PTEN, PI3K) and RAS/MAPK  signaling pathways that are crucial for tumor growth. Interestingly, the frequency of PTEN alterations in gliosarcomas was much higher than in GBMs. Aberrations of PTEN were the most frequent and occurred in 70% of samples. We identified genes differentially expressed in gliosarcoma compared to GBM (including collagen signature) and confirmed a difference in the protein level by immunohistochemistry. We found several novel translocations (including translocations in the RABGEF1 gene) creating potentially unfavorable combinations. Collected results on genetic alterations and transcriptomic profiles offer new insights into gliosarcoma pathobiology, highlight differences in gliosarcoma and GBM genetic backgrounds and point out to distinct molecular cues for targeted treatment.Gliosarcoma is a very rare brain tumor reported to be a variant of glioblastoma (GBM), IDH-wildtype. While differences in molecular and histological features between gliosarcoma and GBM were reported, detailed information on the genetic background of this tumor is lacking. We intend to fill in this knowledge gap by the complex analysis of somatic mutations, indels, copy number variations, translocations and gene expression patterns in gliosarcomas. Using next generation sequencing, we determined somatic mutations, copy number variations (CNVs) and translocations in 10 gliosarcomas. Six tumors have been further subjected to RNA sequencing analysis and gene expression patterns have been compared to those of GBMs. We demonstrate that gliosarcoma bears somatic alterations in gene coding for PI3K/Akt ( Gliosarcoma is a very rare, glioblastoma IDH-wildtype brain tumor that accounts for less than 0.5% of all intracranial tumors and is preponderant in patients aged between 40 and 60 years old, with a slight male predominance ,2. GliosTP53 mutations and, rarely, EGFR and IDH mutations [NF1, RB1 and PTEN alterations. However, TP53 mutations are more frequent and the rate of EGFR amplification/overexpression is lower in gliosarcoma as compared to GBM [Early attempts to characterize the molecular background of gliosarcoma tumors have been based on case reports. In recent years, genetic alterations in primary and secondary gliosarcoma have been characterized ,11,12,13utations ,11,12,13d to GBM ,11,14. SAlthough histopathologically distinct, gliomatous and sarcomatous compartments of gliosarcomas share specific genetic alterations and likely derive from a common clonal origin ,15,16. TDespite certain differences in molecular profile and histological characteristics compared to GBM, gliosarcoma is typically treated similarly to GBM. A number of early phase clinical trials are testing targeted therapies in unique molecularly characterized subsets of patients (baskets ). AvailaTP53 mutation was detected in two gliosarcoma specimens. The mismatch mutation in the mismatch repair gene MSH6 was found in the GSM3. Some mutations have not been detected in the primary Varscan2 analysis, but were found after a secondary manual inspection. In summary, somatic mutations were found in 7/10 gliosarcoma samples. A fraction of detected somatic variants was validated and confirmed by Sanger sequencing. Selection of variants for Sanger sequencing was done primarily for PTEN and NF1 variants as they were found to be the most frequent ones, depending on the availability of samples, some additional gene variants were validated.In search of somatic mutations, we analyzed DNA samples from 10 gliosarcoma specimens and matched blood DNA samples. We sequenced the target-enriched exomic regions of 664 cancer-related genes. We found at least one missense/nonsense, non-tolerated/not-benign somatic mutation with a minimal variant allele frequency (VAF) of 20% in 6 out of 10 gliosarcoma samples . The welNF1 (3/10 gliosarcoma samples), PTEN (2/10), RB1 (2/10) and PIK3R1 (2/10) genes with a minimal penetration of 15% (within a given sample) were found in 5/10 samples , it was recognized as a very confident indel call.Somatic coding indels in  samples . One indNF1 foci was evaluated, and the PTEN focal deletion was detected in two samples . The segmentation analysis of called CNVs revealed five chromosomal gains or losses: chromosome seven gain (four samples), chromosome 10 loss (three samples), chromosome 12 loss (two samples), chromosome 11 gain (one sample), and chromosome 19 loss (one sample) as seen in  samples .PTEN was the most frequently altered gene in gliosarcomas, as 7/10 specimens had somatic mutations, indels or focal deletions in the PTEN gene region. The NF1 gene was altered in 3/10 gliosarcomas due to indels. Somatic mutations in the NF1 gene have been reported in human GBM tumors [EGFR mutations/indels were not detected in gliosarcomas, the chromosome seven amplification (the region where the EGFR gene is located) was very frequent and occurred in 4/10 gliosarcomas, however no focal amplification was observed in the EGFR locus. Somatic alterations in the RB1 were found in 3/10 gliosarcomas, while mutations in the TP53 gene were detected only in 2/10 samples. Activating missense mutations of the PIK3CA gene, occurring in regions coding for the adaptor binding domain (ABD) as well as the C2 helical and kinase domains, have been reported in GBM [PIK3 genes in 4/10 gliosarcoma specimens, with the PIK3CA gene being mutated twice in one sample and the PIK3R1 gene harboring the indel in two samples. Novel missense somatic mutations (based on the COSMIC database) have been found in genes: CDKN2C, HDAC11, PAX6, PTCH1, USP6, and WSC1, novel somatic stop codon mutations have been found in NKX2-1 and OPTN genes. Novel frame shift indels were detected in NF1 and PIK3R1 genes in the patient GSM3, both with a very high penetration above 50%, suggesting that these two mutations are driving neoplastic transformation. A summary of genetic alterations in gliosarcomas is presented in NF1 and PTEN genes were validated by Sanger sequencing; a majority, except one, were positively validated, * details are depicted in PTEN and NF1 genetic alterations in the context of respective protein structures is depicted in In summary, M tumors ,20, amonM tumors ,22. Thoud in GBM . We founWe estimated tumor purity using absCNSeq library in R. We found that the purity of the tested samples varied from 0.35–0.72 , and compared their gene expression profiles to those of GBMs to estimate the similarity between those