Proteobacteria, followed by Actinobacteria, Acidobacteria, and Chloroflexi, while the most abundant bacterial species was Koribacteraceae: NA_01, followed by Cyanobacteria: NA. A network analysis showed that Koribacteracea: NA_01 positively correlated with bacterial groups, including Flavisolibacter sp., Oxalobacteraceae: NA, and Actinomycetales: NA_01. Based on Shannon and Simpson’s diversity indices, the diversity of bacteria was significantly less in the Huangzuishan area than in the Lengshuikeng and Zhuzihu areas. Bacterial assemblages also significantly differed (P<0.05) among the three sites. The present results provide clear evidence to show that environmental variables, including heavy metals, are key factors affecting the bacterial community structure in volcanic soil.Recent studies suggested the presence of magma chambers from the Tatun volcano group under northern Taiwan’s surface, the result of episodic volcanism for 0.2–2.8 million years. However, the microbial community in volcanic soil has not yet been characterized. Therefore, the present study investigated the spatial distribution of microbial communities and their relationships with environmental variables, including heavy metals. Next-generation sequencing was used to analyze the microbial community structures in three areas with different land uses: Lengshuikeng , Zhuzihu , and Huangzuishan (conservation area). High contents of environmental factors, such as nitrogen (0.46–1.14%) and phosphorus (2.01–13.88 ppm), were detected. Large concentrations of heavy metals, such as copper (55.90–127.60 ppm) and zinc (36.13–147.73 ppm), were found among the three sites, whereas those of lead (83.13 ppm) and chromium (48.33 ppm) were higher in the Zhuzihu area. The most prevalent phylum across all sites was  The Tatun volcano group in Yangmingshan National Park, located in northern Taiwan, plays a crucial role in protecting nature and wildlife. It ranges in elevation from 200 to 1,200 meters above sea level and contains many different subtropical and warm climate zones. Underground movement caused massive collisions millions of years ago between the Philippine oceanic plate and the Eurasian continental plate. This event resulted in intense volcanic activity and lifted the Eurasian plate. Hot magma reaching 1,000°C gushed from the erupting volcano and covered tertiary sedimentary rock to form the 20 or more volcanoes that make up the volcano cluster in Taiwan’s northern coastal region. Over thousands of years, volcanic soil, which is formed by the weathering of volcanic materials , Lengshuikeng (LSK), and Zhuzihu (ZIH)—were selected. The soil at each site is classified as volcanic soil. Each site has a specific land use: HZS is associated with a conservation area, ZIH has small-scale agriculture activity, including the planting of vegetables, and LSK has walking/hiking trails for tourists. LSK and ZIH are both used for recreation. The chemical characteristics of volcanic soil, including heavy metals, were investigated to elucidate their influence on soil bacterial communities. The present study assumed that environmental factors and heavy metals significantly altered the bacterial community structure and diversity in volcanic soil in the Tatun volcano group.We herein investigated the spatial distribution of microbial communities in Tatun volcanic soil. Volcanic soil 2. Each replicate was randomly selected, with 5 m of space given between each replicate. The three areas generally have similar weather conditions. LSK and ZIH: partly cloudy, temperature: 25/32°C, wind: 16 km h–1 SE, annual rainfall: 0.8 mm, humidity: 73%, cloud cover: 28%, pressure: 1,009 mbar; HZS is similar, but with less rainfall than the other two sites. Vegetation groups in Yangmingshan National Park are divided into mountain ridge grasslands, temperate evergreen broadleaf forests, and subtropical monsoon rain forests. The national park contains 1,360 vascular bundle plants species, including dark-spotted cherry (Mori cleyera), Taiwan cherry (Prunus campanulata), formosan sweet gum (Liquidambar formosana), Large-leaved Machilus (Machilus kusanoi), and Red nanmu (Machilus thunbergii). LSK is a popular resting place for hiking or touring the national park. ZIH is a typical rural settlement in which clouds and fog often cover the mountain. HZS is a conservation area. Buffaloes often graze around a small pond in the valley of HZS. The total area of Yangmingshan National Park is ~11,455 hectares.Sampling was conducted in the Tatun volcano group  and cove® 50-mL tubes, placed in liquid N for several s, and then transferred and stored on dried ice until their arrival at the laboratory and storage at –20°C. The remaining soil samples were air-dried for subsequent soil chemical analyses.Soil samples were randomly collected from a depth of 0–10 cm with at least three replicates. In LSK, volcanic soil samples were collected from four locations around a service station. Some of the individual soil samples for the microbial analysis were collected in sterilized Falcon–1) bovine serum albumin (BSA), 0.2 M each primer, and 2–5 μg template to a total volume of 50 μL. One hundred nanograms of diluted DNA was loaded into each well to promote amplification. The PCR program consisted of initial denaturation at 95°C for 3 min, followed by 30 cycles at 95°C for 20 s, 52°C for 10 s, and 72°C for 45 s, and then at 72°C for 5 min, with cooling at 4°C. A 1% agarose gel with 1× TAE buffer and the PCR products was examined by SYBR® Green I. The QIAEX II Gel Extraction Kit (QIAGEN) was used to purify target DNA from the gel fragment (approximately 415 bp in size). A NanoDrop spectrophotometer (Thermo Scientific) was used to check DNA quality, purity, and concentrations. Each tag was added to the 5′ ends of the 968F/1391R primer for each sample in the following PCR process. The PCR mixture contained 25 μL Taq Master Mix (MD Bio), 2.5 μL (20 mg mL–1) BSA, 0.4 M each of the tagged primers, and 100 ng V6–V8 amplicon to a final volume of 50 μL. The PCR program for tag addition consisted of an initial denaturation at 95°C for 3 min, followed by 20 cycles at 95°C for 20 s, 52°C for 10 s, and 72°C for 30 s, with a final step at 72°C for 2 min and then cooling at 10°C. The PCR product was purified and 200 ng of the mixture from the tagged V6–V8 region was subjected to the Illumina MiSeq 2000 sequencing system (San Diego) at Genomics (Taipei).The universal primer pair 968F/1391R was used to amplify the V6–V8 region in the 16S rRNA gene and generate an amplicon library. PCR reagents consisted of 25 μL Taq Master Mix (MD Bio), 2.5 μL (20 mg mL2+), magnesium (Mg2+), potassium (K+), and sodium (Na+). In this case, these exchangeable cations are the total number of cations in soil that are available for direct uptake by microorganisms. Soil exchangeable cations  were assessed by atomic absorption spectrophotometry (AAS) using previously reported procedures  (Exchangeable cations are positively charged ions that are loosely attached to the edge of clay particles or organic matter (OM) in soil . Cationsocedures . Volcani1 M HCl) . Availab1 M HCl) . Total s1 M HCl) , soil OM1 M HCl) , and soiDescriptive statistics were used to obtain a preliminary understanding of data, and a one-way ANOVA analysis was then performed to test various hypotheses (Tukey’s test). High-quality and effective sequences that met the requirements were used to elucidate the bacterial community structure. The original line obtained by sequencing for chimeras and removed from ambiguous bases (N), mismatched primers, incomplete barcodes, and overpasses were filtered. Sequences were shortened using the USEARCH (v8.0.1623) system’s built-in settings to merge double-ended rows into a single line, and MOTHUR (v1.35.1) was then used to test sequence quality, keeping sequences with (1) base pair length of 400–450 bp, (2) a homopolymer not exceeding 8 bp, (3) no ambiguous bases, and (4) an average quality score greater than 20. UCHIME was used to detect and remove chimera in USEARCH software with a reference model and minimum ambiguity of 3%. Lines with 97% similarity were classified into operational taxonomic units (OTUs), showing a good representation of diversity. The UPARSE method was used to analyze OTUs  that werSoils were rich in heavy metals, such as As, Cr, Cu, Ni, Pb, and Zn . The consequences from LSK. An analysis of Shannon and Simpson’s diversity indices revealed that bacterial diversity was significantly lower in HZS than in the other areas. Soil in LSK had the highest Shannon diversity index value. The highest Simpson’s diversity index value (0.92) was found in ZIH .Proteobacteria was the most predominant phylum across all three sites, followed by Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes  among all sites. Koribacteraceae: NA_01 positively correlated with Flavisolibacter sp. (2.67%), Oxalobacteraceae: NA (1.49%), and Actinomycetales: NA_01 (1.44%); in contrast, Koribacteraceae: NA_01 negatively correlated with BD7-3 (1.52%) ,relative abundance in LSK was Thermogemmatisporaceae:which negatively correlated with  NA_01 (2.33%), Candidatus Solibacter sp. (2.12%), and Acidimicrobiales: NA (1.03%) (Sinobacteraceae: NA_02 (1.32%) Actinomycetales: NA_01 (1.23%); in contrast, Ellin6513_01and  negatively correlated with Gammaproteobacteria: NA  A. The hi (1.03%) B. The hieria: NA C.NMDS revealed a relationship between the soil bacterial community structure and environmental variables, including heavy metals, across the three sites . Heavy mVolcanism is a significant process of soil formation, providing a model for investigating the role of pioneer microbes. The scientific community has an interest in volcanic environments. The role of microbes in the formation of new soils, followed by the development of terrestrial ecosystems, is one of the main topics of soil biology and general ecology. The Tatun volcano group is considered to have last erupted approximately 20 ka ; it had P<0.05). The Shannon diversity index value was significantly higher in ZIH than in HZS (P<0.05). No significant differences were observed in Shannon diversity index values between ZIH and LSK (P>0.05). HZS had the lowest Shannon diversity index value  increase the stability of microbial communities under changing environments , which was in accordance with previous findings on a grassland ecosystem . The abundance of heavy metals, such as Zn and Cu, positively correlated between ZIH and HZS. Heavy metals, such as As, Cr, Cu, Ni, Pb, and Zn, are potential bioaccumulative toxins from agriculture  and Sinobacteraceae: NA_03 . The bacterial group Xanthomonadaceae is an indicator for agricultural soil . Cyanobacteria are photosynthetic organisms that easily survive on the minimum requirements for light, water, and carbon dioxide (CO2) (P<0.05), and the highest value of N was associated with HZS, which is a good indicator that HZS (the conservation area) remained a natural forest. Based on the results of NMDS, we suggest that one measurement (N) may be the best predictor of the success of a conservation process in the HZS conservation area.NMDS showed that P concentrations were associated with ZIH, similar to previous findings . Althougde (CO2) . NMDS shGemmatimonadetes showed low relative OTU proportions at the phylum level at the three locations. A previous study suggested that the abundance of Gemmatimonadetes was inversely related to soil moisture, and showed that its abundance inversely correlated with OM . Another study identified Actinomycetales as a heterotrophic denitrifying bacteria ; after thousands of years of development, the native soil became suitable for agriculture. HZS is a conservation area. In LSK, soil samples were collected on a grassland away from a hiking trail. Soil in the three locations has the same original rock source and becomes native soil over thousands of years of development; however, land uses differ. The three different sampling sites with different usages were selected for the present study to avoid human disturbance factors on the microbial community. Therefore, the shift in the microbial community originated from natural factors. However, other studies  Environmental Variables Including Heavy Metals Significantly Shape the Soil Bacterial Community Structure in the Tatun Volcano Group, Northern Taiwan. https://doi.org/10.1264/jsme2.ME22005


