NtGSTs.Glutathione S-transferases (GSTs) are large and multifunctional proteases that play an important role in detoxification, protection against biotic and abiotic stresses, and secondary metabolite transportation which is essential for plant growth and development. However, there is limited research on the identification and function of GST gene in tobacco. A total of 59 NtGSTs were identified and classified into seven subfamilies via the whole-genome sequence analysis, with the Tau type serving as the major subfamily. The NtGSTs in the same branch of the evolutionary tree had similar exon/intron structure and motif constitution. There were more than 42 collinear blocks between tobacco and pepper, tomato, and potato, indicating high homology conservation between them. Twelve segmental duplicated gene pairs and one tandem duplication may have had a substantial impact on the evolution and expansion of the tobacco GST gene family. The RT-qPCR results showed that the expression patterns of NtGSTs varied significantly among tissues, varieties, and multiple abiotic stresses, suggesting that NtGST genes may widely respond to various abiotic stresses and hormones in tobacco, including NtGSTF4, NtGSTL1, NtGSTZ1, and NtGSTU40.This study uses K326 and other six tobacco varieties  as materials to conduct comprehensive genome-wide identification and functional characterization of the NtGST gene family, including structures and functions. Many NtGSTs play a critical regulatory role in tobacco growth and development, and responses to abiotic stresses. These findings offer novel and valuable insights for understanding the biological function of NtGSTs and the reference materials for cultivating highly resistant varieties and enhancing the yield and quality of crops.This study provides a comprehensive analysis of the The online version contains supplementary material available at 10.1186/s12864-023-09450-x. Plant secondary metabolites are a class of non-essential small-molecule organic compounds produced by secondary metabolic processes. Despite not being directly involved in the growth and development of plants, they are crucial for ecological adaptation . StudiesGST genes are reflected in the N-terminal and C-terminal domains, which determine the substrate specificity [GST genes, they can be classified into eight typical subfamilies, including Tau (U), Phi (F), Lambda (L), Zeta (Z), Theta (T), elongation factor 1 gamma (EF1Bγ), dehydroascorbate reductase (DHAR), and tetrachlorohydroquinone dehalogenase (TCHQD) [GSTs can be classified into 14 categories, where the remaining six include metaxin, Ure2p, microsomal prostaglandin E synthase type 2 (PGES2), hemerythrin, iota, and glutamate hydroxyquinone reductases (GHR) [GST gene families have become increasingly precise in recent years due to species genome sequencing. Additionally, there are significant differences in the number and classification of GST genes among different species. According to existing reports, there are 79 GSTs in rice [GSTs in Arabidopsis [Each subunit of glutathione transferase contains two N-terminal and C-terminal domains with different spatial structures . The N-tcificity . In high (TCHQD) , 14. Howes (GHR) , 16. The in rice , 55 GSTsbidopsis , 40 in rbidopsis , 22 in Sbidopsis , 73 in Mbidopsis , and 38 GST genes. The function of the GST gene family has been isolated and identified in many species, including Arabidopsis [GSTs enhance plant resistance to salt stress by elevating antioxidant enzyme activity, increasing chlorophyll content, reducing reactive oxygen species accumulation [GST genes are crucial for maintaining the balance between cell turgor and reactive oxygen species [GSTs can catalyze the reaction of GSH with membrane lipid peroxides, reducing the damage that low-temperature stress causes to the cell membrane structure [GST genes also involving the pathogen resistance [TaGSTU6 and TaCBSX3 may positively enhance wheat resistance to powdery mildew [GSTs are crucial in heavy metal stress [The ability of plants to adapt to adversity can be improved by the bidopsis , cotton bidopsis , Chinesebidopsis , etc. Acmulation , and decmulation . Plant G species , reducin species , 30. In tructure , 31. Thesistance , and it y mildew . Furtherl stress , 35, herl stress , high tel stress , light [l stress , and horl stress .GST genes, many studies have focused on the cloning and validation of a single gene [GST gene family. In this study, a comprehensive identification and analysis of the tobacco GST gene family have been conducted, including subfamily classification, gene naming, gene structure and motif, distribution on chromosomes, sequence comparison, phylogenetic tree, and the expression profile of NtGSTs under different stresses have been analyzed. This can provide evidence for analyzing the role of the NtGSTs and cultivating novel resistant tobacco varieties. At the same time, it is crucial to predict the function of the GST genes in other crops and improve the quality traits and stress resistance of crops.Tobacco is a model plant for studying various biological phenomena and is an important leaf cash crop , 41. Forgle gene , 43. HowNtGSTs in seven subfamilies were identified in the genome of tobacco K326 and were named according to their location and order on chromosomes. Simultaneously, the physical and chemical properties and basic characteristics of these 59 genes were analyzed. Table SNtGSTZ2 was the smallest protein, encoding 61 amino acids, and NtGSTL1 was the largest protein, encoding 512 amino acids. Mw varied significantly among different genes, with the mean Mw of 28264.41 KDa, the lowest Mw of 6909.98 KDa (NtGSTU39), and the highest Mw of 59313.6 KDa (NtGSTU1). The isoelectric point (pI) varied significantly among the same subfamilies, ranging from 4.63 (NtGSTL3) to 9.44 (NtGSTL2). Subcellular localization predictions found that there were 43 NtGST proteins located in the cytoplasm, with six NtGST located in the chloroplast, six in the nucleus, two (NtGSTL2 and NtGSTZ3) in the chloroplast thylakoid membrane, one (NtDHAR2) in the mitochondrion, and one (NtGSTZ4) in the organelle membrane.In this study, 59 NtGSTs were divided into seven subfamilies, including Tau subfamily (U), Zeta subfamily (Z), Lambda subfamily (L), Theta subfamily (T), Dehydroascorbate reductase subfamily (DHAR), Tetrachlorohydroquinone dehalogenase-like subfamily (TCHQD), and Phi subfamily (F). Among them, the Tau subfamily was the largest group, representing 67.8% of the number of all NtGST proteins (NtGSTU1–40). Moreover, five proteins (NtGSTF1–5) were classified as Phi subfamily, three proteins (NtGSTL1–3) as Lambda subfamily, four proteins (NtGSTZ1–4) as Zeta subfamily, two proteins (NtGSTT1–2) as Lambda subfamily, three proteins (NtDHAR1–3) as DHAR subfamily, and two proteins (NtTCHQD1–2) as TCHQD subfamily. The phylogenetic relationship showed that the tobacco NtGST proteins had high homology with Arabidopsis AtGST proteins, suggesting that the evolution of GST genes between the two species was conservative and may have the same biological functions.To investigate the phylogenetic relationship of tobacco GST proteins, we constructed an evolutionary tree of 59 tobacco and 56 Arabidopsis GST proteins using the maximum likelihood method Fig. . All ideNtGSTs and named motif 1 to motif 10 to better understand tobacco NtGST proteins’ function. The findings suggested that those genes in the same branch of the phylogenetic tree contained similar motifs, suggesting that these genes might perform similar biological functions. Except for a few genes, the remaining NtGSTs contained conserved motif 3. Most genes also contained motif 2, suggesting that these motifs can be used as markers to recognize NtGSTs  had untranslated region (UTR).The genomic sequences of tobacco NtGST proteins were examined to determine the number and structural information of their introns and exons. The findings revealed that all the NtGST proteins contained introns except for the GST protein was performed to further understand its distribution on the chromosome. The findings suggested that 59 NtGST proteins were localized on 21 chromosomes, and the number of proteins on each chromosome was significantly different , two monocotyledonous plants (Oryza sativa and Triticum aestivum), and three Solanaceae plants  were selected for collinearity analysis to investigate the collinearity relationship between GST gene members and NtGSTs in different species and their evolutionary origins. The findings revealed that 47 NtGSTs displayed a collinearity relationship across the six species, indicating that during the process of evolution, tobacco GSTs were highly conserved on the corresponding chromosomes. A total of 15, 4, 4, 42, 47, and 49 collinear blocks of NtGST genes were identified in Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Capsicum annuum, Lycopersicon esculentum, and Solanum tuberosum, respectively (Supplementary file Oryza sativa and Triticum aestivum), indicating that most GST genes were formed after the differentiation of dicotyledonous and monocotyledonous plants. However, there were more than 42 collinear blocks between tobacco and Capsicum annuum, Lycopersicon esculentum, and Solanum tuberosum, showing a high homology of the GST genes at the same family level.According to the evolutionary relationship and motif composition of GST proteins in different plants, one dicotyledonous plant , salicylic acid (SA), methyl jasmonate (MeJA), and gibberellin acid (GA), stress-related response elements such as light, dehydration, low-temp, and salt stresses, and wound-responsive elements  and different materials . The results showed that the expression patterns of six NtGSTs in the same tissue were significantly different, indicating that NtGSTs served different physiological functions in tobacco  and high temperatures (38 °C), and the expression level of six NtGSTs was measured using qRT-PCR. The results revealed that different NtGSTs expression patterns varied in high and low temperatures, as well as in roots, stalks, and leaves. Under low-temperature treatment, the expression pattern of NtGSTL1, NtGSTT2, and NtGSTZ1 was relatively consistent, whether in the root, stalks, or leaf and indicated stimulated expression in the early stress response stage. Moreover, the expression level was first increased and then decreased  and salicylic acid. The results demonstrated that the expression of NtGSTs was induced by hormones and SA, with significant induction by SA. Most genes responded to hormone stress mainly at 2 h and 12 h after treatment. Under IAA treatment, the induced expression of NtGSTs included up-regulation and down-regulation, and the expression levels of different genes differed  was 4.5 Gb [GST genes. For instance, Arabidopsis has the smallest genome and the fewest GST, whereas wheat has the largest genome and the most GST genes. In Arabidopsis, the GST genes were classified into seven subfamilies. However, compared with the plants belonging to the Solanaceae , none of the genes belonged to the subfamilies of EF1Bɣ1, GHR, MGST, and others. This may be due to the loss of the NtGSTs in the evolutionary process or differentiation into other subfamily members. A total of 40 NtGSTs in tobacco come from Tau (U) subfamily, and Tau subfamily members are the most common in other plants, with the largest number, indicating that NtGSTs conforms to the distribution characteristics of GST gene subfamilies in plants.Based on tobacco genome sequence and RNA seq data, 59 thaliana , 79 OsGSa sativa , 90 SlGSculentum , 85 CaGSm annuum , 90 StGSuberosum , and 346aestivum . The gens 4.5 Gb , 117 Mb s 4.5 Gb , 466 Mb s 4.5 Gb , 900 Mb s 4.5 Gb , 3.5-Gb s 4.5 Gb , 1.67 Gbs 4.5 Gb , and 16 s 4.5 Gb , respectNtGSTs were distributed on twenty-one chromosomes, and no NtGST was found on the other three chromosomes, suggesting an important role of of gene family expansion. During species evolution, gene duplication events can result in the expansion of a gene family, including tandem duplication, segmental duplication, transposition events, and genome-wide duplication [NtGST family. The findings indicated that these gene replication events were crucial in the evolution of the tobacco GST gene family, with the segment duplication event playing a leading role. We hypothesized that this might be because tobacco is allotetraploid, and the results of gene replication events are consistent with those in pepper and wheat [NtGSTs were primarily distributed in the