two entities. Six gliosarcoma samples were subjected to RNAseq analysis and the results were compared to data collected from eight GBM IDH-wildtype specimens. The analysis of TCGA GBM subtypes revealed that most gliosarcoma samples fall into a mesenchymal category (4 out of 6), while one sample falls into a classical subtype category, and one sample (GSM1) is between classical and mesenchymal categories .MLYCD, CFAP54, ATP9B, and TMEM212 were significantly downregulated in gliosarcomas when compared to GBMs  and a collagen COL18A1, in comparison to GBMs  (The Volcano plot A shows a to GBMs B. More iblastoma . When co COL3A1) C.Based on collected RNAseq data, we performed functional  analysis of genes differentially expressed between GBMs and gliosarcomas. Our analysis revealed that many genes coding for proteins involved in the regulation of gene transcription have lower expression in gliosarcomas than in GBMs A. MoreovRABGEF1 and GTF2IRD1P1 genes was present in three samples (PCBP2) was detected (RABGEF1) is implicated in the development of certain human cancers [PCBP2, Poly(C) binding protein 2, plays an important role in post transcriptional and translational regulation of various signaling molecules through direct binding to single stranded poly(C) motifs. PCBP2 has been reported to play a critical role in the development of multiple human tumors [RABGEF1–GTF2IRD1P1 fusion occurring in three gliosarcoma samples was confirmed by nested PCR analysis and Sanger sequencing  and RAS/MAPK (NF1) signaling pathways that are crucial for tumor growth [PTEN somatic mutations/indels were detected in 50% (5/10) of specimens and this frequency is higher than previously reported: 14% (2/14) and 38% (8/21), respectively [PTEN was 70% (7/10), suggesting that the PTEN alteration is crucial for gliosarcoma development , however a small sample size limits the strength of our findings and a larger validation cohort would be desired. If in the future, a therapy targeting PTEN gene will be introduced into clinics, gliosarcoma would be the perfect tumor for a clinical trial.Gliosarcoma is considered to be an IDH-wildtype subtype of GBM and gliosarcoma patients are typically treated according to the standards established for GBM. Previous genomic studies of gliosarcoma have focused on a limited panel of genes known to be mutated in GBM ,14, recer growth ,29. PTENectively ,14. In felopment . PTEN , and no single nucleotide mutation was found. The frequency of NF1 mutations in gliosarcoma was previously described to be 18% (2/11) [NF1 gene is frequently deleted in a mesenchymal type of glioblastoma, but the overall frequency of the NF1 gene deletion/mutation was reported to be around 30% in mesenchymal GBMs [PTEN or NF1 alterations was much higher in gliosarcoma than in GBMs, reported to be 41% for PTEN and only 10% for NF1 in GBM patients [NF1, PTEN, TP53, and PI3K similar as in other studies [PTEN somatic alterations are more frequent than previously reported and they are likely to drive gliosarcoma development.The frequency of % 2/11) . The NF1mal GBMs , similarpatients . We foun studies ,11,14, h . The NFWe also found the BRAFV600E mutation present in two patients , which is in contrast to the reported lack of BRAFV600E immunoreactivity in 48 gliosarcoma tumor sections , but in RB1 gene were found in 30% of samples (3/10). We found a lower frequency of TP53 mutations  than previously reported for the gliosarcoma cases: 64%, 7/11 [TP53 alterations in GBMs (28%) [Somatic alterations in the 4%, 7/11  and the Ms (28%) . These dEGFR amplification/overexpression in gliosarcoma tumors [EGFR amplification [EGFR locus was found, just whole chromosome amplification.CNV analysis showed a frequent amplification  of the chromosome seven region . Previoua tumors ,14, but fication . In our COL1A1, COL1A2), III (COL3A1) and VI  were highly upregulated in gliosarcomas, as seen in We report the results of the transcriptomic analysis of gliosarcomas. The most striking difference between gliosarcomas and GBMs is the collagen gene signature, suggesting a more mesenchymal-like and extracellular matrix (ECM)-rich environment. Collagens type I , University Clinic Heidelberg University  and Ludwig-Maximilians University . Each patient provided a written consent for the use of tumor tissues. Total DNA and RNA were extracted from fresh frozen gliosarcoma tissue samples using Trizol Reagent , following manufacturer’s protocol. Quality and quantity of nucleic acids were determined by Nanodrop  and Agilent Bioanalyzer .More than 90% of known pathogenic mutations registered in the NCBI ClinVar database are located in protein-coding DNA sequence (CDS) regions. We used SeqCap EZ Custom Enrichment Kit, which is an exome enrichment design that targets the latest genomic annotation GRCh38/hg38. Our SeqCap EZ Choice Library covers exomic regions of 664 genes frequently mutated in cancer. A majority of genes (578) were selected from the Roche Nimblegene Cancer Comprehensive Panel (based on Cancer Gene Consensus from Sanger Institute and NCBI Gene Tests). Additionally, we included 86 epigenetics-related genes  based on a literature review ,40,41.DNA isolated from tumor samples was processed for library preparation according to a NimbleGen SeqCap EZ Library SR (v 4.2) user guide. Detailed description in http://bio-bwa.sourceforge.net/, [Fastq files from both tumor and blood samples were prepared as follows. Sequencing reads were filtered by trimmomatic program . Filterege.net/, ). The maBriefly, sorted and indexed BAM files from both tumor and blood DNA samples were used as an input for a Varscan2 analysis . SomaticBased on BAM files as an input VarScan2 called indels and assigned them as Germline, Somatic and Unknown. Somatic indels were passed to further analysis and evaluated by Variant Effect Predictor (VEP) from Ensembl. Only indels assigned by VEP as coding indels were selected. The final list of indels was stringently evaluated to comprise of only the indels located within regions that were at least 20× covered in a tumor sample and a variant itself showed at least 15% penetration (15% or more reads were affected by the indel). Some exceptions from this rule will be discussed separately.https://bioconductor.org/packages/release/bioc/html/GenVisR.html) [Sorted and indexed BAM files from both tumor and blood samples were used as an input for VarScan2 analysis in a CNV mode. Results from Varscan2 