Ischemic acute kidney injury (AKI) has always been a hot and difficult research topic in the field of renal diseases. This study aims to illustrate the safe warm ischemia time of kidney and the molecular network characteristics and pathological features of mild to severe ischemia reperfusion kidney injury. We established varying degrees of renal injury due to different ischemia time  on unilateral (left kidney) ischemia-reperfusion injury and contralateral (right kidney) resection (uIRIx) mouse model. Mice were sacrificed 24 h after uIRIx, blood samples were harvested to detect serum creatinine (Scr), and kidney tissue samples were harvested to perform Periodic Acid-Schiff (PAS) staining and RNA-Seq. Differentially expressed genes (DEGs) were identificated, time-dependent gene expression patterns and functional enrichment analysis were further performed. Finally, qPCR was performed to validated RNA-Seq results. Our results indicated that there was no absolute safe renal warm ischemia time, and every minute of ischemia increases kidney damage. Warm ischemia 26min or above in mice makes severe kidney injury, renal pathology and SCr were both significantly changed. Warm ischemia between 18 and 26 min makes mild kidney injury, with changes in pathology and renal molecular expression, while SCr did not change. No obvious pathological changes but significant differences in molecular expression were found less than 16min warm ischemia. There are two key time intervals in the process of renal ischemia injury, 0 min–16 min (short-term) and 26 min–28 min (long-term). Gene expression of immune-related pathways were most significantly down-regulated in short-term ischemia, while metabolism-related pathways were the mainly enriched pathway in long-term ischemia. Taken together, this study provides novel insights into safe renal artery occlusion time in partial nephrectomy, and is of great value for elucidating molecular network characteristics and pathological features of mild to severe ischemia reperfusion kidney injury, and key genes related to metabolism and immune found in this study also provide potential diagnostic and therapeutic biomarkers for AKI. Acute kidney injury (AKI) is a multidisciplinary disease, at present, the basic research work of AKI is mainly focused on the fields of nephrology, intensive care unit and kidney transplantation . In thesIschemia-reperfusion is one of the main cause of AKI . In urolThe experimental mouse models of renal ischemia/reperfusion injury (IRI) have been widely applied to study the pathogenesis and injury outcome of ischemic AKI . IRI mouWild-type C57BL/6 mice (18–22 g) were purchased from the Animal Center of Chinese PLA General Hospital. These mice were housed in a specific pathogen-free facility under a 12 h light/12 h dark cycle with free access to food and water. Mice were randomly assigned to several groups based on different ischemia time: 0 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, and 30 min. To induce unilateral ischemia reperfusion injury and the contralateral kidney resection AKI (uIRIx), mice were placed on 37°C warm plate  to keep the temperature of kidney all through the surgery; the right kidney was excised, and left kidney pedicle was clipped for indicated time using microaneurysm clamp. Mice were then sacrificed 24 h after uIRIx, blood and kidney tissue samples were harvested for further processing. The animal protocol and all animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Chinese PLA General Hospital, and the permit number is 2019-X15-97.Blood samples were collected from the vena cava at the indicated time points, and the serum was separated by centrifugation at 3,000 rpm for 15 min at 4°C and then sent to the PLA General Hospital Biochemistry Department to detect serum creatinine (SCr). A quarter of the left kidney was fixed in 4% formaldehyde, dehydrated, and embedded in paraffin. Histopathological examination was performed by Periodic Acid-Schiff (PAS) staining of kidney tissue section (2 μm) to evaluate tubular injury.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE192883). The preprocessing of the gene expression profile data, which includes the background correction, the quantile normalization, the median polish summarization, and the log2 transformation, was performed by R software (https://www.r-project.org/) and RStudio software (https://www.rstudio.com/) as we described before . All raw RNA-Seq data were uploaded to the GEO database GSE192883 (d before . Principhttp://www.bioconductor.org/) was applied to find DEGs by comparing expression value between different ischemia groups. Differential expression was calculated using an empirical Bayes model. The criteria for the statistically significant difference of DEGs was | log2 fold change (FC)| > 1 in expression and adjusted p-value  < 0.05. The overlapping DEGs were further analyzed by UpSetR package in R software. We obtained a union DEGs set, which included the combination of DEGs obtained in each ischemia group compared with the ischemia 0min and 16min groups.The linear model for high-throughput data analysis (limma) in Bioconductor  analysis has been widely used to annotate the gene function. The categories of GO include the cellular component (CC), the biological process (BP), and the molecular function (MF) terms (http://www.genome.jp/kegg) is a database containing the information of genes related to metabolic and regulatory pathways  method, and the adjusted p-value < 0.05 was served as the cut-off criterion for selecting significant enrichment pathways. We also used clusterProfiler R package with the same threshold value, as mentioned above, to conduct the gene set enrichment analysis (GSEA) analyses for genes compared between 16 min and 0 min, between 28 min and 26 min, respectively. The criteria for the statistically significant difference of GSEA was |normalized enrichment score (NES)| >0.4 and adjusted p-value < 0.05.Gene ontology  terms . Kyoto Epathways . In the pathways  was usedhttp://string-db.org/) is an online biological resource database that is commonly used to identify the interactions between known and predicted proteins (http://www.cytoscape.org/). In the PPI network, each node stands for a gene or a protein, and edges represent the interactions between the nodes. We then used the plug-in named Molecular Complex Detection (MCODE) by Cytoscape to filter the modules of the PPI network. The first module of each cluster was selected.Considering that proteins rarely work alone, it is necessary to study the interactions among proteins. The Search Tool for the Retrieval of Interacting Genes/Proteins  on an Applied Biosystems 7,500 system PCR cycler . The data were normalized to 18s expression. Primers were obtained from Beijing Genomics Institute . The list of primers was presented in The frozen tissue samples were lysed in TRIzol reagent  and total RNA was extracted according to the manufacturer’s instructions. The levels of transcripts were determined by qPCR using TransStartThe statistical analyses used are specified in the figure legends.p < 0.05), while there was no statistical difference among 16 min, 18 min, 20 min, 22 min, 24 min, or 26 min with 0 min group. The serum creatinine dramatically changed between 26 min and 28 min of ischemia at the condition of 37°C.To explore the pathological characteristics of kidney injury at different ischemia times, we adopted the uIRIx model, which were more stable than bilateral ischemia reperfusion kidney injury AKI model. Mice were randomly assigned to 9 ischemia groups based on different ischemia times of left kidney: 0 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, and 30 min , ischemiPAS staining of kidney  showed tTo further elucidate the expression of injury-related molecules at different renal ischemia time points, we performed immunofluorescence staining on KIM1 . The resIn order to explore the molecular characteristics of kidney injury at above different ischemic times, triplicate left ischemia kidneys in every group were collected to perform RNA-Seq. Before the next analysis, we performed the quality control for every raw expression data to guarantee the quality. The normalized box diagram showed that the data was well preprocessed after normalization. Next, we applied PCA analysis to those 27 samples (different colors) to visualize sample clusters. The results showed that samples at the same time points were clustered , indicatp-value < 0.05. With the increase of ischemia time from 16 min to 26 min, the number of DEGs was gradually but slowly increased, from 863 to 1,448. While in the group of 28 min vs 0 min, the number of DEGs suddenly increased at 2,782. The most pronounced DEGs were in the group of 30 min vs 0 min, with 1,698 upregulated and 1784 downregulated genes  by Cytoscape to filter the modules of the PPI networks. The first module of each cluster was selected .To gain insight into the biological roles of each cluster, KEGG pathway and GO term analyses were performed. The results showed that genes in each cluster were associated with distinct biological processes. Genes in Cluster 1, cluster 4 and cluster 6 were down-regulated with the prolongation of ischemia time. Cluster 1 was mainly associated with metabolic-related processes, such as “fatty acid metabolic process”, “purine-containing compound metabolic process”, “valine, leucine