U-type and F-type subfamilies. Hence it can also be speculated that the expansion of the GST family of tobacco is mainly due to the duplication events of these two families, which is also consistent with that of cotton [The 59 lication , 56. In lication . In thisnd wheat , 58. Thef cotton . Genes wf cotton .NtGSTs were found, and genes in the same branch of the evolutionary tree contained similar motif constitutions, indicating that they may perform similar functions in plants. According to gene function studies, GST family members can participate in various biotic and abiotic stresses, including mechanical damage, heavy metals, drought, temperature, and phytohormones [Cis-acting elements are crucial in regulating plant adaptation to stress, growth, and development by participating in the expression of target genes [cis-acting elements, it was found that the members of the tobacco GST gene family contain various environmental stress response elements, including hormones, stress, MYB binding site, circadian rhythm, and injury induction. This result was consistent with the promoter analysis results of gene families such as SlGSTs and AtGSTs, indicating that these response elements directly determine the transcriptional expression level and response-ability of GST family genes under stress[In this study, ten motifs in 59 hormones , 37. Ciser stress.GST gene family members were found to be expressed normally in different cultivars, and only individual genes, such as NtGSTU40 in the stack of Va116 and NtDHAR3 in the root of GDH88, displayed specific expression. Simultaneously, we found that the expression of NtGSTs have tissue specificity. Tobacco roots had a higher expression level than stalks, followed by leaves. These results were consistent with those of soybeans [NtGSTs are crucial in the growth and development of tobacco. This may be because the root is the key organ for the absorption and transportation of water and mineral nutrients in plants and also the most important organ for recognizing and feeling injuries, which can quickly respond to stress [GST gene family.Analysis of gene expression patterns can reveal crucial information about the physiological function of genes. Most of the tobacco soybeans  and peppsoybeans , indicato stress , 62. MorJrGSTU1 gene in walnut (Juglans regia L.) could enhance the GST activity to regulate the expression of other stress- related genes and thus reduce the levels of reactive oxygen species [NtGST genes and their ability to respond to stress, we examined the expression patterns of six NtGSTs in response to six stresses. The results showed that the expression patterns of different genes varied under different stresses, which were consistent with previous studies[NtGSTF4 expression was up-regulated in the roots, stems, and leaves under cold stress, while NtGSTL1 and NtGSTZ1 were first increased and then decreased. Furthermore, the response times of the same gene to low and high temperatures differed. For example, NtGSTZ1 responded to low temperature at 2 h after treatment, while its maximum response to high temperature was at 12 h after treatment. Interestingly, NtGSTU40 exhibited the maximum expression level in the leaves under low- or high-temperature stresses, and the highest stress response occurred at 12 h after treatment. These results indicated that the NtGSTs might be involved in a complex cross-regulatory network in plants.Several studies have shown that GSTs play multiple functions in many aspects of growth and development in plants. Yang et al. demonstrated that the  species . To evals studies, 50. ForNtGSTF4, NtGSTT2, NtGSTL1, NtGSTU40, and NtGSTZ14, exhibiting an overall upward trend. Previous studies have shown that the heterologous expression of the lambda subfamily, VvGSTF13 or JrGSTT1, in tobacco enhanced the tolerance to salinity and drought stresses[NtDHAR3 showed a down-regulated expression pattern under salt and drought stresses, indicating that the expression pattern of GST family genes would also vary by species. Under the treatment of hormones (IAA and SA), NtGSTU40 was significantly expressed, which was consistent with the expression of TaGSTU62 and OsGSTU4 in rice [NtGSTs might function similarly to other plant GST genes.Salt and drought, the two primary abiotic stresses, have serious impacts on plant growth and crop yield . Under s stresses–65. This stresses claimed  in rice . TherefoNtGSTs was conducted to provide a reference for studying the GST gene function in plants. The potential role of NtGSTs in plant resistance to stress was verified using RT-qPCR analysis. However, the mechanism of action of NtGSTs in response to stress requires further investigation for better application to the cultivation of highly resistant cultivars and thus improve crop yield and quality.In this study, a comprehensive and systematic identification and analysis of the NtGSTs at the genome level were performed in this study. A total of 59 NtGSTs were identified and categorized into seven subfamilies, and information on physicochemical properties and subcellular localization for these genes was provided. The NtGSTs in the same branch of the evolutionary tree have similar exon/intron structures and motif constitutions. And the 59 NtGSTs were unevenly distributed on 21 chromosomes. The analysis of their collinearity with the GST gene in other plants provided an important reference for studying the evolutionary characteristics of NtGSTs. Furthermore, twelve pairs of segmental duplicated genes and one pair of tandem duplication were unevenly distributed on 21 chromosomes, suggesting that gene duplication events are crucial in the evolution and expansion of the tobacco GST gene family. The expression of NtGSTs in different tissues and varieties and the response to multiple stresses and hormone treatment indicated that NtGSTs play a critical regulatory role in tobacco growth and development. These results provide novel and valuable insights for studying the biological function of NtGSTs.In summary, a comprehensive characterization and analysis of the NtGSTs. The K326 was used for different stress treatments, and the remaining six varieties were used to analyze the expression pattern of NtGSTs in different tissues and stages. All materials were provided by Key Laboratory for Tobacco Quality Research Guizhou Province and cultivated in the plant culture laboratory. The collection of plant material, experimental research, and field studies on plants were complied with relevant institutional, national, and international guidelines and legislation. Using floating dish breeding, sowing and seedling raising were performed under environmental conditions, including 75% relative humidity, 16 h/28 °C during the day, and 8 h/20 °C at night. On the 75th day of transplantation, three consistently growing plants in each replicate were sampled from three tissues , and three biological replicates were set.In this experiment, seven tobacco varieties, including K326, Hongda, HG, GDH11, Va116, VG, and GDH88, were selected to determine the function of NtGSTs, combined with the results of their transcriptome analysis in roots, stems and leaves, we focused on exploring the potential biological functions under abiotic stress of six genes with relatively highest or lowest expression levels in different subfamilies located on different chromosomes. When the tobacco plants grew to the sixth real leaf, the same growing plants were selected and treated with high temperature (38 °C), low temperature (4 °C), salt (5% NaCl), drought (15% PEG6000), and hormone (100 µmol/L IAA and SA). Three biological replicates were set for each treatment, and samples were taken at 0 h, 2 h, 12 h, and 24 h after treatment. The collected samples were immediately frozen in liquid nitrogen and stored in a hypothermia refrigerator at -80 °C for real-time fluorescence quantitative PCR (RT-qPCR) experiments.Based on the chromosome location information of 59 https://solgenomics.net/organism/Nicotiana_tabacum/genome), and the hidden Markov model (HMM) profile of the GST domains (PF00043 and PF02798) was obtained from the Pfam protein family database (http://pfam.sanger.ac.uk/). The NtGSTs were searched in the tobacco genome database using the HMMER3.0 software with the default parameters . Meanwhile, using the sequence information in PF00043 and PF02798 files as templates, BLASTP software was used to compare the entire tobacco genome and screen out proteins containing these two sequence information. The results of BLASTP and HMMER 3.0 were integrated and analyzed. Pfam and SMART programs were used to detect candidate genes that may contain GST domains obtained from the BLASTP and HMMER 3.0 analysis results. Each potential gene was then manually examined to ensure predicted conserved sequences in the N and C terminal regions of the GST domain. A total of 59 NtGSTs were identified from the tobacco genome via testing and screening. The molecular weight (MW) and isoelectric points (pl) of the NtGST family members were predicted and analyzed using the online analysis software ExPASy (http:/www.expasy.org/tools/), and BUSCA (http://busca.biocomp.unibo.it/) was used for subcellular localization prediction.The genome sequence was obtained from the tobacco genome project website  [http://meme-suite.org/tools/meme) was used for the conserved domain analysis with the number of motifs set to 10 and the motif width range set to 50–100 amino acids [A multiple sequence alignment of the obtained GST protein sequences and the previously reported Arabidopsis thaliana sequences was performed using the ClustalW default parameters. Subsequently, the amino acid sequence of the deduced GST domain was adjusted using GeneDoc software. To determine the exon–intron structure of the .edu.cn) . The onlno acids .GST genes on the chromosome was obtained. The gene homologous duplication events were then obtained according to the alignment between the pairwise genes. Finally, the chromosomal mapping and alignment results were visually analyzed using online bioinformatics tools. A multiple collinear scanning tool, MCScanX, was used to analyze the gene duplication events [GST collinear analysis atlas was constructed using the Dual Systeny Plotter software, and the GST collinear relationship of tobacco with Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Capsicum annuum, Lycopersicon esculentum, and Solanum tuberosum was determined, respectively.Based on the tobacco annotation data obtained from the Solanaceae database, the data of starting position of the n events . The GSTNtGSTs were obtained from the Solanaceae database. These sequences with cis-acting elements were analyzed using the PlantCare online software and visualized using the bioinformatics software.A total of 2000 bp gene sequences upstream of the https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE233199.The Hongda, HG, GDH11, Va116, VG, and GDH88 were used for transcriptome sequencing analysis. The transcriptome data used in this study is derived from the previous experimental data of our research group. The samples were root, stalks and leaf tissues, and the sampling stage was one week after topping. The sequencing of biological samples was entrusted to Shanghai Majorbio Company. Data analysis was conducted using the method of Mo et al. . The oriNtGST genes under different abiotic stresses. The experiments were performed using the SYBR Premix Ex Taq kit (Takara) and the Applied Biosystems 7500 Real-Time PCR system . Primer Premier 6 was used to design the gene primers. The gene names and corresponding primers used for the qPCR test are listed in Supplementary file −ΔΔCT method was used to determine the relative expression level of each gene [Six genes were randomly selected for RT-qPCR experiments to determine the expression levels of ach gene .P < 0.05 and P < 0.01) using the SPSS (version 16.0) software. Origin 2018 (95_64) and Adobe Illustrator CS6 were used for figure drawing.Duncan’s new multiple range test was conducted to analyze the variation in the gene expression levels (Additional file 1: Table S1. List of the 59 NtGST genes identified in this study.Additional file 2: Table S2. List of the tandem repeat gene pairs of NtGSTs in tobacco.Additional file 3. The lists of Nicotiana tabacum and other species GST synteny gene pairs.Additional file 4: Table S4. Analysis of cis-acting elements in promoters of GST gene family members.Additional file 5: Table S5. Primer sequences of six selected genes used in real-time qPCR analysis.