were segmented and prepared for visualization by DNA copy R library . CNV prosR.html) .Tumor purity estimation  was based on somatic mutations called by a VarScan2 program and CNV called by VarScan2 program in CNV mode. R library absCNseq  was usedSequencing reads (fastq) were filtered by the trimmomatic program, discarding all reads contaminated by the sequencing adapter sequence, as well as discarding reads in a bin of 20 bp reads had mean quality below Q30. The minimal length of the read used for mapping was set to 75 bp. Quality filtered reads were aligned to the human genome (hg38) by Tophat2 program . Evaluatp-value below 0.05). The functional analysis of data was performed using g:Profiler to find statistically significant Gene Ontology terms, pathways and other gene function related terms [https://tcga-data.nci.nih.gov/docs/publications/gbm_exp/) and classified our samples to known subtypes.Functional analysis was performed for the genes that were differentially expressed in GBM and gliosarcomass, as validated by the t-test  were diagnosed as gliosarcomas in Heidelberg and Munich hospitals by combining reticulin and GFAP staining. One sample (GSM2) was diagnosed using a combined VIMENTIN and GFAP staining. For the detection of selected proteins, we performed a staining on 5-μm paraffin-embedded tissue sections. Sections were deparaffinized by incubation in xylene, ethanol , and rehydrated. Epitopes were retrieved by oven boiling in a pH 6.0 citrate buffer for 40 min. Endogenous peroxidase was blocked in 0.3% H2O2 in methanol for 30 min followed by blocking with 10% horse serum. Sections were incubated overnight at 4 °C with mouse anti-COL6A3 (A-5) antibody  or mouse anti-COL3A1 (B-10) antibody , respectively. Next, sections were washed in PBS, incubated with a biotinylated horse anti-mouse immunoglobulin , then with horseradish peroxidase-conjugated avidin  for 60 min and with 3,3′-diaminobenzidine (DAB). Sections were stained with hematoxylin , washed in PBS, and mounted. Immunohistochemical staining for GFAP was performed by an automatic stainer BenchMark Ultra  using an anti-GFAP mouse primary antibody . To visualize the mesenchymal (sarcomatous) component of gliosarcoma, the Gomori’s silver staining was performed using a modified protocol .More detailed methods are described in the PTEN, PI3K) and RAS/MAPK  signaling pathways that are crucial for tumor growth and therapy resistance. Additionally, no gliosarcoma samples carried IDH1/2 gene mutations, placing gliosarcomas among an IDH wild-type subtype of glioblastoma. Our findings demonstrate that gliosarcomas, in terms of somatic alterations, are fairly similar to GBMs, with a higher frequency of NF1 and PTEN alterations, more similar to frequencies observed in mesenchymal GBMs. In terms of the transcriptomic profile, these tumors exhibit more of the mesenchymal signature, with different patterns of focal adhesion and cell invasion related genes. In the context of existing and emerging therapies, the current study shows that PI3K and BRAF inhibitors could be useful in targeted therapy for gliosarcoma patients, and that the molecular profiling of gliosarcoma could be very helpful to find fitting clinical trials or the off-label use of drugs.We demonstrate that most gliosarcoma tumors have somatic alterations of PIK3/Akt (


The highest concentrations of total PFAAs were detected in the western rivers. The effluents from seven STPs were likely important sources of PFAAs in surface water, and the amount of the daily fluxes in the effluent were 132 g for short-chain PFAAs and 109 g for long-chain PFAAs. PFAAs were widely detected in Chinese icefish  collected from Lake Chaohu, with maximal concentrations ranging from 1.79 ng g−1 to 50.9 ng g−1. The logarithmic bioaccumulation factors of perfluorodecanoic acid , perfluorooctane sulfonate  and perfluorononanoic acid  indicated the bioaccumulation of these long-chain PFAAs. This study is useful for enhancing our understanding of the pollution profiles of PFAAs and their environmental health risk in the freshwater lake.The perfluoroalkyl substances (PFAAs) have received growing attention in recent years as emerging contaminants in the aquatic environment. The occurrence, removal and bioaccumulation of fourteen PFAAs  were investigated in Lake Chaohu, China. The concentrations of the selected PFAAs in inflowing river, lake water and sewage treatment plant (STP) samples were analyzed by ultra performance liquid chromatograph (UPLC–MS/MS). The results showed that perfluorohexanesulfonic acid (PFHxS), perfluorobutane sulfonate (PFBS), perfluorobutyric acid (PFBA) and perfluoropentanoic acid (PFPeA), instead of perfluorooctanoic acid (PFOA), were predominant PFAAs in the inflowing river and lake water with maximum concentrations in the ranges of 52.2–1866 and 27–236 ng L Chlorella vulgaris and endocrine disrupting effects in male rare minnow [Perfluoroalkyl substances (PFAAs) are organofluorine compounds in which all the hydrogen atoms on the carbon chain are replaced by fluorine atoms . These Pe minnow ,13,14. TSewage treatment plants (STPs) are considered as a significant source of PFAAs to aquatic environments ,16,17. TNeosalanx tangkahkeii taihuensis in Lake Chaohu.Lake Chaohu is the fifth largest freshwater lake in China, which serves as a drinking water resource, irrigation water, aquaculture farms and a recreational attraction. With the rapid economic growth and urban development during the last several decades, the lake has been seriously polluted by wastewater and waste from industry, agriculture and daily life. Many studies have shown that Lake Chaohu is a significant sink for organic pollutants ,30,31,324HF7O2), perfluoropentanoic acid  and perfluorohexanoic acid , eight long-chain PFCAs of perfluoroheptanoic acid , PFOA (C8HF15O2), perfluorononanoic acid , perfluorodecanoic acid , perfluoroundecanoic acid , perfluorododecanoic acid , perfluorotridecanoic acid  and perfluorotetradecanoic acid , one short-chain perfluoroalkyl sulfonic acid (PFSA) of PFBS (C4F9O3S) and two long-chain PFSAs of PFHxS (C6F13O3S) and PFOA (C8H17O3FS). Perfluoro-n- butanoic acid (13C4-PFHeA), perfluoro-n-octanoic acid (13C4-PFOA), Perfluoro-n-nonanoic acid (13C5-PFNA), perfluoro-n-decanoic acid(13C2-PFDA), perfluoro-n- dodecanoic acid (13C2-PFDoDA), perfluoro-n-undecanoic acid (13C2-PFUnDA), sodium perfluoro-1-hexane(18O2) sulfonate (18O2-PFHxS) and