and isoleucine degradation”, “pyruvate metabolism”, and “tryptophan metabolism”. In addition, immune-related processes such as “complement and coagulation cascades”, “antigen processing and presentation”, and “Th1 and Th2 cell differentiation” were also enriched in Cluster 1. Similar to Cluster 1, Cluster 4 genes were also mainly involved in metabolic-related processes.Genes in Cluster 6 were mainly involved in immune-related processes, such as “antigen processing and presentation”, “cytokine-cytokine receptor interaction”, “response to interferon-gamma”, “leukocyte mediated cytotoxicity”, and “T cell mediated cytotoxicity” . These rGenes in Cluster 2, cluster 3 and cluster 5 were up-regulated with the prolongation of ischemia time. Cluster 2 were mainly involved in cell junction-related processes, such as “tight junction”, “focal adhesion”, “cell junction assembly”, and “cell-matrix adhesion”. In addition, “apoptosis”, “PI3K-Akt signaling pathway”, “MAPK signaling pathway”, and “complement and coagulation cascades” were also enriched in Cluster 2. Genes in Cluster 3 were mainly associated with “wound healing”, “MAPK signaling pathway”, “TNF signaling pathway”, “PI3K-Akt signaling pathway” and “epithelial cell proliferation”. Cluster 5 were mainly involved in “renin-angiotensin system”, “cytokine-cytokine receptor interaction”, “HIF-1 signaling pathway”, “calcium signaling pathway”, “hydrolase activity” .Genes in cluster 7 and cluster 8 showed trapezoidal expression. They mainly enriched in DNA-repair related pathway, such as “DNA replication”, “cell cycle”, “mismatch repair”, “nucleotide excision repair”, and “recombinational repair”. In addition, genes in cluster 8 also involved in “cellular senescence”, “p53 signaling pathway”, “apoptosis - multiple species”, “gap junction”, “tyrosine metabolism” .According to the results of time-dependent gene expression cluster analysis, gene expression changes were most significant in the interval of 0 min–16 min and 26 min to 28 min. Therefore, we further analyzed the GSEA enrichment changes of these two key time intervals in order to find common and characteristic enrichment pathways. GSEA analysis showed that there were many common enrichment pathways in the 16 min–0 min group and 28 min–26 min group, among which DNA-repair related pathways, such as “DNA replication”, “mismatch repair”, and “nucleotide excision repair”, were all up-regulated in both two key time intervals. While metabolism related pathways, such as “propanoate metabolism”, “steroid hormone biosynthesis”, “Valine, leucine and isoleucine degradation”, and “glyoxylate and dicarboxylate metabolism” were all down-regulated in both two key time intervals .In addition, immune-related pathways, such as “antigen processing and presentation”, “Th1 and Th2 cell differentiation” were downregulated only in 16 min–0 min group ; “TNF siThese results were consistent with previous results of GO and KEGG pathway analysis, namely, gene expression of immune-related pathways was down-regulated in short-term ischemia, while in long-term ischemia, metabolism-related pathways were the mainly enriched pathway. Thus, we performed a more detailed analysis of genes in immune-related pathways and metabolism-related pathways . Genes, Furthermore, as indicated above, inflammation related pathways and cell repair related pathways were critical upregulated processes that reflect the kidney injury and repair. Thus, we also performed a more detailed analysis of genes in the pertinent enriched pathways . PathwayIn addition, to verify the expression pattern of the key genes, we performed qPCR assays for the four representative genes in the kidney sample  and compared them with the RNA-seq results. As shown in How many minutes of ischemia time is a safe warm ischemia time of kidney? Different studies have drawn mixed conclusions . What arThe experimental mouse models of renal ischemia/reperfusion injury (IRI) can well simulate renal injury caused by renal artery occlusion in human partial nephrectomy. However, there are various surgical methods for IRI model reported in different studies: bilateral IRI (bIRI) , contralIn this study, combined with the experience of previous studies, we established a relatively stable uIRIx model to explore the pathological features, and molecular network characteristics of mouse kidney with different degrees of ischemia. We found that there was no significant difference among 0 min–26 min of ischemia in SCr, while the SCr was dramatically changed between 26 min and 28 min ischemia groups at the condition of 37°C. However, the pathological changes were not exactly the same as SCr. Although there was no change in SCr, renal damage such as cellular casts and renal tubular obstruction could be observed from the group with more than 20 min of ischemia.Soranno et al. reported that SCr may be poor predictor of long-term histological and functional outcomes after uIRI . Slocum Some experts reported that both pathological and urea nitrogen (BUN) changes occurred after 18 min of renal ischemia , while oOther than SCr and BUN, many other injury-related molecules, such as Kim1 (havcr1), Lcn2 , and Il6, were also determined to predict kidney damage. Our results showed that the increase of Kim1 and Lcn2 expression emerged at 16 min and 22 min of ischemia respectively, and gradually enhanced according to the ischemia time. However, the expression of Il6 was significantly increased at 30 min of ischemia, with no significant changes in less than 28 min. As can be seen, the molecular expression patterns of each injured molecule are not the same under different ischemic time. Therefore, we further performed high-throughput RNA-Seq.High-throughput RNA-Seq results also confirmed that the molecular levels of kidney genes had started to change at 16 min–26 min of ischemia whereas SCr showed no difference. The pathology of the 863 DEGs in the 16 min ischemia group presented no alterations. Those results indicate that molecular alterations were faster and earlier than pathology changes and SCr. It is also worth noting whether there will be changes under electron microscopy at the organelle level rather than cellular level. Among the 863 DEGs in the 16 min–0 min group, Kim1 (Havcr1) presented the highest expression DEGs, and the log(fold change) was 4.99. Since Kim1 can be detected in blood and urine , and itsFor a long time, AKI was considered to be a self-recovery disease, while recent studies have highlighted that AKI can lead to fibrosis and CKD . When kiThe expression of genes in Cluster 2 and Cluster 3 were both enhanced with the increase of ischemia degree. Genes in Cluster1, Cluster 4, and Cluster 6 presented reduced expression after ischemia. Inhibiting the function of continuously increased expression genes in Cluster 2 and Cluster 3 or promoting the function of the continuously decreased expression genes in Cluster1 and Cluster 4 may have a potential renal protective effect on AKI. For example, Cdk4 and Cdk6 were members of Cluster 2, Pabla et al. found that G1/S-regulating cyclin dependent kinase 4/6 (CDK4/6) pathway is activated in nephrotoxic AKI , and tarTargeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival . Kim et The expression of genes in cluster 7 and cluster 8, which mainly enriched in DNA-repair related pathway, increased in mild injury and decreased in severe injury. It was reported that epithelial cell cycle arrest plays an important role in the development of fibrosis . Thus, wAccording to the results of time-dependent gene expression cluster analysis, gene expression changes were most significant in two key time intervals, 0 min–16 min and 26 min to 28 min. GSEA enrichment analysis of these two key time intervals were consistence with above results. These results revealed that gene expression of immune-related pathways was down-regulated in short-term ischemia, while in long-term ischemia metabolism-related pathways were the mainly enriched pathway. This finding suggests the important role of metabolic pathways in ischemia-reperfusion.Recently, single-cell transcriptomic data showed that metabolic processes are critical for maintaining the cellular identity of fully differentiated PT cells, and dysregulation of these pathways underlies the failure of damage-associated PT cells to redifferentiate into normal PT cell state . Matrix-As validated by qPCR assays, the expression patterns of four representative genes, Stat1, Lcn2, Pgam2, and Ptgs2 were highly consistent between the RNA-seq and qPCR results. These key genes were very important in the process of AKI, which could be used to explore some new diagnostic and therapeutic strategies. Signal transducer and activator of transcription 1(STAT1) is a member of STATs, which are important transcription factors that regulate the expression of inflammatory genes . JAK-STAThe present study has some limitations and drawbacks. First, because we mainly focused on observing molecular network characteristics and pathological features of mild to severe ischemia reperfusion kidney injury, the main experimental observation time was 1d after AKI. Therefore, we did not monitor the long-term prognosis in mild to severe AKI. Second, this study lacks human kidney tissue sample data, which may result in not full applicability in human. Third, the above results, such as the gene expression level and gene function, should be validated by further mechanism experiments. These issues will be illustrated in our future research.In summary, our results indicated that there was no absolute safe renal warm ischemia time, and “every minute counts”, which means every minute of ischemia increases kidney damage. Warm ischemia 26 min or above in mice makes severe kidney injury, renal pathology and SCr were significantly changed. Warm ischemia between 18 and 26-min leads to mild kidney injury, with changes in pathology and renal molecular expression, while SCr did not changed. No obvious pathological changes under 16 min warm ischemia were observed, while significant differences were found in molecular expression. The results also showed that SCr could not completely reflect the severity of kidney injury. There are two key time intervals in the process of renal ischemia injury, 0 min–16 min and 26 min to 28 min. Gene expression of immune-related pathways were most significantly down-regulated in short-term ischemia, while metabolism-related pathways were the mainly enriched pathway in long-term ischemia.Taken together, this study provides novel insights into safe renal artery occlusion time in partial nephrectomy, and is of great value for elucidating molecular network characteristics and pathological features of mild to severe ischemia reperfusion kidney injury, and key genes related to metabolism and immune found in this study also provide potential diagnostic and therapeutic biomarkers for AKI.