Finally, the lightweight DL model, which was designed to focus on the top five important features, demonstrated superior predictive capabilities, highlighting its potential in improving patient outcomes and minimizing treatment-related complications with secured reliability.This study developed a prediction model for late bladder toxicity in patients with uterine cervical cancer undergoing radiation therapy. A deep learning (DL) model was trained on data from 281 patients and compared its performance with a multivariable logistic regression model. The DL model outperformed the regression model, achieving higher accuracy, recall, F1-score, and area under the receiver operating characteristic curve. Specifically, based on the feature importance analysis, the DL model identified the doses for the most exposed 2 cc volume of the bladder (BD2cc) as the most important feature, followed by BD5cc and the ICRU bladder point. In the case of the lightweight DL model, the F-score and AUROC were 0.90 and 0.91, respectively. (4) The DL models exhibited superior performance in predicting late bladder toxicity compared with the statistical method. Through the interpretation of the model, it further emphasized its potential for improving patient outcomes and minimizing treatment-related complications with a high level of reliability.(1) In this study, we developed a deep learning (DL) model that can be used to predict late bladder toxicity. (2) We collected data obtained from 281 uterine cervical cancer patients who underwent definitive radiation therapy. The DL model was trained using 16 features, including patient, tumor, treatment, and dose parameters, and its performance was compared with that of a multivariable logistic regression model using the following metrics: accuracy, prediction, recall, F1-score, and area under the receiver operating characteristic curve (AUROC). In addition, permutation feature importance was calculated to interpret the DL model for each feature, and the lightweight DL model was designed to focus on the top five important features. (3) The DL model outperformed the multivariable logistic regression model on our dataset. It achieved an F1-score of 0.76 and an AUROC of 0.81, while the corresponding values for the multivariable logistic regression were 0.14 and 0.43, respectively. The DL model identified the doses for the most exposed 2 cc volume of the bladder (BD Cervical cancer is the third most commonly diagnosed malignancy in women worldwide, as reported in 2022 . With eaExternal beam radiation therapy (EBRT) with concurrent chemotherapy followed by intracavitary brachytherapy represents the standard treatment for locally advanced cervical cancers ,3. Owing2cc) [51.43Gy [8.5Gy/w [Predicting the occurrence of bladder toxicity before treatment is crucial for improving patients’ long-term health outcomes and quality of life, as it reduces the probability of treatment-related complications or interruptions. Previous studies have focused on investigating prognostic factors associated with radiation-induced bladder toxicity. These studies have examined various dose volumetric parameters, such as the doses for the most exposed 2 cc volume of the bladder (BD2cc)  and the [51.43Gy  and V8.5[8.5Gy/w .Statistical methods and deep learning (DL) models have been introduced to predict the toxic effects of radiation therapy on the gastrointestinal ,13,14,15A machine-learning-based prediction model of fistula formation after interstitial brachytherapy for locally advanced gynecological malignancies achieved an accuracy of 0.901 using Support Vector Machine (SVM) .A feasibility study utilized a deep convolutional neural network (CNN) with transfer learning to predict rectum toxicity in cervical cancer radiotherapy, achieving an AUC of 0.89 .An observational study predicting radiotherapy impact on late bladder toxicity in prostate cancer patients used univariate logistic regression, achieving an AUC of 0.626 [Various studies have focused on predicting urinary toxicity in prostate cancer radiotherapy using different models, such as the international prostate symptoms score model, logistic and Cox regression, the edited nearest neighbor algorithm together with the regularized discriminant analysis classifier, and others ,18,19.Predicting late organ-at-risk toxicity after prostate radiation therapy has been explored using statistical analysis, cox regression, and random forest models ,22,23.In radiotherapy for cervical cancer, radiomics analysis of 3D dose distributions has been employed to predict toxicity rates, achieving AUCs ranging from 0.57 to 0.89 .Related studies that have used predictive models for toxicity in various cancer types and radiation therapy techniques:The differences between the statistical and DL methods can be described based on three factors: (i) model complexity, (ii) feature importance, and (iii) model transparency. Statistical methods tend to exhibit relatively low complexity, and, compared with DL models, they operate based on clear governing principles. Statistical methods often demonstrate relatively low accuracy; however, they offer relatively high model transparency, which simplifies the interpretation of feature importance. DL models tend to outperform statistical methods. However, they are more complex ,26,27 anTherefore, in the present study, we propose an interpretable DL model for predicting late bladder toxicity in patients with cervical cancer who have received definitive radiotherapy. We compared the performance of the statistical method and the DL model. In addition to achieving a high level of reliability, we conducted a feature-importance analysis and validated the performance of a lightweight DL model.We identified 545 patients with primary uterine cervical cancer who underwent definitive RT with curative intent at our institution between February 2006 and December 2017. Follow-up evaluations were performed every three months in the first two years, every four months in the third year, every six months in the fourth and fifth years, and annually thereafter. In this study, we included patients with more than three years of follow-up after treatment completion. The radiation-induced bladder toxicity was evaluated during the regular follow-up visits based on the European Organization for Research and Treatment of Cancer late radiation toxicity criteria, which represent a structured scoring schema developed by the Radiation Therapy Oncology Group (RTOG) .In total, 281 patients with cervical cancer were included in this study . ClinicaThe patients received concurrent chemoradiotherapy using either three-dimensional (3D) conformal EBRT or an intensity-modulated RT technique and high-dose-rate brachytherapy. The clinical target volume of EBRT included gross disease, the entire uterus, a margin of 2.0 cm around the tumor extent at the level of the vagina, and the entire parametrial tissue. In addition, regional lymphatics, including the common, internal, and external iliac nodal regions and the presacral and para-aortic lymph nodes, were considered. Prior to 2008, brachytherapy planning was CT-based; afterwards, MRI-based planning has been employed . BrachytThe patients received a combination of 45–50 Gy of pelvic EBRT and 30 Gy of high-dose-rate brachytherapy with daily fractions of 5 Gy over 3 weeks. The biologically equivalent dose to point A in 2 Gy fractions was previously calculated as approximately 82 Gy for cervical tumors (α/β = 10) and 91 Gy for normal tissue (α/β = 3) . Concomi, the dose delivered to 100% of the primary GTV (GTV-D100), International Commission on Radiation Units and Measurements (ICRU) bladder point (BPICRU), BD0.1cc, BD1.0cc, BD2.0cc, and BD5.0cc. Features that were not continuous were dummy-coded. All features were deliberately collected in accordance with IRB guidelines and based on clinical experience and relevant literature [We used four groups of the feature set as input data for the statistical analysis and DL method: (i) “patient” feature group (n = 2), including age and pathology; (ii) “tumor” feature group (n = 3), including the federation of gynecology and obstetrics (FIGO) stage, tumor-node-metastasis (TNM) category, and maximum tumor length on axial T2-weighted magnetic resonance image (MRI); (iii) “treatment” feature group (n = 4), including concurrent chemoradiotherapy (CCRT), CCRT regimen, number of CCRT cycles, and adjuvant chemotherapy; (iv) “dose” feature group with the equieffective dose (EQD2) at 3.0 Gy (n = 7), including the total dose of external beam radiation therapy (EBRT)terature ,37,38,39Late bladder toxicity was graded according to the scoring schema of the RTOG, with scores in the range of 0–5. The median durations from the initial start of EBRT to the occurrence of late toxicity are 37.7 months in the current study population. Owing to the imbalance of grades, the grade of late bladder toxicity was binarized by considering the occurrence of late bladder toxicity only. When late bladder toxicity is absent, the occurrence status is set to 0; otherwise, the occurrence status is set to 1. t-test or Wilcoxon rank-sum test, whereas categorical variables were compared using the Chi-square or Fisher’s exact tests. The dataset was randomly divided into two sets containing 199 and 82 cases for training and testing the statistical model, respectively. The ratios for the training and test sets were 70% and 30%, respectively. A statistical model was built using a training set with univariable and multivariable logistic regression. A univariable logistic regression was performed on the input data, considering patient, tumor, treatment, and dose features. The results of the univariable logistic regression were used as the input for the multivariable logistic regression. The target was the occurrence of binarized late toxicity. The developed multivariable logistic regression model was tested using the test dataset. Statistical analyses were performed using SAS version 9.4  and R version 4.2.1 . We assumed that statistical significance is achieved for p-values below 0.05.Patient characteristics were indicated in terms of mean ± standard deviation or median (min–max) for continuous variables; frequencies and percentage values were used for categorical variables. Continuous variables were compared using the x is the input data, y is the output data, iW and ib are the weight matrix and bias vector for the i-th layer, respectively, if is the activation function for the i-th layer, and ih is the output of the i-th hidden layer.To implement the DL model by using Pytorch version 1.12.1 , we used an MLP. MLP is a type of artificial neural network that consists of multiple layers of nodes with batch normalization and nonlinearity functions such as rectified linear unit (RELU), leaky RELU, and sigmoid. Each node in one layer is connected to all nodes in the next layer, forming a fully connected network. The input layer takes the input data, and the output layer produces the final predictions. The layers between the input and output layers are called hidden layers. The DL model was built using six hidden layers. The architecture can be represented mathematically as follows:As a preprocessing step, 16 features were used as input data, and binarized late bladder toxicity