sodium perfluoro-1- octanesulfonate (13C4-PFOS), were used as internal standards. All of the analytical standards (purity ≥ 95%) were purchased from J.T. Baker. Ammonium acetate (purity ≥ 98%) and methanol (HPLC grade) were obtained from TEDIA .Fourteen PFAAs were selected as target chemicals, including three short-chain PFCAs of perfluorobutyric acid  and the lake . Grab samples of influent and effluent water from seven sewage treatment plants  were collected to evaluate the mass loads of PFAAs from major STPs in Hefei City. Furthermore, fish  samples were collected from a fishing boat in Lake Chaohu to assess the potential levels of PFAAs to fish. This fish species is one of the dominant and main economic species of Lake Chaohu [Water and unsexed fish samples were collected in November 2015. The detailed locality of the sampling sites is shown in e Chaohu . In addie Chaohu . Fish tr® WAX cartridges . The cartridges were preconditioned with 1% ammonium hydroxide in methanol and followed by pure water. A flow rate of 5 mL min−1 was maintained through the cartridges. The cartridges were then washed with pure water and were dried completely under vacuum. Afterwards, the target compounds were eluted in 4 mL of methanol and 4 mL of 1% ammonium hydroxide in methanol into a PP tube. The eluent was concentrated to approximately 20 µL under a gentle nitrogen stream and reconstituted using 1 mL of the sample diluents (50% methanol in water).River water, lake water and wastewater samples were extracted in triplicate according to previous methods ,41. BrieFish samples were extracted and cleaned up according to a method previously reported by He and Chen  with som−1. The gradient elution started with a 0.2 min isocratic step at 10% of solvent B, then was ramped to 90% in 0.5 min, held at 95% for 1 min and brought back to 90% at 6.5 min. Quantification of PFAAs was performed on a triple quadrupole mass spectrometer (Waters TQs) with electrospray negative ionization in multiple reaction monitoring (MRM) modes. Two MRM transitions were used to identify all the target PFAAs compounds (except for PFBA and 13C4-PFBA), while one transition was chosen for the quantification of target compounds. For PFBA and PFHxA (as they can only provide one MRM transition), the confirmation was performed based on one MRM transition in addition to the retention time. The instrumental parameters were optimized to reach the maximum response . The UPLC-MS/MS method was modified from a previous study . Chromatresponse .A sample blank was routinely included in each batch of samples to eliminate any external source of contamination. Method blanks, prepared with 0.5 L Milli-Q water according to the same sample pretreatment procedures, were used to assess potential contamination during sample extraction, cleanup and analysis. In addition, an isolator column  was equipped between the autosampler and the pump to eliminate the potential effect of polyfluoroalkyl substances on the solvent or pipe system.−1 in surface water, 0.04–0.6 ng L−1 in influent water, 0.02–0.30 ng L−1 in effluent water and 0.05–0.5 ng g−1 (wet weight) in fish samples, respectively. The final concentrations of PFAAs reported in this study were not recovery corrected.Matrix-spike recoveries of individual PFAAs were obtained by spiking the 14 target analytes (5 ng for each compound) into randomly collected surface water, influent water, effluent water and fish samples. The recovery tests were performed in quintuplicate, with maximum relative standard deviations (RSDs) of less than 16%. Recoveries of PFAAs  spiked i−1 and 17–215 ng L−1 in influent water and effluent water, respectively. Among the detected PFAAs, PFOS (C8), which belongs to long-chain PFSAs, was the predominant PFAA in influent water with a maximal concentration of 333 ng L−1 found in STP6 followed by PFOA (C8) with a maximal concentration of 163 ng L−1 found in STP6. For all influent samples, the total concentrations of short-chain PFAAs (ΣPFAAs) in STP1 to STP6 ranged from 10.8 to 19 ng L−1 and were obviously lower than long-chain ΣPFAAs (94 to 515 ng L−1), while short-chain ΣPFAAs (214 ng L−1) displayed higher levels than long-chain ΣPFAAs (126 ng L−1) in STP7. This finding may be attributed to the larger use of short-chain PFAAs in the area of STP7, which is near the car manufacture industry and chemical industry area. According to the concentration of PFAAs in effluent samples, the long-chain PFAAs were obviously eliminated in STP1, STP2, STP3, STP4 and STP6, with removal rates ranging from 67% to 95%. Furthermore, PFOS was not detected (or the concentration was below the LOQ) in the effluents of STP1, STP2, STP3, STP4 and STP5, while concentrations ranged from 27.3 to 57.7 ng L−1 in influent water. Similar results were reported by Zhang et al. [Koc values with increasing carbon chain length [STPs play an important role in the protection of the aquatic environment around the city cycle. Influents of STPs are likely to receive PFAAs from industrial and household sources . Howeverg et al. , and then length .−1) in effluents × the daily discharge (m3/d). As shown in The daily mass flux of PFAAs (g/d) in Lake Chaohu from effluents in STPs was calculated using the following equation: Mass flux (g/d) = the concentrations of PFAAs  and PFSA (4 C) and long-chain (7–14 C) PFCAs and PFSAs (4–6 C) in inflowing rivers were 32.6–101 ng L−1, 11.5–29.7 ng L−1 2.44–2576 ng L−1 and 1.87–49.4 ng L−1, respectively  were generally higher than those in eastern rivers  and southern rivers (Baishishan River and Zhaohe River). The highest pollution level was observed in the Paihe River with a maximum total concentration of 2743 ng Ll ethers  and antil ethers  were fou−1), PFBA (12.7–46.2 ng L−1) and PFBS (4.39–27 ng L−1). With regard to the long-chain PFAAs, high levels were found for PFHxS (3.44–168 ng L−1) and PFOA (17.1–33.28 ng L−1).All 14 selected PFAAs were widely detected in Lake Chaohu  with fre−1 at S13. The source of the Paihe River (2742 ng L−1 in river water) resulted in a higher concentration of PFAAs (420 ng L−1) in the estuary site (S5) compared to the Zhaohe River estuary site (82.1 ng L−1 at S7), suggesting a greater contribution of input of PFAAs by cities compared to rural areas. Furthermore, the PFAAs at sites of villages and towns near the North Lake (S9–S12) clearly showed the effects of industrial activities on Lake Chaohu.The detailed concentrations