Physiologically, αS may populate disordered monomeric, helically folded tetrameric, or membrane-bound oligomeric states. Pathologically, αS may assemble into toxic oligomers and subsequently fibrils, the prion-like transmission of which is implicated in a class of neurodegenerative disorders collectively termed α-synucleinopathies. Notably, αS does not adopt a single “amyloid fold”, but rather exists as structurally distinct amyloid-like conformations referred to as “strains”. The inoculation of animal models with different strains induces distinct pathologies, and emerging evidence suggests that the propagation of disease-specific strains underlies the differential pathologies observed in patients with different α-synucleinopathies. The characterization of αS strains has provided insight into the structural basis for the overlapping, yet distinct, symptoms of Parkinson’s disease, multiple system atrophy, and dementia with Lewy bodies. In this review, we first explore the physiological and pathological differences between conformational states of αS. We then discuss recent studies on the influence of micro-environmental factors on αS species formation, propagation, and the resultant pathological characteristics. Lastly, we review how an understanding of αS conformational properties has been translated to emerging strain amplification technologies, which have provided further insight into the role of specific strains in distinct α-synucleinopathies, and show promise for the early diagnosis of disease. Decades of work have illuminated the remarkable conformational plasticity of αS essential for its physiologic functions, but which also underlies its pathological properties in diseased states. In this mini-review, we seek to illustrate the unique conformational properties of αS and how they have been exploited by emerging technologies for the early diagnosis of disease. We begin by discussing the physiologic conformational states populated by αS and their functional properties . We thenαS is a 140-amino acid protein consisting of an N-terminal amphipathic repeat region, central hydrophobic region, and C-terminal acidic region . In contIn particular, recent reports have provided a structural basis for αS’ functions at membrane surfaces. These studies largely rely on acidic small unilamellar vesicles (SUVs) as a mimetic of synaptic vesicles . Upon SUInterestingly, lipid binding by the N-terminal and central regions is decoupled, such that each region may not necessarily be associated with the same membrane. Specifically, the N-terminal anchor and central anchor of a single αS molecule may, at times, interact with different membranes  9]. Sim. Sim9]. Although these aforementioned reports have provided mechanistic insight into the lipid-bound αS state, they relied largely on artificial SUVs, which may not fully capture the dynamic and heterogeneous composition of membranes in vivo . Indeed,Promisingly, a recently reported “cellular unroofing” approach has enabled the biophysical characterization of lipid-bound αS with native plasma membranes . In thisAn additional finding from the “cellular unroofing” study was the localization of αS to exocytic sites, suggested by colocalization with SNARE proteins , which cThe existence of a helically folded αS tetramer was first reported nearly a decade ago by two research groups using distinct approaches ,20. ThesIn 2011, Bartels et al. demonstrated, using multiple approaches, the possible existence of a tetrameric αS species. Initial evidence came from native polyacrylamide gel electrophoresis (Native-PAGE) of several cell lines, murine cortical samples, and human erythrocytes, which all contained a band corresponding approximately to the size of an αS tetramer. As Native-PAGE migration does not depend solely on molecular weight, the group developed an alternative in vitro crosslinking approach that is now frequently used to study the putative αS tetramer. In this technique, chemical crosslinking is performed prior to SDS-PAGE to preserve native assemblies. Lastly, the authors developed a method to purify natively folded αS directly from human erythrocytes. Both scanning transmission electron microscopy and analytical ultra centrifugation of purified αS supported the existence of a tetrameric species. Interestingly, tetrameric αS had a circular dichroism (CD) spectrum characteristic of an α-helical protein, was resistant to aggregation, and bound membrane phospholipids with substantially higher affinity than the monomer.Shortly thereafter, a second group reported similar findings in bacterially expressed αS . When puSeveral reports have since delineated the basic properties of the αS tetramer, including the sequence features governing its formation. Intriguingly, tetramerization appears to be a general property of the synuclein protein family, which contains several imperfectly conserved KTKEGV repeat motifs throughout the protein sequence . AlthougFurthermore, αS mutations implicated in familial PD (fPD) cause a shift in the populations of αS species in favor of the monomer in vitro and in vivo . One sucThese findings suggested that the stabilization of the tetrameric state may have therapeutic potential for PD, and led to the subsequent identification of cellular regulators of tetramer abundance ,26,27. IMiglustat also appeared to be protective in an E3K model, suggesting the general importance of lipid content in the regulation of αS conformational states . Indeed,The initial report of a physiologic tetramer  was contNevertheless, multiple experiments by Burré et al. support the absence of a soluble tetrameric αS conformation . Specifi15N-enriched αS was exogenously introduced into five mammalian cell lines, and approximately 90% of the introduced αS was detectable as unstructured monomers by NMR. This suggests that either (1) at most, a small population of αS could exist in a tetrameric state, in contrast to reports that three-quarters of αS are tetrameric in the healthy brain . Wi. Wi98]. Prior studies focused mainly on using seeding aggregation assays to distinguish synucleinopathies from non-synucleinopathies, but recent developments in our understanding of kinetics, fibrillary aggregate structures, and advancements in technology have improved the discriminating ability of these assays to distinguish between synucleinopathies.Shahnawa et al. of the Soto group were the first to establish an αS-PMCA assay to discriminate between samples of CSF from patients diagnosed with PD or MSA with high sensitivity and specificity. PMCA analysis indicated differences in the maximal ThT fluorescence and aggregation kinetics between CSF samples from PD versus MSA patients, which was confirmed by post-mortem brain specimens of the same subjects. Additional spectroscopic, biochemical, and toxicity studies also showed differences in structure and toxicity between MSA and PD αS aggregates .The assessment of PMCA aggregation kinetics was conducted by the Soto group, as measured by ThT fluorescence. MSA samples aggregated faster, but reached a lower maximum fluorescence than PD samples. The group confirmed with post-mortem brain specimens of both PD and MSA patients that the activity of aggregates in CSF reflects that of aggregates in the brain. PMCA analysis distinguished PD from MSA, as well as PD and MSA from non-synucleinopathy controls . After PAdditionally, in 2020, Singer et al. of the Low group, in collaboration with Soto, used PMCA to differentiate MSA from other Lewy body synucleinopathies in CSF samples . The groOverall, PMCA was shown to maintain the biochemical and structural properties of disease-specific αS strains during the amplification process, and thus may serve as an important tool in distinguishing between MSA and PD, which may present with similar early clinical signs, despite their distinct progressions and treatment plans . WithoutA comparison of αS-PMCA and RT-QuIC was performed by Kang et al., who provided the Soto and Green groups with separate aliquots of CSF from the same subjects. Between the two techniques, there was a high concordance of results of 92%. Among the discrepant findings, no systematic explanation was found .PMCA and RT-QuIC differ slightly in their protocol conditions, but both techniques apply a maximum ThT fluorescence threshold to identify a positive case of disease. The ThT dye is specific to aggregated fibrils, and is used in real-time to measure the kinetics of αS aggregation. Cutoff values for maximum ThT fluorescence are chosen to differentiate a sample with synucleinopathy from controls. Both assays were sensitive and specific, but the sensitivity and specificity were improved when both assays were performed together for concordant results . In prinIn summary, the self-aggregation assays, PMCA and RT-QuIC, show promise for the screening and diagnosis of Parkinson’s disease and other α-synucleinopathies. Recent advances in these techniques have enabled the identification of the conformationally distinct αS strains that underlie the pathophysiology of different α-synucleinopathies. They are objective tools with the capacity for early diagnosis, raising the possibility for enhanced patient care through early interventions targeting pathologic αS transmission or other neuroprotective measures. An enhanced understanding of the kinetics of αS aggregation, structural properties of αS fibrils, and coupling to additional biomarkers may further enhance the accuracy of these assays in the diagnosis and prognosis of synucleinopathies.The αS protein is remarkable for its ability to populate a diverse set of conformational states—both physiologic and pathologic. Physiologically, the αS monomer is largely disordered in the cytosol, but adopts a highly ordered α-helical conformation upon membrane binding. Membrane-bound αS plays important physiological roles in synaptic vesicle clustering, fusion, and tethering at plasma membrane surfaces . In addiStrain amplification assays, PMCA and RT-QuIC, are powerful emerging techniques that can detect misfolded αS to diagnose and differentiate synucleinopathies. The prion-like seeding activity of αS in CSF, skin, and GI biopsy samples is a highly sensitive and specific biomarker for the presence of a synucleinopathy . The uti