values were considered the output data. Z-score normalization was applied to the input data so that the DL model could rapidly converge to the optimal solution. The normalization parameters (mean and standard deviation) were determined from the training set.The standardized input data were passed through an input layer, and output data were obtained on the output layer. In each hidden layer, except for the last one, batch normalization and dropout techniques were applied to avoid overfitting the training data. The probability of a node being zeroed for dropout was set to 0.2. In addition, a leaky ReLU was adopted as the activation function in the hidden layers to improve model complexity and performance. A sigmoid function was used as the activation function of the last layer only. These functions introduce nonlinearity into the model, allowing it to capture complex relationships within the data. The detailed architecture of the proposed model is presented in N represents the total number of samples in the batch, iy represents the true label of the i-th sample (either 0 or 1), and ip represents the predicted probability (between 0 and 1) of the i-th sample belonging to the positive class. The DL models were trained to minimize the loss.The loss between the ground truth and predicted toxicity occurrence was calculated using binary cross-entropy as follows:To avoid overfitting, the synthetic minority oversampling technique (SMOTE) was adopted during the training procedure as oversampling technique and augmentation strategy, which adds random noise to the input data in every training epoch . MoreoveK-fold cross-validation (CV) was employed to ensure the generalizability of the DL model. The value of k was set to 5, resulting in the training set being divided into five folds. Furthermore, an early stopping strategy was implemented, where the model achieving the lowest validation loss during the 500 epochs was saved as the best-performing model. Each fold yielded independent results as separate models were trained. The final output data were determined using the voting method, with a threshold of 3 sets to obtain the ultimate decision.The permutation analysis is used to calculate the feature importance of a DL model. It involves randomly permuting the value of a single feature and evaluating the model’s performance on the test dataset . The anaTo measure the importance of each feature, the mean squared error (MSE) is calculated between the reference dataset and a corrupted dataset, where the value of a single feature has been randomly permuted. This is compared with the reference MSE, which is the MSE calculated on the uncorrupted reference dataset. i is used to index the features, j represents the total number of features, j-th feature, K is the number of repetitions or permutations, S is the reference MSE, k-th repetition, and K repetitions for j-th feature. The variation in model performance resulting from the permutation of a feature is used to compute the importance of each feature.The value of K was set to 500, indicating that the permutation analysis was repeated 500 times. Finally, the permutation feature importance was calculated by averaging the feature importance values obtained from DL models trained using different training folds.In order to develop a lightweight deep learning model, we utilized the importance values derived from permutation feature analysis and selected the top 5 features as input. This approach enabled us to focus on the most influential features while reducing the computational complexity of the model.When designing the hidden layers, we adhered to the commonly employed standards of multi-layer perceptron (MLP) models. Specifically, our model was designed with a decreasing number of neurons in each hidden layer, starting from the number of input features and gradually reducing to 5, 4, 3, 2, and finally 1 neuron.The activation functions, preprocessing steps, loss function, optimization technique, cross-validation, and final decision-making process of the lightweight deep learning model remain the same as those of the deep learning model described previously.To compare the performance of the DL model and multivariable logistic regression, four metrics were adopted: accuracy (Equation (10)), precision (Equation (11)), recall (Equation (12)), F1-score (Equation (13)), and the area under the receiver operating characteristic curve (AUROC) (Equation (14)). To compute these metrics, we counted the number of true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) in the test set. Precision is defined as the number of positive predictions  expressed as a fraction of the total number of predictions. Recall indicates the fraction of positive instances that the model could identify. The F1-score is the harmonic mean of precision and recall. Therefore, a high F1-score indicates that the model exhibits a good balance between precision and recall. If a model performs well, it has a high AUROC value (close to 1.0).ICRU, BD0.1cc, BD1.0cc, BD2.0cc, and BD5.0cc. Patients’ characteristics are summarized in p < 0.2: CCRT cycle, GTV-D100, BD2.0cc, and BD5.0cc. The prediction metrics were calculated using a multivariable logistic regression model on the test data. Multivariable logistic regression achieved accuracy, precision, recall, F1-score, and AUROC of 0.85, 0.08, 0.5, 0.14, and 0.43, respectively. Although multivariable logistic regression yielded an accuracy of 0.85, the regression model did not exhibit acceptable performance on the test dataset. Univariable and multivariable logistic regression analyses were performed to identify risk predictors for adverse events in the training set . Based oPermutation feature analysis determines the importance of features by quantifying the extent to which the performance of a trained model changes when the dataset is permuted (Equation (7)).An independent permutation feature importance analysis was conducted on the DL model, separately for each training fold. To determine the overall feature importance, the mean and standard deviation of the calculated permutation feature importance were computed by averaging the importance values across folds. 2cc, followed by BD5cc and BPICRU. Additionally, the features with the highest importance within each group were age, TNM category, number of CCRT cycles, and BD2cc for the patient, tumor, treatment, and dose feature groups, respectively. Our findings reveal that the feature with the highest importance was BD2cc, BD5cc, BPICRU, TNM category, and tumor size. We made the decision to select tumor size instead of FIGO stage as a feature, considering the possibility of redundancy between FIGO stage and TNM category. This decision was based on the scientific rationale that tumor size provides valuable and distinct information for the prediction model [We identified the top five features with high importance as BDon model .The prediction performance of the DL model for late bladder toxicity was evaluated using the voting method. Finally, both the DL model and the lightweight deep learning model were evaluated using the voting method with a threshold of 3. For the DL model, the evaluation metrics were as follows: Accuracy (0.91), Precision (0.85), Recall (0.69), F1-score (0.76), and AUROC (0.81). For the lightweight deep learning model, the evaluation metrics were as follows: Accuracy (0.93), Precision (0.94), Recall (0.88), F1-score (0.90), and AUROC (0.91). However, because the AUROC could not be calculated from the labels predicted by each of the five models, the average of the AUROCs of all the models was considered as the overall AUROC value.This study aimed to (1) compare the ability of multivariable logistic regression and DL models to identify those patients who, having received radiation therapy, are at risk of bladder radiation toxicity, and (2) interpret the results of DL models to understand the significance of input features. To the best of our knowledge, no previous study has attempted an interpretation to ensure the reliability of a DL model in predicting the occurrence of late bladder toxicity.Several statistical methods and DL models are available for predicting clinical outcomes, including radiation toxicity. In many instances, DL models can effectively find near-optimal solutions for nonconvex optimization problems using gradient methods and nonlinear activation functions. However, statistical methods can also achieve high accuracy and reliability in specific cases. Despite the rapid development of DL models and their relatively high performance, their clinical utility is still a topic of controversy due to concerns about their reliability, particularly in terms of how clinicians interpret the results and features.In this study, we utilized an MLP with relatively low model complexity to enhance the interpretability of deep learning models. The performance of the MLP relies on the configuration of the hidden layer, and Muhammad et al.  suggesteConsidering the experiment on our dataset, the DL model proved superior compared with the statistical model. To address the class-imbalance problem in the DL method, we adopted the SMOTE oversampling technique. Mylona et al.  suggesteThe precision and recall of the DL model exhibited a tradeoff relationship depending on the value of the threshold used for voting, as shown in 2cc, BD5cc, and BPICRU for the accurate prediction of late bladder toxicity occurrence, which is also consistent with the prognostic factor for urinary toxicity suggested in previous studies, such as BD2cc in [Permutation feature importance is a model-agnostic technique that provides a comprehensive and computationally efficient assessment of feature importance by considering feature interactions and avoiding the bias caused by collinear or redundant features. In this study, permutation feature importance is utilized to calculate the relative importance of features while also employing a feature reduction technique like principal component analysis ,43,44. TOur study has certain limitations that need to be considered. Firstly, our analysis was based solely on structured data (patient and treatment dose features), which may not fully capture the complexity of bladder toxicity. Recent studies have demonstrated the potential of more sophisticated DL models that incorporate 3D dose distributions, medical images, contours, etc. to predict clinical outcomes ,46. RadiSecondly, although the DL model outperformed the statistical model in our dataset, it is important to note that the performance of DL models can vary depending on the specific dataset and problem domain. Further validation using larger and more diverse datasets through multi-institutional studies is required to confirm the generalizability of our findings.Thirdly, interpreting DL models remains a challenging task, especially concerning feature importance. Permutation feature analysis is a useful technique for assessing the importance of features in DL models. However, it should be noted that this method has limitations. It tends to assign higher importance to continuous variables and can produce different feature rankings depending on the choice of evaluation metric. Additionally, applying this technique to 3D input is challenging. While permutation feature analysis