of 14 PFAAs from S1 to S14 are shown in The profile of PFAAs in rivers and the lake is illustrated in In addition to the changes in the composition of PFAAs in Lake Chaohu, the levels of almost all target compounds were elevated in comparison with those sampled in 2011 . In partThe concentrations of PFAAs in fish samples are shown in −1) was found to be the dominant PFAA in fish samples. This observation is consistent with several previous studies, which found that PFOS was predominant in marine and freshwater fish samples from China, Greece and US [−1), PFDA , PFPeA  and PFUnDA  were the four dominant PFCAs detected in fish. The predominance of C10 and C11 PFCAs is similar to that reported for fish samples previously collected from Lakes Chaohu and Taihu [−1, indicating the low bioaccumulation potential of this compound in fish.All target PFAAs were detected in fish samples. PFOS, PFOA, PFBA, PFPeA, PFDA, PFUnDA and PFDoDA were detected in all the samples, while PFTriDA (92%), PFNA 85%) and PFTDA (85%) were found in the majority of the samples. PFHxA, PFHpA, PFBS and PFHxS were less frequently detected (<55%). PFOS (0.44–50.6 ng g5% and PFe and US ,49,56,57nd Taihu , the higBioaccumulation is a process in which chemical substances are absorbed in organisms via multiple exposure pathways including water and food . The bioThe present study showed that the target PFAAs were widely distributed in rivers, surface waters, wastewater and biota samples in Lake Chaohu. The prevalence of PFAA effluents of seven STPs demonstrated that traditional secondary treatment processes were not sufficiently efficient to completely remove target compounds. PFHxS and short-chain PFAAs were predominant in surface water samples, suggesting that they are being increasingly produced and applied due to regulations on PFOS and PFOA. Western inflowing rivers, such as the Paihe and Naifei Rivers, were the primary import routes of the PFAAs to Lake Chaohu. Additionally, the contamination levels of PFAAs in the western area of Lake Chaohu were generally higher than those in other areas, suggesting the remarkable effects of human activities on the residues of PFAAs in the aquatic environment. The high bioaccumulation potential in fish species was observed for PFDA, PFOS and PFNA. Further work is needed to study the bioaccumulation of PFAAs in organisms at different trophic levels in Lake Chaohu.


Today, with the existence of genes resistant to every natural and synthetic antimicrobial compound, national surveillance systems track AMR in human and animal populations to deepen our understanding of resistance and find ways to circumvent it. Although we have established surveillance systems across North America and Europe, pathogens do not respect international boundaries, and the emergence of resistance in any country poses a worldwide threat. In this issue of the AJTMH, Hedman et al.2 report spillover of AMR to developing world settings with no prior history of agricultural antimicrobial use. We are reminded that surveillance must become a global “One Health” effort if we are to solve one of today’s most significant threats to human, animal, and environmental health.In 1940, one year before the first administration of penicillin in man, two members of the team who discovered the drug revealed that resistance to penicillin already existed.3 spread of metallo-beta-lactamase-1 in India,4 and the emergence of plasmid-mediated carbapenem-resistant Enterobacteriaceae in swine for the first time in the United States.5 Leading world health agencies consider the threat of AMR as paramount and recognize its complex causation: expanding human and domestic animal populations; increased globalization, international trade, and demand for animal source foods; and all-too-easy access to antimicrobials in both developed and developing countries. The proficiency of genome evolution via horizontal gene transfer and the emergence of new forms of resistance have compounded the lack of new antibiotic discovery and development, while intensifying the threat posed by drug-resistant pathogens. By 2050, an estimated 10 million human lives per year will be at risk if we fail to attenuate the rise of drug resistance, and critical medical procedures such as administration of cancer chemotherapy, joint replacement, and gastrointestinal surgery may be associated with increasing morbidity.6 The increase in AMR burden correlates with a 65% increase in antimicrobial consumption in humans between 2000 and 2015 in 76 countries7 and administration of 63,000 tons in animals in 2010, with a projected 67% increase in consumption by 2030.8Antimicrobial resistance has reached its tipping point, and some are saying we are now in the post-antibiotic era. Recent reports have highlighted this trend, including the emergence of multiple plasmid-mediated colistin resistance genes in human and animal pathogens,Antimicrobial resistance poses a particularly significant threat to low- and middle-income countries. This is due not only to the health-care challenges these countries face, but also to an increase in small-scale intensive animal production, exacerbated by poor sanitation infrastructure. The findings reported by Hedman et al. in this issue exemplify this problem and the difficulty of understanding the complicated dynamics of AMR transmission between humans and animals sharing the same environment. The researchers investigated the prevalence of CTX-M extended-spectrum beta-lactamases in chickens from small-scale poultry farms and in children living on the farms in rural Ecuador. CTX-M-mediated cephalosporin resistance was seen in bacteria both in commercially bred “broiler” chickens treated with high levels of antibiotics and in free-grazing animals that had no direct exposure to antibiotics. Resistance was also detected in bacteria from children in the community. After phylogenetic analysis, the authors reported a shared evolutionary history between chicken and human samples. Hedman et al., thus, provide valuable insight into the rise of phenotypic resistance and avian-to-human spillover in areas that have previously reported low AMR levels in both poultry and humans.9Altogether, the data provided by Hedman et al. support a familiar narrative: gene exchange is a property of bacteria that efficiently enables the transmission of resistance between animals and humans. Of particular importance to surveillance systems, the