COVID-19 is a challenge for education systems around the world. This study aimed to evaluate the perceived impact of the COVID-19 pandemic on nursing students, by assessing their emotions, the level of concern in contracting the virus and their perceived stress. We conducted an observational cross-sectional study. A total of 709 nursing students completed an anonymous questionnaire. The levels of anxiety and stress were assessed using the generalized anxiety disorder scale and the COVID-19 student stress questionnaire, respectively. In total, 56.8% of the sample often or always found it difficult to attend distance-learning activities. The main difficulty referred to was connection problems (75.7%). The mean generalized anxiety disorder score was 9.46 (SD = 5.4) and appeared almost homogeneous among students across the three years of study; most of the students showed mild (35%) to moderate (27%) levels of anxiety; 19% had severe anxiety. The overall COVID-19 stressor mean scores were 11.40 (SD = 6.50); the majority of the students (47.1%) showed scores indicative of moderate stress, 25% showed low stress levels, and 28% showed high-stress levels. Improvements and investments are needed to ensure high-quality distance learning, adequate connectivity, technical support for students, as well as strategies to promote mental health. Coronavirus Disease COVID-19) is an infectious disease caused by a newly discovered coronavirus that was identified in Wuhan, the largest metropolitan area in China’s Hubei province, in December 2019 [9 is an iThe COVID-19 epidemic spread rapidly globally; on 11 March 2020, the World Health Organization (WHO) declared a pandemic state . The firIn this sense, the aim of this study was to evaluate the perceived experience of the COVID-19 pandemic on nursing students, by assessing their emotions, the level of concern in contracting the virus during university activities, and their perceived stress during the pandemic lockdown.This study included an online survey involving students enrolled in the bachelor’s degree course in Nursing from two universities in the Sardinia region, Italy. University of Sassari (UNISS) and University of Cagliari (UNICA) with its branch in Nuoro city were the participating universities. The total number of students enrolled in the three years of course was 912 students.The survey used the free-access Google Forms application in order to administer a self-reported structured questionnaire. The link to the online questionnaire was sent through the students’ WhatsApp groups. Data collection took place in about ten days, from 17 March to 27 March 2021.The survey was designed in Italian, and the questions were developed after reviewing the relevant literature and international guidelines ,21,22,23According to Italian law, studies recruiting students and not including sensitive data such as critical health condition, do not require permission from the Ethical Review Authority. Faculty Directors of the Universities of Sassari and Cagliari authorized the study.All procedures in the study were in accordance with the ethical standards of the 1964 Helsinki Declaration and its later amendments, and with the General Data Protection Regulation (EU) 2016/679 (GDPR). Informed consent was obtained from students before to complete the questionnaire. Students were informed that their participation was voluntary and anonymous, according to Italian Data protection law . Additionally, students were informed that they could leave the study at any time without any adverse consequences for their university program.2) test was applied to compare subgroups of the sample. Frequencies and percentages for the study variables were compared between students of first, second, and third year of study to detect possible differences between groups. ANOVA analysis with post hoc multiple comparisons was performed using Bonferroni’s procedure to analyze differences between mean values of the study variables. Confidence intervals (95%) of the differences were calculated.Data were analyzed using the SPSS  version 26.0 statistical software. Descriptive statistics such as frequencies, percentages, mean and standard deviation, were performed to analyze the characteristics of the sample for the study variables. The independence chi-square . Specifically, 61.8% (n = 438) were enrolled at the University of Sassari, 30.7% (n = 218) at the University of Cagliari, and 7.5% (n = 53) at the branch in Nuoro.Almost all of the students  were Italian. Most of the sample were first-year students  and eight (1.1%) were students who had not completed university exams within the established time period (outside the prescribed time). Most of the students  were single, 50 students (7.1%) were married, and 10 students (0.7%) were divorced or separated. Almost the entire sample  lived with their families, and 131 students (18.5%) lived alone.Almost the entirety of the sample  indicated that their universities organized distance learning during COVID-19 lockdown, and 93.3% of the students (n = 664) declared that they attended the distance learning. Among them, 56.8% (n = 403) reported that they often or always encountered difficulties in attending the distance-learning activities. The main difficulty referred to was connection problems , followed by the difficulty in managing applications by students or teachers . The application used for distance learning was described by 58.7% (n = 416) of the participants as barely functional and 32.3% (n = 229) referred a lack of technical support. Nevertheless, to the question “Do you think distance learning can replace face-to-face teaching?”, most of the students  answered “partially”, 25% (n = 177) answered “yes”, and 18.1% (n = 128) answered “no”.To the question “Can the COVID-19 emergency negatively impact your education career?”, 236 students (33.3%) answered “extremely” or “a lot”, 252 (35.5%) “moderately”, and 221 students (31.1%) “little” or “not at all”.Regarding the question “Can the COVID-19 emergency negatively impact the training for your future career?”, 39.7% (n = 281) of students answered “extremely” or “a lot”, 38.3% (n = 272) answered “moderately”, and 22% (n = 156) said that the COVID-19 emergency can influence “little” or “not at all” the training for their future career. For 261 (36.8%) students, the COVID-19 emergency will change for the worse how students will be trained for their future careers; 60 students (8.5%) said that there will be no change; and 267 (37.7%) students do not know. For 121 (17.1%) students, the COVID-19 emergency will change for the better how students will be trained.Among the participants, 164 students (23.1%) had one or more relatives who contracted coronavirus, 641 students (90.4%) said that one or more acquaintances were infected, and 68 students (9.6%) declared that they did not know anyone who contracted the virus. Furthermore, 38.8% (n = 275) of responders stated to be “extremely” or “a lot” worried that their families might contract coronavirus, 41.9% (n = 297) were “moderately” worried, and only 19.3% (n = 137) were “little” or “not at all” worried for their families.Regarding the concern to become infected during daily activities, 275 students (38.8%) indicated being “extremely” or “a lot” worried, 297 (41.9%) were “moderately” worried, and 137 (19.3%) were “little” or “not at all” worried.Regarding clinical placement, 491 students (69.2%) reported being “extremely” or “a lot” worried that the clinical learning activities would expose them to the risk of contracting the virus, 167 (23.6%) were “moderately” worried, and 51 students (7.2%) were “little” or “not at all” worried.Face-to-face teaching is considered for 38.2% (n = 271) of students as an activity “extremely” or “a lot” at high risk for contracting the virus, for 36.8% (n = 261) of students it is considered as “moderately” at high risk, and “little” or “not at all” at high risk for 25% (n = 177) of the students.The perceived risk for nurses contracting the virus during their work activity is “extremely” or “a lot” probable for 69.7% (n = 494) of the participants, “moderately” probable for 25.8% (n = 183), and “little” or “or not at all” probable for 4.5% (n = 32) of the participants.To the question “During clinical activity, which measures do you think might prevent COVID-19 infection risk?”, the prevention strategies more frequently reported by students were “Environmental sanitation” , “Using individual protective equipment” , “Providing disinfectants and surgical masks to all patients in the waiting room”  “Environmental ventilation” , and “Reducing the number of patients in the waiting room” . Measures less frequently reported by students were “Body temperature measurement”  and “Telephonic screening/anamnesis to identify possible critical cases” . Other prevention strategies freely suggested by students included implementing vaccinations .Among the emotions experienced by students regarding the COVID-19 emergency, 42.2% (n = 296) of students perceived a low sense of fear, 32.1% (n = 225) moderate fear, and 25.7% (n = 180) intense fear. Concerning sadness, 46.1% (n = 323) of students perceived this emotion intensely, 31.9% (n = 224) poorly, and 23% (n = 154) moderately. With regard to the sense of worry with emergency, 45.5% (n = 319) of students indicated intense worry perception, 29.7% (n = 208) moderate worry, and 24.8% (n = 174) experienced low worry. About anxiety, 38.7% (n = 271) of participants perceived this emotion intensely, 35.8% (n = 251) poorly, and 25.5% (n = 179) felt moderate anxiety. Finally, regarding anger, 43.4% (n = 304) of students declared feeling intense anger, 23.5% (n = 165) moderate anger, and 33.1% (n = 232) experienced low perceived anger.A higher percentage of students  indicated mild levels of general anxiety disorder, compared to 27.1% (n = 190) moderate levels, and 18.8% (n = 132) minimal levels. Nevertheless, 19% (n = 133) of students showed general anxiety disorder scores indicative of severe anxiety.With regard to students’ stressors related to the coronavirus pandemic, the majority of the students 47.1% (n = 330) showed scores indicative of moderate stress, 25% (n = 175) showed low stress levels, and 28% (n = 196) referred high stress levels.p = 0.285; F = 0.55; p = 0.575, respectively) and the average values were almost homogeneous among the three years of study.With regard to the perceived impact of COVID-19 on both students’ university career and their preparation for the profession, the ANOVA results and multiple comparisons from Bonferroni’s test showed that there was no significant difference between student groups . Specifically, multiple comparison revealed a difference between first-year students (M = 3.10 ± 0.98) and second- and third-year students  who showed to be more concerned than the former. There was no difference between second- and third-year students. The percentages of students who are “extremely” or “a lot” worried about contracting COVID-19 infection during daily activities were significantly higher among second- and third-year students  than first-year students (31.7%) .Regarding students’ worry about contracting COVID-19 infection during daily activities, the results showed a significant difference between groups . Post hoc comparisons showed a significant difference between first-year students who perceived a lower risk (M = 3.70 ± 1.04) than second- and third-year students . Additionally, in this case, there was no difference between students enrolled in years subsequent to the first academic year (second and third year). The percentage of student “extremely” or “a lot” worried significantly differed across year of study ; the highest percentages were for second-year (79.4%) and third-year (80.9%) students, and lowest percentage was for first-year students (57.1%).The same can be said for the perceived risk of infection during clinical learning . The chi-square test supported these results  by showing that the percentages of students who considered face-to-face teaching as “extremely” or “a lot” risky of getting COVID-19 significantly differed across year of study. Specifically, highest percentages were observed among second-year students (50.5%) with respect to first-year (31.1%) and third-year (36.8%) students. About face-to-face teaching activity as a risk of infection, the only significant difference we found was between first-year (M = 3.04 ± 0.97) and second-year students (M = 3.42 ± 1.13) who perceived a lower risk of contracting the infection than the former. There was no difference between first-year students and third-year students, as well as between second-year students and third-year students  and worry  emotions. About anxiety, multiple comparison revealed a difference between first-year students (M = 1.91 ± 0.85) and both second- and third-year students  who showed to be more anxious than the former. There was no difference between second- and third-year students. As emerged from the chi-square test, the percentages of students who perceived high levels of anxiety significantly