provides valuable insights, it needs to be supplemented with other interpretive techniques to gain a more comprehensive understanding of the behavior and functional importance of DL models. Therefore, as a further study, it is necessary to apply various analysis methods, such as LIME and its variants, input gradient-based methods, CAM and its variants, etc., to the DL model to ensure the reliability of models with relatively higher performance ,48.Furthermore, in order to address the limitation of a small sample size, we established an extra-validation set consisting of 17 individuals. Utilizing a lightweight DL model, we conducted predictions for late bladder toxicity. The results revealed an accuracy of 0.81%, a precision of 0.99%, a recall of 0.61%, an F1-score of 0.92, and an AUROC of 0.93. These performance metrics indicate that the lightweight DL model had limited accuracy in forecasting late bladder toxicity. While the precision was high, indicating few false positives, the recall was relatively low, meaning it missed many true positive cases. It is crucial to exercise caution when interpreting these findings due to the relatively small size of the extra-validation set. The restricted sample may not adequately encompass the full range of late bladder toxicity in radiation therapy. For instance, the longest observed duration of late bladder toxicity in our institution was 107.7 months from the initiation of external beam radiation therapy (EBRT) to the occurrence of late toxicity. As a result, the performance metrics on the extra-validation set obtained may not truly reflect the predictive capabilities of the DL model. Thus, future research should strive to incorporate a larger patient cohort from multiple institutions to validate and enhance the predictive capabilities of DL models in accurately anticipating late bladder toxicity.Overall, this study contributes to the ongoing discussion on the clinical utility of DL models in predicting radiation toxicity and emphasizes the importance of interpretability to enhance the reliability and practical applicability of these models. By addressing the limitations and conducting further research, we can advance the field and ultimately improve patient outcomes in radiation therapy.2cc as the most important feature for risk prediction. In this study, we compared the performance of logistic regression and DL models for the prediction of late bladder toxicity in patients with cervical cancer. Logistic regression did not show acceptable performances, whereas the lightweight DL model achieved an accuracy of 0.92 ± 0.03 and an AUROC of 0.91 ± 0.03. Moreover, the permutation feature importance analysis identified BD


Non-motor symptoms (NMS) are an important and ubiquitous determinant of quality of life in Parkinson’s disease (PD). However, robust evidence for their treatment is still a major unmet need.This study aimed to provide an updated review on advances in pharmacological, non-pharmacological, and exercise-based interventions for NMS in PD, covering the period since the publication of the MDS Task Force Recommendations.st of January 2017 to the 1st of December 2021 and limited our search to randomised and non-randomised (including open-label) clinical trials.We performed a literature search to identify pharmacological, non-pharmacological, and exercise-based interventions for NMS in PD. As there are recent reviews on the subject, we have only included studies from the 1We discuss new strategies to manage NMS based on data that have become available since 2017, for instance, on the treatment of orthostatic hypotension with droxidopa, several dopaminergic treatment options for insomnia, and a range of non-pharmacological and exercise-based interventions for cognitive and neuropsychiatric symptoms, pain, and insomnia and excessive sleepiness.Recent evidence suggests that targeted non-pharmacological treatments, as well as some other NMS management options, may have a significant beneficial effect on the quality of life and need to be considered in the pathways of treatment of PD. In his 2st of January 2017 up to the 1st of December 2021. We have limited the review to (randomised) clinical trials and non-randomised (including open-label) studies where NMS have been measured by validated outcome measures, as outlined in section 3, and excluded case reports. To keep the search as broad as possible, we did not have a priori restrictions regarding the language in which a study was published.We performed literature searches in electronic databases, including Medline and Cochrane Library, and checked references of included manuscripts to identify further relevant studies. We have included all pharmacological, surgical, and non-pharmacological studies describing treatments for NMS in PwP, regardless of whether the NMS was a primary or secondary outcome in the study. As extensive previous reviews have been published on the topic of NMS treatment in PwP, in order to reduce overlap with previous reviews, we have only included studies from the 1As many studies described multiple non-motor outcomes with the same intervention, in order to describe these findings, we have in this review grouped NMS together based on the Non-Motor Symptoms Scale (NMSS) where possible . For eac31). These studies will be described in the below sections.Using the above strategy, we identified over 100 studies focusing on the treatment of cardiovascular and autonomic symptoms, sleep, excessive daytime sleepiness and fatigue, neuropsychiatric symptoms, and pain  and constipation are common, with overall prevalence rates ranging from 10% to 70% and 11 to 83%, respectively -20. OH ce.g., in a small RCT with 13 PwP where fludrocortisone showed a 37% improvement in diastolic blood pressure drop on the orthostatic challenge (level of evidence 2B), while pyridostigmine bromide had no effect on symptom severity related to orthostatic hypotension and quality of life with both interventions [et al. provided level 2B evidence showing that in 30 PwP with Hoehn and Yahr stages 2 and 3, resistance exercises decreased normalised low-frequency components of heart rate variability and reduced systolic blood pressure fall during orthostatic stress [Lower level evidence is available for other treatments, ventions . Finallyc stress .5.2Sialorrhoea, and subsequent dribbling of saliva, is a common and disabling NMS in PwP and affects 19-29% of newly diagnosed PD patients , 25. Dat1).Since 2017, several studies have confirmed the efficacy of salivary gland injections with botulinum toxin (BNT) for the treatment of sialorrhoea in PwP. Tiigimae and colleagues reported that in 12 PwP injected with BNT-A for treatment of sialorrhea, a significant decrease in salivary flow rate at 4 weeks was noted, and there was a significant increase in salivary levels of Lactobacilli, but not Streptococcus mutans . Two subAnother treatment with level 2B evidence, which has been investigated recently is oral dihydroergotoxine mesylate (2.5mg BID) reduced sialorrhoea scores, measured through the UPDRS sialorrhoea subscore and the Sialorrhea Clinical Scale for PD, in 20 PwP with a two-thirds response rate being defined as at least 30% reduction in sialorrhoea scores [5.3Constipation is one of the earliest symptoms of PD and is commonly present in the prodromal stage with two different subtypes: slow transit and outlet constipation . For theet al. [For the pharmacological treatment of other autonomic symptoms in PwP, such as sexual dysfunction and overactive bladder, there have been very few additions to those already described in the reviews by Seppi and colleagues  and Quaret al. . There het al. , 34. Foret al. .5.4Although we have identified a number of efficacious treatments for cardiovascular and autonomic symptoms in PwP, there is insufficient evidence for many interventions to make adequate conclusions for clinical use. Indeed, for several indications, further studies, preferably in the form of RCTs, are required. This is particularly true for gastrointestinal symptoms in PwP. On the other hand, level 1 evidence is available for the treatment of OH with Droxidopa and for sialorrhoea in the form of botulinum toxin injections to the salivary glands. Nonetheless, some treatments show promising results, even though currently insufficient evidence is available. This is, for example, the case with Opicapone for urinary and gastrointestinal symptoms and probiotics for constipation , 33. Mor6Sleep dysfunction in PwP is an umbrella term comprising multiple symptoms and conditions, ranging from excessive daytime sleepiness (EDS) and insomnia to fatigue. The prevalence of EDS in PD patients is 2-3 fold higher than in the general population, with controlled studies showing EDS in 34-54% of PD patients compared to 16-19% in healthy subjects , 38. In 3.An overview of currently investigated treatments for sleep and related problems in PwP is provided in Table 6.1Well-established pharmacological treatment for sleep disorders in PD with the most robust evidence is Rotigotine transdermal patch, with level 1A evidence and a strong recommendation for its use. Examples of recent studies include the PD0013 trial, in which, it significantly improved PDSS-2 scores with no worsening of ESS scores , 48. A mOther treatments interacting with dopaminergic neurotransmission, both with level 2B evidence, include Rasagiline and Opicapone. In a study with 30 patients (2:1 randomisation to treatment vs. placebo), significant improvements in sleep maintenance after eight weeks of Rasagiline treatment were observed using polysomnography which showed a decrease in wake time after sleep onset and number of arousals in addition to improvements in daytime sleepiness as measured by the Epworth Sleepiness Scale . The OPTCircadian interventions, such as melatonin given at four mg daily for eight weeks, on the other hand, were unable to improve REM sleep behaviour disorder in 30 PwP enrolled in a recent double-blind RCT . This se6.2Rutten and colleagues compared the effects of 10,000 lux bright light therapy and placebo (200 lux light) in 83 PwP in a double-blind RCT and reported no benefit on mood as measured by the Hamilton Depression Rating Scale, while subjective sleep quality  improved significantly, along with total salivary cortisol secretion . VidenovIn another RCT comparing acupuncture versus sham acupuncture in 40 PwP, both the general Fatigue score of the Multidimensional Fatigue Inventory after 5 weeks of treatment along with the Geriatrics Depression Scale and Epworth Sleepiness Scale ESS remained stable with no improvement . These f6.3et al. reported that, in 35 moderate to advanced PwP, an exercise programme comprising 30 minutes of conventional rehabilitation, including stretching, strengthening, and balance exercises, followed by 30 minutes of treadmill exercise five days a week for six weeks improved fatigue by up to 65-80%. In addition, pain improved by nearly 90% when measured by the Nottingham Health Profile [Since 2017, a few studies have been published on exercise-based treatments for fatigue in PwP. For example, eight weeks of yoga treatment in 15 PwP, compared to 12 PwP not receiving this intervention, improved Parkinson’s fatigue scale-16 scores . Atan et Profile . However6.43). On