study also highlights the pivotal role of the environment in AMR transmission. The ability of the environment to act as a reservoir for resistance is not a new concept and may have promoted the potential spillover event described by Hedman et al. in Ecuador. Indeed, the environmental AMR resistome consists of more than one million distinct bacterial species, which markedly exceeds the number of species that infect human and animal populations.Salmonella serotypes across commercial swine farms following manure application.10 In addition, numerous studies have reported very little difference in the shedding of drug-resistant bacterial strains between animals raised under organic or antimicrobial-free production systems.14 Combined with studies such as that conducted by Hedman et al., these findings demonstrate the need to apply a One Health approach and study environmental reservoirs more closely, rather than focusing only on the resistance that arises following antimicrobial administration.18Despite the knowledge of environmental influences on AMR, current surveillance systems often neglect environmental sampling. It is now crucial that we re-emphasize the role that the environment plays as a reservoir and in maintaining AMR genes as we establish surveillance systems to combat AMR. We know that many of the resistance mechanisms we see in veterinary clinics and animal production systems likely have environmental origins. Recently, we have reported horizontal dissemination of resistance determinants in multiple 19 At the country level, the authors suggested that improving sanitation, ensuring good governance, better access to clean water, increasing expenditure on improving public health care, and better regulation of the private health sector were all required for reducing AMR. Factors including poor sanitation, warmer temperatures, and higher corruption levels were consistently associated with a higher prevalence of AMR strains. Without harmonizing surveillance between nations, we will never know the extent of the AMR challenge, nor will we be able to combat it effectively. We are already taking strides to ameliorate this problem. In 2015, the World Health Organization launched the Global AMR Surveillance System (GLASS) to establish a standardized GLASS. So far, 40 countries are participating,20 and by collecting and analyzing AMR, epidemiological, clinical, and population-based data from these countries, systems such as GLASS can generate actionable data, improve analysis, influence policy decisions, and ultimately reduce the burden of AMR worldwide.Importantly, combating AMR will also require global cooperation because decreasing the use of antimicrobials only in one population or in one country will not necessarily attenuate the spread of resistant strains. Collignon et al. conducted a multivariate analysis based on antimicrobial consumption data from 63 countries to determine the role of anthropological and socioeconomic factors in the global spread of AMR.


Four different self-made free surface electrospinning (FSE) techniques, namely, modified bubble-electrospinning (MBE), modified free surface electrospinning (MFSE), oblique section free surface electrospinning (OSFSE) and spherical section free surface electrospinning (SSFSE), designed for high-throughput preparation of high-quality nanofibers, are presented in this paper. The mechanisms of fiber preparation of the corresponding four FSE devices were studied by simulating the electric field distribution using the Maxwell 3D software. The properties of the electric field in the device are very important for the FSE process. The effects of the particular technique on the morphology and the yield of nanofibers were experimentally investigated. The experimental data agree well with the results of the simulations and show that all four FSE devices can be used to prepare large quantities of high-quality nanofibers. A comparison of the spinning mechanisms of these four FSE devices illustrates that the SSFSE device performs best, providing the highest quality and yield of nanofibers. The SSFE device could yield 20.03 g/h of nanofibers at an applied voltage of 40 kV. Due to their excellent properties, such as high surface-to-volume ratio and high porosity, nanomaterials have become more and more important in industrial manufacturing. As one of the most important methods for preparing nanomaterials, electrospinning (ES) –3 has reAccordingly, in recent years, many needleless electrospinning methods were presented to obtain the high-throughput production of nanofibers –23. Niu In our previous work –22, a moAs shown in The effects of the MBE, MFSE, OSFSE and SSFSE device design on the morphology and the yield of the produced nanofibers were experimentally investigated. The differences between them were explained based on simulations of the electric field distribution using the Maxwell 3D software. Our results show that all four FSE devices can be used to prepare large quantities of high-quality nanofibers, whereas the SSFSE aparatus is the optimal FSE device, providing the highest quality and the highest yield of nanofibers.MW = 150,000) was purchased from Beijing Lark Branch Co., Ltd. . SDBS was purchased from Sinopharm Chemical Reagent Co., Ltd. . N,N-Dimethylformamide (DMF) was provided by Shanghai Chemical Reagent Co. Ltd. . The spinning solution was obtained by dissolving 10 wt % PAN and 1 wt % SDBS in DMF under magnetic stirring at 60 °C for 4 h using a thermostatic magnetic stirrer  to yield a transparent liquid.Polyacrylonitrile , as shown in The jet initiation in the OSFSE process viewed from the side by a high-speed camera is shown in F1 produces a centripetal force resulting in the shrinking of the radius of the whipping circle. The resulting vertical force component F2 provides the kinetic energy of the jet, impelling the stretch of the jet towards the copper mesh collector. The force components are calculated as follows:v is the velocity of the jet, a and b are constants to be further determined theoretically or experimentally, qA is the signed magnitude of the charge at point A that is determined by the nature of the spinning solution, and E is the electric field intensity that is calculated using the applied voltage and the working distance.where According to the above equations and figures, the distribution of the electric field intensity plays a very important role in the FSE