changed across years of study ; percentages were higher for second-year (42.7%) and third-year (48%) students than for first-year (31.7%) students.Regarding students’ emotions about the COVID-19 emergency, ANOVA analysis showed significant differences between groups only for anxiety , although the percentage of students who perceived high worry about the pandemic was higher among second-year (48.2%) and third-year (51.3%) students than first-year (41.1%). Regarding the emotions such as fear, sadness, and anger, no significant difference was found between groups for both mean values and frequency distribution of the variables. As regards worry, the only one significant difference we found was between first-year (M = 2.12 ± 0.83) and third-year students (M = 2.32 ± 0.78), who perceived higher worry than the former. There was no difference between first- and second-year students and between second- and third- year students. However, this result is not supported by the chi-square analysis that revealed no significant difference between groups (χp = 0.44). In fact, the mean score of GAD appears almost homogeneous among students across the three years of study . The overall mean score for GAD was 9.46 (SD = 5.41) and all the student groups reported scores referring to moderate levels of GAD. The chi-square test supported the ANOVA results by showing uniformity in percentages of students with severe general anxiety across year of study  .With regard to the general anxiety disorder (GAD) scores, the ANOVA results showed nonsignificant differences between groups . Specifically, multiple comparison revealed a difference between first-year (M = 10.30 ± 6.52) and both second-year and third-year students  who perceived higher levels of COVID-19-related stressors than the former. There was no difference between second- and third- year students. The overall mean score for students’ stressors was 11.40 (SD = 6.50) and all the student groups reported scores referring to moderate levels of perceived stressors. The chi-square test supported the ANOVA results  by showing that the frequency distribution of students with high COVID-19-related stressors significantly differed across year of study with higher percentages for second- and third-year students . Regarding students’ stressors related to the coronavirus pandemic, ANOVA analysis highlighted that there were significant differences between groups , probably because of the “technical” difficulties encountered “many times or always” by more than half of the students , and due to the lack of social interactions as well. On the one hand, the perception about the poor quality of distance learning finds support from Oducado and Soriano’s study , by showAt the time of the study, students were not attending clinical or internship activities. This could be the reason why the negative impact of COVID-19 on the preparation for the future profession was rated from moderately to extremely high by the majority of the students. ANOVA results showed that there was no significant difference between student groups and the average values for the variable were almost homogeneous among the three years of study. A similar situation is reflected in the results from Ilankoon et al.’s study  that shoAbout 40% of the students indicate that the COVID-19 emergency will change for the worse the way they are trained for their future profession. This is an important percentage, but it is considerably lower than that emerged from Bellini et al.’s study among dental hygiene students (53.9%) . Such a Regarding students’ knowledge about preventive measures to contrast the infection, the results showed a good knowledge of the recent guidelines concerning COVID-19 prevention procedures. This result is in line with Clements’s study . MoreoveWith regard to both clinical learning and frontal teaching, our students reported a high risk of getting coronavirus during these activities; nearly the entirety of the participants were either “extremely”/”a lot” or “moderately” concerned about contracting COVID-19 infection.As regards clinical training activities, first-year students perceived a lower risk for contracting coronavirus than second- and third-year students. It is probable that students enrolled in the years following the first have reached higher awareness of the practices to be performed on patients during the internship. In addition, at the time of the survey, first-year students had not yet started the clinical activity and probably they did not have a real perception of the risk they were exposed to.The most common feelings experienced by students at high levels are sadness, worry, and anger; fear and anxiety are instead experienced at mild to moderate levels. Similar results were found among Turkish nursing students , where cAnother relevant result involves general anxiety disorder for which severe anxiety is perceived by 19% of the students. This result is higher than that revealed in previous research .Regarding psychological health and emotions related to the pandemic, we found that irritability, fear of contagion, problems with relaxation, and depressed mood were the central aspects that emerged also among nursing students in China  and mainRegarding the students’ stressors related to the pandemic, the majority of the students showed scores indicating moderate and severe stress. The high prevalence of stress among university students was also supported in recent research . MoreoveThis study presents some limitations that should be discussed. First, although we recruited a copious sample of nursing students, we used a convenience sample from one Italian region, which reduces the possibility of generalizing the results to all Italian students. Second, the study sample mostly included first-year students compared to second- and third years students. First-year students typically are in the process of adjusting their learning styles and habits and transitioning from high school to university; this could potentially have affected the results of our study. Third, the data were collected using a self-administrated questionnaire, thereby rating bias might be present. However, all the variables analyzed in the study refer to students’ perception about their experiences during the pandemic. In this way, collecting data using self-reported measures is adequate for perception data.Despite the limitations, our findings provide an overview of the status of undergraduate nursing students after the COVID-19 lockdown and suggest possible intervention strategies to protect their well-being. The universities will need to capitalize on this experience and reorganize their e-teaching and e-learning approaches. When social distancing is required for public health issues, the peculiar moments of the traineeships need to be replaced by virtual reality systems. In addition, learning programs including synchronization techniques and simulation software should be implemented to facilitate interactive learning among students and increase access to feedback. It is essential that teachers who are not familiar with technology be trained in using online and virtual learning environments. The nursing course staff should stay in continuous contact with the students beyond online teaching, by identifying and supporting their needs. Moreover, it is important to maintain a stable educational framework that includes reducing to minimum any changes in the teaching schedule, announcing information about changes as soon as possible, and supplying updated information about the continuance of the academic activities and exams . In addiThe results of the present study indicate that nursing students at the studied universities perceive the impact of COVID-19 on their study career as high. Students indicated a high level of stress related to the suspension of all clinical training activities, fear of contracting COVID-19, and dealing with the challenges of distance education. University has an important role in developing a sense of control and providing a stable educational structure for the students.


Over the last decades, antipsychotic prescriptions in children have increased worldwide. However, adverse events are frequently observed, with some such as psychiatric adverse events remaining poorly documented.The French ETAPE study is a 12-month naturalistic prospective multisite study that included 190 antipsychotic-naïve pediatric patients (mean age = 12 ± 3 years), treated by antipsychotic for psychotic or non-psychotic symptoms. From the ETAPE database, we performed additional analyses focusing on psychiatric adverse events.p < 0.001). 63.2% (n = 120) of the 190 children and adolescents presented at least one psychiatric adverse event. The most frequent were externalized behaviors such as aggressiveness or agitation (22.7%), mood changes (18.4%) and suicidal ideas or behaviors (11.8%). Half of psychiatric adverse events occurred during the first quarter, 49.46%, compared to 23.79% during the second, 15.77% during the third, and 10.96% during the fourth.Children received mainly second-generation antipsychotic for conditions out of regulatory approval, with risperidone and aripiprazole being the most frequent (respectively 52.5% and 30.83%). Clinicians reported 2447 adverse events, mainly non-psychiatric , including neuromuscular, metabolic, gastroenterological, and  psychiatric. 55.88% of psychiatric adverse events were attributable to antipsychotic by the clinician, compared to 89% of non-psychiatric adverse events (This additional analysis from the French ETAPE study emphasizes that psychiatric adverse events might be more frequent than expected in the pediatric population. Also, the potential risk of psychiatric adverse events should be part of the benefit-risk evaluation and sub-sequent follow-up.The online version contains supplementary material available at 10.1186/s13034-022-00517-3. All over the world, the use of antipsychotics (AP) is widespread in the pediatric population for psychotic and non-psychotic disorders –4. AmongIn light of the high frequency of AEs reported in the literature, the frequent use of APs remains a concern . These AHowever, several AEs remain poorly documented in AP-naïve pediatric patients , includiETAPE is a French multisite, naturalistic and observational study aiming to determine the incidence of AEs potentially attributed to AP treatment prescribed for psychotic or non-psychotic symptoms in AP-naïve pediatric patients. Standard definitions and terminology for key aspects of clinical safety reporting are taken from the Clinical Safety Data Management of the European Medecines Agency . Our resThis manuscript describes the additional analysis of the French ETAPE study data focusing on psychiatric AEs. Patient characteristics entering the study are presented in Table Descriptive results of continuous variables are expressed as means (± SD) and as absolute numbers and relative frequencies for categorical variables. The comparison of characteristics between patients with or without at least one psychiatric AE potentially attributable to the AP was performed using the χ² test . Concerning CGI and CGAS scores, the number of AEs and psychiatric AEs were compared using the Kuskall-Wallis test. The incidence rate of psychiatric AEs was evaluated by dividing the number of patients with a new AE during the entire follow-up period by the number of person years at risk. The 95% confidence intervals (CI) are also presented. The comparison of psychiatric AEs according to those different parameters was performed using the χ² test . If necessary, post hoc tests were performed . Statistical analyses were conducted using SAS Enterprise Guide 7.1 (Copyright (c) 2017 by SAS Institute Inc., Cary, NC, USA).