the other hand, options that might seem more intuitive, such as the use of Melatonin, including its use for RBD, are currently not supported by high-level evidence (Table 3). In relation to the latter, potential contributors should be identified and addressed, such as antidepressant medication, which has been associated with causing or worsening RBD [3). Nonetheless, for most explored options, either insufficient evidence is available or treatments seem inefficacious, whereas the use of BLT seems promising to treat EDS in PwP [In spite of some evidence for the treatment of sleep and related disorders in PwP, improving PD‐related sleep problems and daytime somnolence is usually complex due to the heterogeneity of their causes , 48, 59.ning RBD . For theS in PwP , 55.et al. showed that several small open-label studies had explored the potential utility of istradefylline in the management of non-motor symptoms, including sleep and mood disorders, although the evidence thus far is limited [Further developments are expected, although the field of sleep-related problems is currently receiving less attention than some other NMS. Some examples of currently ongoing studies include adenosine A2A receptors, most notably istradefylline, used for the treatment of motor fluctuations in PwP. Here, Jenner  limited . In addi limited , 63.74.Depression can affect between 11% and 45% of patients with PD -68. With7.14 and Fig. 1).Perhaps the most convincing evidence (level 1A) for the pharmacological treatment of depressive symptoms in PD is for Rotigotine. In a meta-analysis of 10 studies with 1,844 patients, compared to placebo, Rotigotine significantly improved apathy scores  and depressive symptoms (Beck Depression Inventory and the NMSS mood domain) . Similar4).In addition to this, studies have focused on antidepressant treatment in PwP, providing evidence ranging from level 3B to 2C. An example here includes a study involving 27 mildly to severely depressed PwP where both SSRIs  and duloxetine improved depression scores, measured by the Quick Inventory of Depressive Symptomatology scale . ExenatiOne of the better studied pharmacological interventions for neuropsychiatric symptoms, with a level of evidence 2A, is pimavanserin, a selective serotonin 2A receptor inverse agonist. In PwP with psychosis, an analysis of a 6‐week randomised, double‐blind, placebo‐controlled, phase 3 trial of pimavanserin 34mg  showed that in 185 PwP, it was well-tolerated and showed an improvement in PD‐adapted Scale for the Assessment of Positive Symptoms scores. Changes were most pronounced in those with additional cognitive impairment . The ope7.2Since 2017, three studies have focused on the effects of transcranial stimulation (TMS) in PwP, providing level 2B evidence for its use; the results, however, have been varying. One study showed that transcranial Direct Current Stimulation (tDCS) over the left dorsolateral prefrontal cortex in 22 PwP who received tDCS and computerised cognitive training for two weeks significantly reduced depressive symptoms and improved cognitive performances . Another4 and Fig. 1).Other treatments, with the level of evidence ranging between levels 3B and 2A, including cognitive behavioural therapy (CBT) and implementation intention training, have been investigated for use in PwP. Compared to verbal rehearsal, CBT improved self-reported depressive symptoms, but not anxiety, following a 10-week intervention period. In addition, the carer symptom burden was also reduced . In a la4).Several studies have also reported non-effective interventions for depression, anxiety, and cognitive performance. Since 2017, these include studies on bright light therapy with 10,000 lux light , acupunc7.3et al. were unable to demonstrate the effectiveness of resistance training in 33 PwP on depressive symptoms  [Over the last few years, multiple exercise-based interventions for neuropsychiatric symptoms have been explored in PwP, but not all were effective, such as a 12-week RCT of 60 minutes improvisation theatre sessions for anxiety and depression in 12 PwP . Also, d scores) .1 and Fig. 1).On the other hand, agility exergaming and stationary cycling showed beneficial effects (level of evidence 3B) in an RCT with improved Beck Depression Inventory scores . Moreove7.44. The best evidence for treating depressive symptoms in PwP is for Rotigotine, Pramipexole, and Venlafaxine (Table 4). This would suggest that optimising dopaminergic treatment in PwP could be the first step when trying to address depressive symptoms. In fact, part of the depressive symptoms might stem from non-motor fluctuations, which often go unrecognised [e.g., cognitive behavioural training, various forms of dance therapy, and transcranial magnetic stimulation (Table 4). For apathy, less supporting evidence is available, and only in the form of pharmacological treatments, but especially Rotigotine appears efficacious [4).The neuropsychiatric symptoms in PwP are complex, and their underlying pathophysiology is heterogeneous, including dopaminergic, serotonergic, and noradrenergic systems . Nonethecognised -111. Evicognised  improvescognised , 114. Foicacious   studies on PwP with dementia had been published, with one study showing a positive signal for rivastigmine and another with equivocal results for donepezil [For treatment of cognitive dysfunction and related problems, much of the available evidence had already been reviewed elsewhere. For example, Seppi onepezil . Moreoveonepezil , 85 and onepezil , 102 seeFor psychosis, little has changed in terms of treatment advice over the last few years. The best way forward seems to consider dose reductions of dopaminergic drugs to a level that would lead to a resolution of psychotic symptoms while maintaining sufficient symptomatic motor control first . Unfortu88.1et al. showed in a post-hoc analysis of pooled data from 352 PwP enrolled in two randomised trials that Safinamide, as add-on therapy in moderate to advanced PwP with stable doses of levodopa and motor fluctuations, after 24 weeks of treatment induced a reduction of the number of concomitant pain treatments of 23.6% compared to those receiving placebo (evidence level 1A). In addition, the PDQ-39 items related to musculoskeletal and neuropathic pain, as well as bodily discomfort, improved significantly [Among the recently explored non-oral pharmacological treatments for pain in PwP, Botulinum toxin A injections reduced numeric rating scale pain scores after 4 and 12 weeks of treatment, although not superior to placebo, perhaps related to the small number of patients (12) included in the study and the insufficiently enriched study population (evidence level 2B) . On the ficantly . These eficantly . Similarficantly .In a group of 93 PwP, of whom 70 completed the study, the introduction of Rotigotine improved the four 'affective dimension' items of the 12-item Pain Description List of the German Pain Questionnaire by 30% after at least 25 days of treatment. The study was, however, non-interventional, and the introduction of Rotigotine was based on standard clinical practice . Also, R8.2st of January 2017, necessitating further dedicated research on this topic.Aquatic therapy (‘aquatic Ai Chi’) in twice-weekly sessions for 10 weeks, compared to exercise on dry land, was able to improve VAS scores for pain from 5.5 to 4.0 in 15 PwP, with no change in those doing exercise on dry land (5.8 pre-intervention and 5.3 post-intervention) . This wa8.3et al., who summarised that, to date, only three drugs  had been investigated specifically for their analgesic effects in PwP in double-blind RCT setting [https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001175-32/GB), and the CAN-PDP study evaluating the effects of cannabidiol (https://www.parkinsons.org.uk/research/can-pdp-cannabidiol-parkinsons-psychosis-0).Treatments for pain in PwP have been very recently reviewed by Rukavina  setting . None of setting , 127. In setting , 128. Fu9e.g., in the Manage-PD tool [2.The therapeutic effects of the device-aided therapies, including deep brain stimulation (DBS), apomorphine subcutaneous infusion (Apo), and intrajejunal levodopa infusion (IJLI), will be discussed separately due to their indications for use, limiting them to the more advanced stages of PD. In addition, the initiation of device-aided therapies is still determined by motor symptoms, especially uncontrollable motor fluctuations -131, alt-PD tool , 132-134-PD tool . An over9.1et al. showed that after 36 months of DBS-STN in 87 PwP, compared to 84 PwP on best medical treatment, significantly improved NMSS total scores with specific improvements in sleep/fatigue, urinary and miscellaneous NMSS domains [1).Several studies have shown an overall NMS burden improvement. Recently, Jost  domains . Overall domains . Similar domains . In anot domains  et al. who showed that mean bladder volumes at desire and urge point to void seemed to improve, as well as the quality of life associated with recurrent urinary tract infection, while urinary retention and incontinence seemed to increase [Non-motor areas that have been specifically addressed in recent reviews and meta-analyses include mood, with an overall improvement in depressive and anxiety symptoms after STN-DBS , 139, anincrease . Nonetheincrease , 145. Fiincrease .9.2et al. [Prakash and Simuni  have recet al. . From thet al. , 132. Aset al. , 149.9.3et al., demonstrating improvements in six of nine NMSS domains [https://clinicaltrials.gov/ct2/show/NCT02549092). As such, further improvements might be achieved by adding COMT-inhibitors to IJLI [The non-motor effects of IJLI have been recently reviewed by Prakash and Simuni  and by A domains . However to IJLI , and suc to IJLI .9.4Device-aided therapies are now established worldwide for the management of advanced Parkinson’s disease, but the emphasis remains on the motor profile of PwP, even though NMS have also been shown to play a part in the prognostic aspects of the successful delivery of these therapies . ConsideThe present review summarises the available randomised and non-randomised clinical trials based on evidence published since 2017 regarding the treatment of NMS in PwP, including pharmacological, non-pharmacological, and exercise-based trials. Although we have been able to identify a number of efficacious treatments, most treatments and treatment strategies described here do not exceed level 2 evidence. Therefore, it is unlikely that such treatments will be widely used in clinical practice at this moment in time. Nonetheless, it is encouraging to see that NMS are being increasingly used as an endpoint in studies and not just as a secondary outcome measure but primary in some cases with the increasing use of NMSS, a robust, validated outcome tool since 2007 . A furth1). New molecules in pharmacological treatments for NMS in PwP, although further evidence for their usefulness is needed, include Opicapone, Safinamide, Exenatide, and possibly Istradefyline [Compared to previous reviews and meta-analyses on the topic of NMS treatment in PwP, we have not been able to identify many novel pharmacological candidates. Many of the pharmacological treatments described in the current review had already been described up to 2017 , but thedefyline , 119.


Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite The online version contains supplementary material available at 10.1007/s10096-023-04597-7. Toxoplasma gondii is a zoonotic protozoan parasite that uses domestic cats and other felids as definitive hosts and causes clinical disease in both humans and animals ) was included  and two archetypal strains (n = 2), provided in duplicate  or one sample (second and third parts) of bovine carrier DNA alone was included as negative controls. The trial was blinded for all participants, including the organizing laboratory (E); only in the third part, the operators knew about the non-archetypal nature of some of the isolates included, but were unaware of the identity and order of the samples.For each part, Irrespective of laboratory-specific protocols, each laboratory was asked to use 5 μL template DNA per reaction in the multiplex typing PCR.After completing each part, interlaboratory divergences were assessed and discussed among the participants. All laboratories tried to improve their protocols and procedures for the subsequent part. The aim was to improve the individual typing results of each of the participating laboratories and to harmonize MS typing by using and extending the internal guidelines of laboratory B.A questionnaire was distributed to collate the technical and methodological details in each laboratory. During online meetings, further details on individual protocols, such as use of particular rules to analyze sequencing profiles, were recorded.https://cran.r-project.org/) was used for linear regression and specifically, the R packages “binom,” “ggpubr,” “ggplot2,” and “cowplot” for calculating confidence intervals and preparing graphical representations of the results.After reception of the data, all results were computed in tables for each part and studied separately  had replaced NEDFl with TAMRAFl, and two laboratories  had replaced NEDFl with Atto550Fl, for three or two of the MS marker regions, respectively  to 100% (laboratory B) . The proportion of correct identifications of the canonical types I, II, and III and a type II × III recombinant decreased depending on the dilution of the samples. If a participant had added a question mark to the result or provided an ambiguous typing result, NA was recorded. There were no differences in the typing results between the two software tools used by laboratory A and the two operators from laboratory E.T. gondii DNA, but also the proportion of undetermined types, i.e., from 18% (5/28) or 14% (4/28) for the 1st and 2nd dilution to 41% (23/56), 62% (69/112), or 88% (98/112) for the 3rd, 4th, and 5th dilutions, respectively  and 75% (21/28) of the results provided by all participants were correct. At the two following concentrations (3rd and 4th dilution), 52% (29/56) and 32% (36/112) of the typing outcome reported were correct. In contrast, the 5th dilution analysis revealed no (0/112) correct identification  were often recorded. In 27 cases, minimum or maximum values exceeding 2 bp were observed Table . Most 66.Table 6Fl instead of HEXFl to label fragments  of the major differences  occurred in particular markers, for which participants employed primers labeled with different fluorophores compared to reference laboratory B. In lineage typing, major differences were only observed between laboratory A and the reference laboratory . In the affected marker regions, laboratory A had used VIC/23 of thn = 17), the laboratory had used an alternative to the originally reported fluorophore, i.e., HEXFl was replaced with VICFl, NEDFl with Atto550Fl, or NEDFl with TAMRAFl  can be used to differentiate strains within the same lineage. In the second part of the ring trial, laboratories B and E reported identical fingerprinting results in all regions of the duplicates of 10 different strains, i.e., on 70 occasions . While laboratory A reported non-existing differences in two (3%), laboratories C and D reported non-existing differences in seven (10%) or 18 (25%) of 70 occasions, respectively . The results for one marker (IV.1) were not available for analysis in the case of one isolate (FRENCHGUIANA15) in laboratories A, C, and E, because they failed to amplify this marker  fragments were 2 bp longer and Atto550Fl (ME) fragments were 4 bp longer than reference NEDFl fragments.The Atto550ts Table . In the T. gondii strains is important to study the global population structure of the parasite. Genomic diversity of T. gondii may influence the epidemiology of the parasite, affecting, for example, definitive host and intermediate host adaptation [T. gondii genotypes are reported to have a higher virulence for particular hosts than other genotypes [Typing of aptation , 33, 34.enotypes , 36. Sucenotypes , 38.T. gondii strains at the level of lineages globally [Multilocus MS typing was established more than one decade ago  and has globally , 15, 39 globally , 40. EssT. gondii DNA prior to typing, different fluorophores  onwards, the proportion of samples increased, in which laboratories were no longer able to determine the lineage type. However, the proportion of reporting incorrect lineage typing results did not increase with decreasing DNA content. The results were consistent among the participating laboratories and relative to the results of the reference laboratory only up to dilution 2 (0.1 ng/μL T. gondii DNA). Thus, it seems to be important to estimate the level of T. gondii DNA concentration in samples prior to MS typing, and to use this information to select those samples, for which lineage typing (and subtyping) is most likely possible, or to optimize DNA concentration for typing  than in dilution 2 (0.1 ng/μL T. gondii DNA) samples. It has been noted previously that an overrepresentation of target DNA may cause so-called minus-A peaks during capillary electrophoresis [It should be noted that not only a limited concentration of DNA may negatively influence the accuracy of determining the correct fragment size, but also an excess of phoresis . Minus-AFl and TAMRAFl, are rhodamine dyes, while NEDFl belongs to the fluorescein dyes. The previous study reported that TAMRAFl-labeled fragments tended to be larger than NEDFl-labeled fragments and that this effect depended on the fragment size, i.e., the smaller the fragment, the stronger the retardation in capillary electrophoresis of TAMRAFl relative to NEDFl-labeled fragments [Fl in the original protocol. The strongest effects of a 4–5-bp retardation in Atto550Fl relative to NEDFl-labeled fragments were observed in the smallest fragment N60 (149–151 bp) and a 2-bp retardation in the larger fragments M102 (168–192 bp) and AA (265–267 bp). TAMRAFl-labeled fragments also appeared to be 2 bp larger relative to NEDFl-labeled fragments, but size-dependent differences could not be determined.Major differences, mainly affecting fingerprinting markers, were observed in results reported in all parts of the ring trial, especially among those laboratories that used different fluorophores for labeling forward primers Tables , and 11.ragments . ResultsFl and Atto550Fl, VICFl (used in laboratory A instead of HEXFl) belongs to the fluorescein-like dyes, similar to HEXFl, so no effects on fragment size were expected. This was confirmed in our analysis.In contrast to TAMRAFl-labeled primer pairs, used to amplify N60, M102, or AA, had been purchased from the company ME and not from the companies AB or EU  obtained results that differed in determined fragment sizes relative to results provided by the reference laboratory by up to 28 bp Table , includiory Fig. .So-called stutter peaks are frequently reported in MS typing .In conclusion, the results of this interlaboratory ring trial suggest that harmonization of MS typing appears to be possible, which might allow the combination of larger data sets on ESM 1(PDF 1.44 MB)ESM 2  (XLSX 87.2 KB)


Canine morbillivirus, also known as canine distemper virus (CDV), is the causative agent of canine distemper (CD), which is a serious contagious disease of canines, large felids, and, occasionally, raccoons. This study included seven raccoons from the Timisoara Zoological Garden, Romania. CDV was detected using RT-qPCR on blood samples, but several other exams were also performed—clinical, bacteriological, immunohistochemistry (IHC) and histopathology, toxicological screening, and necropsy—which confirmed CDV infection. Severe digestive disorders (diarrhea and frequent hematemesis) were observed. The necropsy findings included pseudo membranous gastroenteritis, congestion, and pulmonary edema in two raccoons. Immunohistochemistry showed immunolabeled CDV antigenantibodies on the viral nucleocapsid. Histopathology revealed lymphocyte depletion in mesenteric lymphnodes and intranuclear and intracytoplasmic inclusions in the enterocytes of the small intestine. Based on the RT-qPCR assay, laboratory tests, and the lesions observed, it was established that the raccoons were infected with CDV, which was the cause of death in two cases. The results from the necropsy, histology, and immunohistochemistry in the raccoons are comparable with reported CDV lesions in dogs. In conclusion, several exams may be performed to establish the etiology of possible interspecific viral infection, but only very specific exams can identify aCDV infection. Laboratory analyses must be completed by RT-qPCR assay or IHC to establish infection with uncommon viruses in raccoons with high accuracy. Procyonidae, which includes 18 species. Procyon lotor lives in a large part of North America, from southern Canada to most of continental North and Central America [Procyon lotor), a species that might play a role in the epidemiology of Morbillivirus canis, known as canine distemper virus (CDV) in domestic dogs [Raccoons belong to the family tic dogs . In EuroMorbillivirus. It was first isolated by Carré in 1905 [Canidae [Felidae, Mustelidae [Procyonidae, Ursidae, Viverridae, and Hyaenidae. The infection has also been described in captive and free-ranging large felids [Primates [Phocidae [Artiodactyla, and Proboscidea. The epidemiology of CDV in raccoons has been described by many authors in recent decades [The infection commonly known as CD is caused by an enveloped single-stranded RNA virus of the genus  in 1905  and rema in 1905 . It affe in 1905 ,7,8,9. C in 1905  as evide[Canidae ,6,11, Festelidae , Procyone felids , PrimatePrimates , PhocidaPhocidae ,15, Arti decades ,17,18 anThe primary sources of CDV are infected and carrier animals, which shed large amounts of the virus in all secretions and excretions starting as early as day 5 after infection, before the onset of clinical signs ,21,22. VCD is highly contagious and probably transmitted mainly through aerosols . Direct The incubation period ranges from 3 to 6 days. The virus initially infects lymphoid tissue (monocytes) in the upper respiratory tract and tonsils, subsequently being disseminated via the lymphatics and blood to the entire reticuloendothelial system. In the second stage of cell-associated viremia and fever (8 to 9 days after infection), when CDV infects the cells of the respiratory tract, gastrointestinal tract, central nervous system, urinary tract, and skin, as well as white blood cells .The risks are lower if the infection is diagnosed early and if the immune status of an animal is in good condition. The only way to prevent infection in dogs and non-domestic captive hosts is to vaccinate them, according to routine practice ,24,25. MThis study was performed to establish the cause of infection in seven raccoons from Timisoara Zoological Garden, Romania, and the cause of death in two raccoons. Ancillary laboratory tests and necropsies were performed to verify the hypothesis that infection and death were caused by a possible interspecific infection with CDV.The study started upon identifying the first clinical signs and included the following: necropsy, laboratory analyses  to identify the etiological agent. Other exams would follow upon approval of Ethical Statement no. 73/2020; however, the animals had meanwhile been displaced from the Zoo.The seven raccoons from the Timisoara Zoological Garden, Romania lived in a separate area, isolated from other domestic or wild animals while being visible to visitors. The Zoo veterinarian reported that two out of seven raccoons, aged 7 months, showed clinical signs of digestive system disorders, characterized by severe diarrhea and hematemesis, which are common in CDV infection, but also in other microbiological infections or other causes. No clinical signs were detected in five of the animals. None of the raccoons had been vaccinated against CDV, as the infection had not been reported in zoological parks either in raccoons or in other susceptible species in this type of habitat. The blood samples were collected in EDTA vacuum blood collection tubes at the beginning of antibiotic treatment and transported to the laboratory for screening in an ice box at approx. 