire the skills needed to deliver intervention strategies to their children with DD , 23. EduWith the introduction of the CST program in Serbia, local partners build the capacities of relevant institutions and professionals. This type of knowledge and skills will be applicable in working with all families of children with disabilities, so that professionals educated through this program remain a permanent resource for participating institutions. The pilot project is implemented in three cities in Serbia , in four different health care institutions (primary and tertiary) dealing with the DD of children and their mental health. Steps have been taken to train facilitators from other cities in Serbia, making the program available to more families. With the support of partners from the respective governmental institutions, the program will become a regular service in institutions dealing with early intervention and developmental delay in children, and to will be cost free for the families.Findings from the application of CST in Serbia indicate that the program could potentially aid a wider variety of parents and caregivers, and thus that the form of its application  can encompass a greater number of parents, providing them adequate support for working with children and as well as for accepting their child's DD. Our indicative results provide the argument for its wider implementation, but it requires consistent advocacy efforts in order to secure the support of decision-makers. Only in that way the CST in Serbia can be widely implemented and sustained over time.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Institute of Mental Health. The patients/participants provided their written informed consent to participate in this study.NG contributed with technical support, data collection, literature research, writing and reviewing of the article, and statistical analysis. SP, RG, and SS contributed with data collection, literature research, result analysis, interpretation, writing, and reviewing process. MP-M contributed in the writing and reviewing process, the interpretation of the results, and expertise. All authors contributed to the article and approved the submitted version.The implementation of the CST Program and this research was supported by UNICEF Serbia and HELP—Mission to the Republic of Serbia. All authors received funding from these two organizations.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.