± 3.1) years, with a predominant proportion of boys (70.8%). The main clinical indications for AP treatment in AP-naïve children and adolescents included were “Schizophrenia and other psychotic disorders” (30%), “disruptive, impulse-control, and conduct disorders” (19.2%), “autism spectrum disorder” (10.8%) as well as “personality disorders” (10.8%). We found no statistically significant difference between attributable and non attributable groups for age (p = 0.40), sex (p = 0.39), pubertal status (p = 0.73) and clinical diagnosis .The characteristics of the ETAPE population are shown in Table ± 0.92, a score of 4 “moderate illness” and 5 “marked illness”), consistent with the burden of severe mental illness in the study population. This was also reflected in the measurement of social functioning with a mean CGAS score of 46.2 (± 9.9) (score of 50–41 representing a “moderate” and 60 − 51 “variable functioning with sporadic difficulties). No statistically significant difference was found between the different CGI scores and the number of overall psychiatric AEs; and also, between the different CGAS scores and the number of overall psychiatric AEs.At the beginning of the study, patients presented a mean CGI-S score of 4.85 (p = 0.42). On inclusion, 26% of patients were with comedications .Risperidone and aripiprazole were the most prescribed, respectively, 52.5% and 30.8%, among patients exposed to the same AP molecules throughout the study (n = 125 patients). No statistically significant difference was demonstrated between the molecule AP drug and the distribution of psychiatric AEs (p < 0.001).Within the ETAPE sample of 190 pediatric patients, a total of 2447 AEs were reported. Patients with at least one psychiatric AE were 63% (n = 120). Among the different clinical dimensions of AEs, psychiatric AEs represented 15.28% (374/2447). The overall psychiatric AEs incidence rate was 1.70 per person-year . Moreover, 55.88% of psychiatric AEs were attributable to SGA by the clinician, compared to 89% of non-psychiatric AEs (The Table p < 0.005) when comparing the distribution of the types of psychiatric AEs according to imputability to AP drugs. In fact, psychiatric AEs “Mood changes” and “Apathy / restricted range of emotion/lack of interest” were significantly more frequently attributable to AP treatment following the clinical investigator’s judgment (respectively p = 0.0021 and p = 0.0025), contrary to “Aggressiveness / agitation / challenging behaviors” that were declared significantly less frequently attributed to AP (p = 0.0001).The most frequent observed psychiatric AEs were externalized behaviours such as “Aggressiveness, agitation or challenging behaviours” (22.7%), “Mood changes” (18.4%) and “Suicidal ideas or behaviours” (11.8%). Moreover, we found a statistically significant difference (p = 0.0008), contrary to severe AEs more frequently described in the non attributable group (p = 0.0001). There have been five psychiatric AEs of “extreme severity”, three attributable to AP treatment , and two non attributable (tantrum and suicide attempt).The severity of the psychiatric AEs is presented in Table p = 0.6156).Table p = 0.47 and p = 0.10. However, during Q2, the distribution is statistically different according to their imputability (p = 0.01): “Mood changes” are more frequent within attributable AEs, contrary to “Aggressivity/agitation/challenging behaviors” and “Anxiety” which are more related to non attributable AEs.The distribution of the occurrence of the different groups of psychiatric AEs during the quarterly follow-up is presented in Supplementary Table This manuscript describes psychiatric AEs in AP-naïve children and adolescents treated with AP over 12 months during the ETAPE study , 28. TheDespite a limited list of approvals, APs have taken a central place in the treatment of mental health disorders in the pediatric population. Within APs, risperidone and aripiprazole are the most AP prescribed , 30. TheIn our prospective naturalistic study, the overall psychiatric AEs incidence rate was 1.70 per person-year, and psychiatric AEs were 15.28% of all AE reported. The very few studies to compare our results to come from pharmacovigilance databases . MoreoveIn our pediatric population, three types of psychiatrics AEs were the most represented with “Aggressiveness, agitation or challenging behaviors”, “Mood changes”, and “Suicidal ideas or behaviors”,  (Table p > 0.05); but some studies made other observations [We didn’t show any association between diagnoses , disease severity (CGI) or social functioning (CGAS), sex and the presence of psychiatric AEs . Likewise, some internalized AEs (such as mood change or negative symptoms) are attributed to AP drug by the on-site investigator; however, the externalized symptoms  are less attributed to the AP drug. This may suggest that they are related to the underlying mental health disorders; whereas these symptoms were not present before the introduction of the AP. These observations raise the issue of whether the type of AE might influence the clinician’s judgment about imputability to the AP molecule . In the Regarding mood swings, well-designed clinical trials, carried out in the adult population, suggest that AP-induced mania/hypomania is a marginal phenomenon , 32.Suicidal behaviors as AEs during AP treatment as observed in our pediatric study population, have also been reported by other authors , 23, 33.Nevertheless, even if some evidence points to psychiatric AEs, including suicidal behaviors in AP-treated pediatric populations presenting mental health disorders, more specific studies, including control groups, are required to verify the imputability of AP treatment for those AEs . The intConcerning the onset kinetics of the first AEs in the pediatric population Table , more thMoreover, it is also necessary to underline the risk of polypharmacy which makes it challenging to understand the occurrence of AEs and can lead to severe AEs , 40.In ETAPE study, we have noted these limitations: (1) the small number of patients in the sub-groups does not allow generalizing the results; (2) all categories of AEs were systematically screened using the PAERS, which may have led to higher detection of AEs compared to studies with spontaneous reporting; (3) psychiatric AEs were not specifically researched; (4) there was no control group and (5) causality assessment was completely dependent on expert judgments.ETAPE results show that psychiatric AEs are observed in pediatric patients treated by AP. Thus, clinicians need to be aware of the possibility of occurrence of psychiatric AEs while prescribing APs. Furthermore, the relation to AP treatment should systematically be assessed and monitored for any novel or worsening psychiatric symptoms, also considering the evolution of the underlying psychiatric condition. The risk of psychiatric AEs should be part of the benefit-risk revaluation when prescribing an AP to pediatric patients. AEs should be monitored, especially during the first months after introducing an AP drug to this population. Further studies are needed in this area on larger samples to better understand the occurrence of psychiatric AEs, with the mechanisms underlying them and the role of AP drug treatment.Below is the link to the electronic supplementary material.Supplementary Material 1: Table S1: Distribution of psychiatric Adverse Events during the quarterly follow-up


The demographic trend of an ageing society is mirrored in the rising number of hospitalized geriatric patients in Germany. However, there is still a wide gap of knowledge regarding the dermatological diseases, comorbidities and performed procedures within this growingly important group of patients. The study was conducted as a retrospective monocentric data analysis of all patients 65 years or older from the Department of Dermatology, Medical Center—University of Freiburg, Germany. In total, 10,009 individual hospitalisations were included from 2009 to 2017, and there was a notable increase of geriatric patients in the study period. This study illustrates the following: leading major diagnoses included malignant neoplasm of the head and neck, ulcerated and non-ulcerated inflammatory spectrum of chronic venous insufficiency, whereas angina pectoris, type 2 diabetes and cardiac diseases were noted most frequently as secondary diagnoses. Patients with venous diseases had considerably more often cardiopulmonary minor diagnoses, whereas endocrine diagnoses peaked in the cohort of patients with psoriasis and psychiatric and muscululoskeletal disorders in patients with bullous dieseases. Moh’s surgery, dressings and multimodal dermatological treatments were the most often encoded procedures.The online version contains supplementary material available at 10.1007/s00403-021-02244-9. In Western Europe, the increase in life expectancy due to an increasingly higher health status and continuously improving medical care results in a growing number of elderly people, a trend that can also be observed in the age distribution of hospitalized patients –3. This Current geriatrics literature focuses mainly on neuropsychiatric, orthopaedic or internal diseases –10, but From a health planning and management perspective, characterising the demands and requirements of geriatric patients (65 years of age and older) in relation to specific challenges accompanying hospitalization and treatment, is crucial. Our work illustrates the burden of diseases severe enough for inpatient treatment.http://www.drks.de, DRKS-ID DRKS00016865).The study was conducted retrospectively from January 1st, 2009 to December 31st, 2017 at the Department of Dermatology, University of Freiburg, Germany. It included all hospitalized patients who were 65 years or older at the time of the admission. The study was approved by the University of Freiburg Ethics Committee Board and registered on German Clinical Trials Register -10 codes in cooperation with the Department of Medical Controlling. If patients were hospitalized more than once in the investigated time period, each admission was included separately.Subgroup analysis of the main diagnoses were done by clustering primarily surgical patients (nonmelanoma skin cancer (NMSC) and Merkel cell carcinoma (MCC) , and melanoma ), as well as non-surgical patients (psoriasis (L40), mature T/NK-cell lymphoma (C84), (autoimmune) bullous dermatoses (L10–L14), lupus erythematosus (L93), erysipelas (A46), zoster (B02), dermatitis and eczema (L20–L30), and venous disease with or without ulceration (I83)), and others.Performed procedures were grouped according to OPS (‘Operationen- und Prozedurenschlüssel’)-301 categories .t test as well as anova tests followed by a Tukey multiple comparison of means test was performed for inductive statistics. Detailed calculations of significance can be found in Online Resource 2. Figures were created using R (v.4.0.2) and CorelDraw (v.21).Descriptive statistics . The Dermatological Department ranked fourth out of 26 of all inpatient departments regarding the percentage of geriatric patients (1192 patients or 53%), whereas the departments anaesthesiology (66%), ophthalmology (63%), and palliative medicine (54%) had the eldest patient collective.Between 2009 and 2017, 10,009 individual geriatric hospitalizations were confirmed, of which 5346 patients presented once, while 1148 patients were hospitalized twice or more than twice (603 patients) during this timeframe.p < 0.01). Considering different age subgroups, the number of inpatients between 75 and 84 years as well as 85 and 94 years increased