4 °C. Although the veterinarian isolated the animals in individual cages and attempted treatment with Enrofloxacin (5 mg/kg body weight) and lactated Ringer solution for rehydration (3 mL/kg and hour) twice a day, two raccoons died 2 and 3 days after the onset of clinical signs.CDV was detected in the raccoons by RT-qPCR assay. Total genome extraction was performed from blood samples using a QIAamp Viral RNA Kit  following the manufacturer’s data sheet protocol. Viral antigen was detected using the IVD virus detection kit for veterinary practice One-Step Distemper . The qualitative real-time PCR assay was performed using an Mx3005P real-time PCR probe-based technology , according to the data sheet protocol. Each sample was analyzed in triplicate: a negative control, a positive control, and an internal reaction control were used for each run. The results were interpreted following the kit producer’s data sheet protocol according to Ct values, as follows: a value lower than 11 and higher than 40 was considered negative, Ct values between 12 and 35 were considered positive, and values higher than 35 and lower than 40 were considered inconclusive.The raccoon cadavers were transported under biosecure conditions in sealed plastic bags from the Timisoara Zoo to the Laboratory of Pathological Anatomy within the Faculty of Veterinary Medicine in Timisoara to establish the cause of death. The necropsy was performed according to mammalian autopsy protocol after skinning the carcasses, opening the thoracic and abdominal cavities, and examining the tissues and organs, looking for macroscopically visible lesions.Since the toxicological examination required a short turnaround time, it preceded the PCR test. The purpose of this rapid intervention was to ensure the survival of the remaining raccoons, in case intoxication/poisoning was the cause of the clinical signs and deaths. To exclude the possibility of intoxication/poisoning, prior to obtaining the results from RT-qPCR, and after the necropsy, the liver samples were analyzed to identify the main anticoagulant raticides on the Romanian market . The technique used for this purpose involved high-performance liquid chromatography (HPLC). An Agilent HPLC 1100 was used to analyze the anticoagulant rodenticides with a C18 analytical column, 300 mm × 4.5 mm, 5 µm particle size, and a 5 µm guard column. The analysis was performed using 50 mM of sodium acetate (pH 6.5) as solvent A and methanol as solvent B at a constant flow rate of 1 mL/min in a gradient run. Linear gradient elution was used by means of solvent B from 30% to 65% over 0–17 min, followed by linear gradient elution of solvent B from 65% to 90% over 17–24 min. The column was washed with 90% solvent B over 5 min, followed by 4 min equilibration to the initial conditions. The fluorescence detector was set to 318 nm excitation and 400 nm emission, and the excitation spectrum was recorded at 240–360 nm. The injection volume was 30 µL and the column temperature was adjusted to 25 °C.E. coli bacterial infections, samples from the small intestine , liver, and lymph nodes were analyzed to isolate the bacterial strains. The samples were incubated in nutrient broth for 12 h at 37 °C. Subsequently, the primary cultures were cultured on TBX medium  and EMB agar . The colonies showing characteristic figures on TBX and EMB agar were subjected to biochemical tests  for confirmation. The hemolytic activity was evaluated on Columbia agar. Biofilm formation as a virulence factor was detected by the Congo red agar method.To exclude Candida spp.Sabouraud glucose agar gel  was used for mycological examination with chloramphenicol and gentamicin (50 mg of each antibiotic/1000 mL). Inoculation was performed in aerobic conditions for 48 h at 37 °C. Any grown fungal colony was subjected to methylene blue staining and lactophenol cotton blue staining. The morphological characteristics of the isolated strains on smears stained with methylene blue revealed the presence of For the histopathology, samples were collected from the lung and intestine to highlight the inclusions produced by the virus. The techniques applied in staining intestinal and lung samples included fixation , embedding , sectioning (6 µm with the Slee Mainz microtome), and hematoxylin and eosin staining (H&E stain). Tissue was collected from each intestinal portion for the tissue processing protocol. Each sample was fixed in 4% paraformaldehyde for 24 h, after which the samples were washed in tap water and kept in successively increasing volumes of alcohol solutions. The samples were then embedded in paraffin blocks, which were sectioned using a (4 µm thick) microtome and placed on glass slides. The samples were subjected to paraffin removal, rehydration, washing, neutralizing endogenous peroxidase, protein block incubation, washing with TBS 1, and the addition of a conjugate consisting of the primary antibody coupled with peroxidase in a dilution of 1:100 for each sample. After being kept for 12 h in trays with water in the refrigerator, the slides were subjected to the following: washing with TBS 1, incubation with post-primary antibody, washing with TBS 1 and incubation with Novolink polymer containing the primary antibody for 30 min, rinsing with TBS 1, treatment with 3,3′-Diaminobenzidine for 5 min, and washing with distilled water. Subsequently, hematoxylin staining of the nuclei was performed for 40 s. Finally, the slides were washed with distilled water (two successive water baths for 5 min), followed by afinal washing of the slides with Unyhol, Unyhol Plus, and BioClear.Immunohistochemistry enabled the fixation of the primary antibodies coupled with peroxidase by the viral nucleocapsid. For this purpose, portions of intestines with macroscopic lesions were collected from the carcasses. The paraffin-embedded sections were deparaffinized, rehydrated, and incubated with the primary antibody  overnight at 4 °C. Primary antibody labeling was performed using a polymer detection system  and DAB  as a chromogenic substrate. Hematoxylin staining was also performed prior to dehydration and mounting. For the negative control, the primary antibody was substituted. Images were acquired with aCX 41 microscope with a 3MP CMOS digital color camera (Olympus).g for 20 min at +40 °C. An amount of 50 µL of suspension was taken from the resulting supernatant, covered with 150-mesh electrolytic grids (copper mesh grids) with a double membrane (formvar and carbon) for 1–2 min. The grids were contrasted with 2% uranyl acetate in distilled water, followed by an examination by electron microscope. The lung and intestine segments collected were fixed for 30 min in cold water in PBS, with 2.5% glutaraldehyde. Subsequently, the parts were post-fixed in osmium tetroxide (OsO4) solution, dehydrated by successive passages in ethyl alcohol baths in increasing concentrations, followed by propylene oxide clarification, and included in Epon 812. Ultrafine preparations sectioned by LKB III ultramicrotome were placed on electrolytic grids and double contrasted in Reynolds solution. The transmission electron microscopy (TEM) investigation was performed with a Hitachi-7100 microscope  at 75 kV. Viral particles were visualized in a suspension (sample) according to their specific adsorption on the surface of a double-membrane electrolytic network (formvar and carbon), followed by fixation, washing, and negative staining  by transmission electron microscopy. For the electron microscopy (EM-DNSM) by direct negative staining, lung and intestine segments were placed in a suspension of quartz sand  and phosphate-buffered saline  in sterile conditions. The suspension was harvested and subjected to clarification by centrifugation at 400× The five raccoons did not show any clinical signs, suggesting that the infection hadnot affected all the animals. After treatment, both dead raccoons displaying clinical signs were in good body condition  before dFollowing analysis by RT-qPCR ,31, the E. coli. An examination of the small intestine revealed nematodes of the genus Ascaris spp. Most probably, the death was caused by hypovolemic shock.Gross pathology was characterized by hemorrhagic gastroenteritis a, pulmon2 = 0.997), the limits of detection (LOD) and limits of quantification (LOQ) obtained from blank samples spiked with anticoagulant rodenticides were LOD = 0.003 mg/kg and LOQ = 0.010 mg/kg; with difenacoum, brodifacoum, and bromadiolone, LOD = 0.010 mg/kg and LOQ = 0.030 mg/kg with warfarin. No detectable anticoagulant rodenticides were found in samples over the LODs, and thus intoxication/poisoning was excluded.The HPLC results were negative. For the linearity assessment parameters  or abiotic fomites . This may be the case, given that the zoo is located in a wooded area on the outskirts of the city of Timisoara, and many stray dogs can reach the area; . Unvaccinated dogs have a high prevalence of CDV infection (according to unpublished data from a retrospective unpublished study) and they may be sources of infection. Moreover, the raccoons in our study could have come into contact with animal lovers  who later entered the zoo and came into contact with them. One limitation of the study is due to several factors pertaining to the specificity of the research conducted. The intention was to later take other samples from the raccoons that survived to detect CDV-specific antibodies after convalescence, but while waiting for the Ethical Statement to be issued, the zoo in Timisoara, Romania was closed down by the local authorities, and the animals were relocated. Therefore, the RT-qPCR data reported in The clinical signs of CD vary dramatically and are highly dependent on the virus strain, age, and immune status of the host. A serological survey has proved that many free-ranging carnivores, puppies, and dogs experience subclinical infection, followed by death . The virThere is no specific treatment to cure this infection, but there are methods of treating clinical signs. Depending on the severity of the condition and how advanced the infection is, IV fluid infusions are recommended if the animal is dehydrated and antimicrobial drugs are recommended for secondary bacterial pneumonia, as well as oxygen supplementation and nebulization . The resCDV in raccoons may be tested with nasal swabs, as well as PCR on tissue samples, as it is done in dogs. Based on our experience with PCR analysis, an ideal sample type varies based on clinical signs. A PCR exam is recommended after observing the following clinical signs: gastrointestinal signs (whole blood and/or feces), respiratory signs , or neurologic signs . RT-qPCR can be used to detect CD in raccoons, as it is a high-sensitivity (98.9%) and high-specificity (100%) method used in CDV diagnosis in dogs ,31,38. DAscaris spp., which may explain the eosinophils detected by the histopathology.The presence of bacteria and fungi has also been reported as secondary bacterial infections ,40. As iThe histopathology characteristic of CDV infection, namely intracytoplasmic and intranuclear inclusions, correspond to those mentioned by other researchers in raccoons  as well As stated in the literature, during the evolution of CDV, immunity played an important role in triggering the infection and causing its severe evolution. Due to the high morbidity and mortality rates and broad host range , understanding the epidemiology of CDV is important for its control in both domestic animals and wildlife ,52,53. FAlthough CDV is not believed to cause disease in humans, two decades ago, it played a controversial role in Paget’s bone disease . CDV RNAAs seen above, the clinical signs , as well as the histopathology (including immunohistochemistry) and electron microscopy images obtained in raccoons are comparable with observations reported in dogs ,48. BaseIn conclusion, zoological parks may help reduce CVD outbreaks by minimizing contact with infected animals, prohibitingvisitors’ pets, controlling wild host animals, and vaccinating captive animals , which m