significantly in an even greater extent  , and males represent the majority in the age cohort 65- to 85-year-olds (54% of patients), whereas female patients make up for more than 61% of all patients aged older than 85 years.The most frequently encoded individual diagnoses identified were “other and unspecified malignant neoplasm of skin” (C44), “malignant melanoma of skin” (C43) and “varicose veins of lower extremities” (I83) (see Online Resource 1: Table 1A and B).A subgroup analysis clustering indications in “surgical”, “non-surgical” and “other diagnoses” revealed a steady distribution over the study period Fig. , but witp < 0.05); 2.6% vs. 1.2% (p = 0.086)), whereas diagnoses like “varicose veins of lower extremities” (I83), “non-pressure chronic ulcer of lower limb, not elsewhere classified” (L97) or “transepidermal elimination disorders” like reactive perforating collagenosis (I87) were far more often encoded in 2017 .Differences in the encoded main diagnoses in the year 2009 compared to 2017 showed the following: We had more hospitalizations for “atherosclerosis of arteries of extremities” associated ulcerations (I70.2), “atopic dermatitis” (L20) or “secondary and unspecified malignant neoplasm of lymph nodes” (C77) in 2009 (1.6% (2009) vs. 0.41% (2017), 3% vs. 0.9% (both p < 0.01), whereas older patients were less often treated for malignant melanoma , or psoriasis  .Regarding gender, our data reveal more males hospitalized for melanoma  and NMSC  than females . Cutaneous malignancies localized on the skin of scalp and neck  and the ear/external auricular canal  were significantly more prominent in men than in women . Vice versa, there is a slight trend towards a more frequent occurrence of malignancies on the skin of the face  in women than in men . On the contrary, “pemphigoid” (L12), “bullous erythema multiforme” (L51.1), “pyoderma gangraenosum” (L88), and “discoid lupus erythematosus” (L93.0) are seen more often in women than in men ; 0.57% vs. 0.25%; 0.16% vs. 0.02% (both p < 0.05), respectively).Overall, some diagnoses are unevenly distributed: “Sézary syndrome” (C84.1) and “rhinophyma” (L71.1) occur in 1.49% and 0.42% of all male cases and are almost never registered in women , “type 2 diabetes mellitus” , “skin changes due to chronic exposure to nonionizing radiation”  and “atrial fibrillation and flutter”  (see Online Resource 1: Table 2).Correlating the different minor diagnoses with groups of the main diagnoses, we found that patients with venous diseases have a higher frequency of circulatory and respiratory illnesses 44%) when compared to all diagnoses combined (27.5%), whereas patients with bullous disorders suffer more often from musculoskelettal (6.8%) as well as psychiatric diseases (10%), and patients diagnosed with psoriasis were diagnosed with more endocrine disorders (9.6%) than other diagnoses. Also, patients hospitalized due to NMSC/MCC or melanoma more often reported a comorbidity related to other neoplasms (11.4%) than patients with other major diagnoses (5.9%).  , “tendency to fall” , “(pre)senile dementia” , “Alzheimer disease”  or “presbycusis”  were registered quite frequently  and dementia (F03) became far more frequent with older age (R32: 2.6% vs. 23.5% (65 to 74 years vs. aged 95 years or older); F03: < 1% vs. 12.7%). On the contrary, patients between 65 and 84 years regularly suffer from “obesity” , while only 5.2% of patients aged 85 years or older suffered from this complaint (Table 2 and demp < 0.001)). Also, the numbers for “mental and behavioural disorders due to use of alcohol” (F11) or “tobacco” (F17) or “sleep disorders” including sleep apnoea (G47) are doubled in the male patient clientele.A gender-specific comparison reveals that male patients have far more often a “personal history of malignant neoplasm”  or “chronic ischemic heart disease” . Also, diagnoses like “volume depletion” (E86) or “other disorders of fluid, electrolyte and acid–base balance” (E87) occurred almost twice as much in women . Psychiatric disorders such as “depressive episode” (F32) or “recurrent depressive disorder” (F33), “other anxiety disorders” (F41) and “reaction to severe stress, and adjustment disorders” (F43) were almost three times more frequent in women (p < 0.001).Vice versa, women are affected more prominently from diagnoses like “seropositive and other rheumatoid arthritis”  or “other noninfective disorders of lymphatic vessels and lymph nodes” , “dressing of extensive and severe skin diseases” , “phototherapy” , “multimodal dermatological complex whole body treatment” , and “regional skin flap”  .Between 2009 and 2017, there were only few differences in the most often encoded procedures. In 2009, more patients were encoded with procedures like “paraffin gauze dressing with antiseptic ointments without debridement or bath” , “psoralen and ultraviolet A (PUVA) treatment” , and “extensive local tissue expansion skin flap on the head” . On the contrary, “paraffin gauze dressing without debridement or bath” , “extensive debridement on the lower leg” , “biopsy on the facial skin and the scalp” , and “Moh’s surgery on the nasal skin”  listed more frequently in 2017  and very old (≥ 90 years) patients is expected to be rising, as the “baby boomer generation” (born in Germany between 1955 and 1969), is reaching the geriatric age and their needs will become a more important socioeconomically topic throughout all medical disciplines . Whereas 21% of the German population in 2016 was 65 years or older, about 53% of the registered hospitalized patient cases belonged to this age cohort. In the next 15 years, the proportion of elderly is expected to increase to around 28% and the hypothetic rising number of geriatric inpatients requires more complex health care coordination.A Germany study from Makrantonaki et al. exemplarily investigated all dermatological findings in inpatients of a geriatric unit and thereby gives an all-encompassing picture of the skin diseases older people suffer from, independent of the severity or need for inpatient treatment . Since GThe observation that the top three registered diagnoses in men are all NMSC of different localisations confirms their increased risk for cutaneous malignancies with an additional clear surplus of all inpatient cases between 65 and 84 years , 15. TheThe discrepancy of a clear surplus of hospitalized women in the group over 85-year-old in the gender distribution of our inpatients is clearly mirrored in the demographic distribution in Germany. Besides less risky health behaviour, this gender gap can be led back to male soldiers lost in the second world war, and may equally adapt due to missing social cuts in the meantime.Nevertheless, the registered procedures match the found main diagnoses: With NMSC and melanoma being the most common reasons for hospitalization, Moh’s surgery was the most frequently encoded procedure, respectively. Similarly, dressings essential after both surgical procedures and for ulcerations are often recorded, as are specific dermatological therapies needed for disorders like dermatitis or psoriasis. But, as mentioned above, the incomplete documentation of the performed procedures makes it impossible to validly describe occurring trends throughout the years. That said, the observed shift in the frequencies of the registered measurements might be partly due to revisions of guidelines for melanoma, squamous cell carcinoma, pemphigus and bullous pemphigoid, and atopic dermatitis in the last years , 21–24.For example, the comparably low number of very old patients with a hospitalization diagnosis of melanoma may, amongst other reasons like the overall lower incidence than NMSC or a higher mortality in earlier life, be associated with a decision against excessive treatment options such as lymph node extirpation and thus a more frequent ambulatory therapy.Analysing the registered comorbidities, we saw that most frequently encoded diagnoses coincide in both sexes. These included cardiovascular diseases, venous diseases, diabetes mellitus type 2 or obesity, and malignancies, which represent the major burdens resulting from an unhealthy lifestyle with an excess in caloric intake and little exercise. Gender-specifically, we found that men enlist far more often with a “personal history of malignant neoplasm”, an ongoing observation of which Nicholas provides an excellent overview of possible reasons , 26. OthAstonishingly, age-related comorbidities are underrepresented in comparison to clinical reality, which may be attributed to tight time schedules, agitating clinical environment, “geriatically” untrained physicians or less relevant financial reimbursement, although these factors also contribute to the length of stay, complications, medical transfers and consultative support by other disciplines (data not shown).In general, all registered diagnoses as well as the encoded procedures strongly link to the experience, continuity and consistency of the person responsible for coding. Yet, a structural bias was evident to the effect that not all, but foremost those for financial reimbursement needed diagnoses were recorded. Procedures performed at the ward that are intended for good clinical practice, are time-consuming and costly, but did not have OPS coding, were not encoded at all. This includes small biopsies or caretaking of a postoperative bleeding, blood workup in various areas, urine or pregnancy testing, microbiological testing or histological examinations, laser therapy, ultrasound of lymph node or duplex ultrasound for venous diseases. While this is a monocentric study, differences between individual medical facilities surely exist.Benefitting from a steep improvement and innovations in the health care system and, in comparison to former generations, better nutrition and less physical work, this age group is expected to reach a high age in considerable fitness. Therefore, it will be crucial not only to adapt to the physical need by senior-friendly modifying buildings, but also to create a modernized structured comprehensive all-encompassing registration system that serves as a foundation for data registration, transfer and extraction of diagnoses and procedures mentioned in medical treatment. New balances between an adequate financial recompensation for the highly complex in- and out-patient treatment of the aging society and the lack of temptation for health care providers to “sicken” their patients. Education of the medical staff for age-related changes and development of disorders, which includes recognition and diagnosing as well as the attendance to the specific requirements. In our opinion, a sensibilisation and medical education of the general population, which medical indications can still be treated meaningfully in high age, is crucial.Supplementary Material 1 (Online Resource 1): A document with three tables and one figure. Table 1A lists the most common main diagnoses for male and female geriatric patients hospitalized in the study period. Table 1B lists the 20 most common main diagnoses grouped by main ICD-10 codes of the same patients. Table 2 lists the 20 most common minor diagnoses grouped by main ICD-10 codes for the same patients. Table 3 lists the frequency of minor diagnoses for certain main diagnoses for all patients of the study. Figure 1 compares the number of individual hospitalizations of elderly patients to the age distribution of Germany in 2017 (PDF 391 KB)Supplementary Material 2 (Online Resource 1): A document that lists the results of statistical tests used to test for significance of results reported in the main document. (PDF 103 KB)Below is the link to the electronic supplementary material.