process since multiple jets form on the entire spinning area. To optimize the electric field distribution while keeping the applied voltage constant, the solution reservoir of the FSE apparatus was modified yielding the MBE, MFSE, OSFSE and SSFSE devices. It is necessary to simulate the distribution of the electric field intensity in the different FSE processes to illustrate and compare their spinning performance.11 µs/cm, the electric conductivity of the polymer nozzles in the MBE was 0 µs/cm, the electric conductivity of the PAN-DMF solution with a concentration of 10 wt % PAN and 1 wt % SDBS was 2372 µs/cm, the working distance was 180 mm and the applied voltage was 40 kV.The distribution of the electric field between the solution reservoir and the collector in the four different self-made FSE devices was simulated using the Maxwell 3D software. The simulations were carried out using the following experimentally realized parameters: the copper reservoir as positive pole was a cylinder with a diameter of 40 mm and a height of 30 mm, the bulk conductivity of copper was set to 5.8 × 10The morphology of the PAN nanofibers was characterized by scanning electron microscopy (SEM) . The matrix morphology and the diameter of the fibers were characterized using the Image J software  to study 100 randomly chosen nanofibers in 50 SEM images. The mass of the PAN nanofibers was measured as follows using a precise electronic balance :W is the yield of nanofibers, W0 and W1 are the weights of the aluminum foils before and after spinning and t is the spinning time.where The results of the simulations of the electric field inside the MBE, MFSE, OSFSE and SSFSE devices are illustrated in To further compare the spatial distribution of the electric fields of the MBE, MFSE, OSFSE and SSFSE devices, a parameter ƒ is introduced as follows:Emax is the maximum electric field intensity and Eav is the average electric field intensity.where Emax, Eav and ƒ are listed in f-values of the four FSE devices. Hence, the axial distributions of the electric field are similar for the four FSE processes. The Emax values are the largest for the SSFSE device.Emax, Eav and ƒ are are given in ER) along the radial axis (0–20 mm) of the four self-made FSE devices.f-value of the MBE device is the largest, while that of the MFSE device is the smallest. This means that the electric field distribution inside the MBE device is the least uniform due to the influence of the polymer nozzle. In fact, most of the jets develop mainly on top of the bubble produced by the nozzle and near the top edge of the copper reservoir, as shown in f-value of the SSFSE apparatus is relatively small, and its electric field intensities are the largest. Hence, multiple jets are produced on the entire solution surface, as displayed in From The morphology of the PAN nanofibers produced by the four self-made FSE devices was investigated by SEM. f-value of the FSE device, the more uniform the electric field distribution, resulting in a more uniform diameter distribution of the nanofibers. Indeed, the diameter distribution of the nanofibers produced by the MFSE and SSFSE methods is more uniform as a result of the corresponding smaller f-value. The quality of the nanofibers prepared by the MBE, MFSE, OSFSE and SSFSE methods was compared to each other, and it could be found that the quality of the nanofibers obtained by the SSFSE approch was highest, in line with the results of the electric field simulation.The results from In this paper, we presented four different self-made FSE devices, namely the MBE, MFSE, OSFSE and SSFSE devices, designed to obtain high-quality PAN nanofibers in large quantities. Using the Maxwell 3D software, the spinning mechanisms of the four FSE devices were studied by simulation of the electric field distribution, which is very import in the FSE process. The simulation results showed that the electric field distribution of the MBE device is the most nonuniform, and the electric field intensities inside the MFSE device are the smallest. The SSFSE device showed the best spinning performance owing to the uniform distribution of the electric field inside the device, as indicated by the ordered electric field vectors, and the largest resulting electric field intensity.Furthermore, the impact of the device design on the morphology and the yield of nanofibers was experimentally investigated. We found that the average diameter of the nanofibers prepared by the MFSE method was the largest. The size distribution of the nanofibers produced by the MBE device was the least uniform. In contrast, the size distributions of the nanofiber generated by the of MFSE and SSFSE devices were more uniform. These experimental data are in accordance with the results of the simulations and the theoretical analysis. Our results show that the SSFSE is the best performing FSE device since it provides the highest quality and the maximum yield of nanofibers. Its yield could reach 20.03 g/h at an applied voltage of 40 kV. Compared with the performance of other spinning devices reported in the literature ,17,20, t


The current treatments of Parkinson disease (PD) are ineffective mainly due to the poor understanding of the early events causing the decline of dopaminergic neurons (DOPAn). To overcome this problem, slow progressively degenerating models of PD allowing the study of the pre-clinical phase are crucial. We recreated in a short ex vivo time scale (96 h) all the features of human PD (needing dozens of years) by challenging organotypic culture of rat substantia nigra with low doses of rotenone. Thus, taking advantage of the existent knowledge, the model was used to perform a time-dependent comparative study of the principal possible causative molecular mechanisms undergoing DOPAn demise. Alteration in the redox state and inflammation started at 3 h, preceding the reduction in DOPAn number (pre-diagnosis phase). The number of DOPAn declined to levels compatible with diagnosis only at 12 h. The decline was accompanied by a persistent inflammation and redox imbalance. Significant microglia activation, apoptosis, a reduction in dopamine vesicle transporters, and the ubiquitination of misfolded protein clearance pathways were late  events. The work suggests inflammation and redox imbalance as simultaneous early mechanisms undergoing DOPAn sufferance, to be targeted for a causative treatment aimed to stop/delay PD. Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease . The corNowadays is clear that PD is a multifactorial pathology. Several genes have been identified as risk-factors, but very few cases (about of 5–10%) are related to genetic mutations, and the majority of Parkinson cases are classified as sporadic ,8. OlderFrom a molecular point of view, an incredible amount of work has been done in the past decades in every available experimental model  to unravel the molecular determinants of dopamine demise. Demonstrated components are oxidative stress, inflammation, microgliosis, excitotoxicity, apoptosis, autophagy, mitochondrial dysfunction, alterations in the endosomal-lysosomal functioning, vesicle trafficking, and the ubiquitination mediated clearance of unfolded protein, until the most recent involvement of epigenetics ,16,17,18Nevertheless, the etiology of PD is still elusive ,8: a comTo unravel the onset of DOPAn demise, a dynamic (time-course) slowly progressing approach comparing multiple potential causative mechanisms is crucial . This isFor this reason, we decided to address the time course of the molecular events ongoing to DOPAn demise by developing an ex vivo chronic (96 h) model of PD characterized by a slow and progressive degeneration of dopaminergic cells of the SN, reproducing all the features of the human pathology (needing dozen of years) . The priTo gain access to the early, causative molecular mechanisms undergoing DOPAn sufferance, possibly preceding diagnosis, it was crucial to set-up a slow progressive PD model. To reproduce all the features of human pathology in an in vitro time scale (96 h), we combined the rat brain organotypic cultures of substantia nigra, maintaining in vitro the architecture, cellular heterogeneity, and metabolic features of the in vivo tissue, with low dose of rotenone. Rotenone is a pesticide known to induce PD in human ,25,26, aBecause the cutting procedure to generate brain slices determines an important tissue sufferance, as first we followed the LDH release  during the first days in culture, without any challenging. Because challenging already suffering slices may introduce bias in the model, we waited the normalization of LDH (indicative of slices recovery). As shown in The concentration of rotenone required to develop a slow progressive demise of DOPAn was experimentally determined. After recovery from the stress due to the slicing procedure It has been established that most of the diagnoses occur when the degenerative process is advanced, with an estimated death of 40–60% of DOPAn, leading to the loss of 80% of putamen dopamine content ,11,29,30p < 0.01) in challenged slices. This decrease is considered not sufficient to lead to symptom [p < 0.001), the reduction of the number of neurites stabilizes to values of about of 50–60% of controls. The results at 12–96 h agree with Cheng [The first sign of DOPAn sufferance was the reduction of the number of neurites b, droppi symptom , thus itth Cheng , estimatth Cheng . Moreoveth Cheng ,32 of 1–th Cheng . No chanp < 0.01, p < 0.05, p < 0.05, respectively). This picture well reproduces the human pathology as described by the Braak’s staging [The number of TH+ DOPAn was not significantly affected at the early times 3–12 h, c, reachi staging  and HY s staging . Specifi staging  and Braa staging ,33,35, w staging ,35,36, cp < 0.001, p < 0.05 and p < 0.01, respectively). No changes in the length of neurites were detected in control OBCs all long the experimental period.Similarly, the neurite length d startedAltogether, the morphological analysis confirms we reached the goal of creating a slow progressing ex vivo system, supporting its use to represent the human pathology and allowing the analysis of the early (possibly causative) mechanisms ongoing DOPAn sufferance. As it is, the 3 h experimental time will offer the opportunity to focus on an early temporal window (pre-diagnosis) actually not accessible in a clinic. To identify the earliest (causative) molecular events undergoing DOPAn sufferance and demise, we performed a time-dependent comparative study of selected markers for the principal possible mechanisms underlining DOPAn degeneration and PD . To thisHmox1), noticed up-regulated in PD patients and considered a redox sensor [Srnx1), an adaptive response to redox stress, potentially protecting DOPAn from ROS toxicity [Redox stress has been mentioned as the first-hint in PD development ,37. To mx sensor ,39,40,41toxicity ,42,43,44Tnfα [Cox2 [Il6 [Il1 β); and Cd68, the marker of microglia activation [Inflammation is a recognized component of PD. A heterogeneous panel of inflammatory molecules have been reported in literature, possibly representing different stages of the inflammatory cascade activated in the numerous models, as well during the progression of the diseases in patients. For this reason we chose multiple markers: Tumor necrosis factor alfa α Tnfα ,46,47), ,47, Tnfαfα [Cox2 ,49,50,51ox2 [Il6 ), interltivation ,54,55. Bdnf), reported decreased in serum of PD patients and correlating with motor impairment an DOPAn death [The brain-derived neurotrophic factor  [Uchl1/Park5), responsible for the clearance of misfolded/aggregated proteins , and reported as reduced in the cerebrospinal fluid of PD patients [Vmat2) participating in the intracellular vesicular system regulating the cytosolic level of free dopamine, with consequent ROS generation [We analyzed also the risk factors described to be involved in the development of PD: nuclein) ,60,61, cnuclein) ,62. The patients ,63,64,65neration ,67. Anxa5).Finally apoptosis ,68, consThe release of glutamate in the medium (Glu) was used to monitor glutamate neurotoxicity, leading to excitotoxicity and suggested enhancing DOPAn loss ,70.As reported in the previous section, at that time the only sign of DOPAn sufferance was the decreased number of neurites, in absence of changes in the number of TH+ DOPAn or neurite length . This exHmox1 (redox marker), Tnfα and Cox2 (inflammation). Hmox1 is up regulated of about 1.7 fold (p < 0.01), returning to basal levels at 12 h. Tnfα and Cox2 experience an increase of about 1.7 fold (p < 0.05) and 1.9-fold (p < 0.05), reverting rapidly thereafter to control levels (Tnfα), or picking (Cox2) at about 2.5-fold at 24 h, (p < 0.001) before reverting. The results suggest that redox imbalance and inflammation are the early and simultaneous effectors (causes) of DOPAn demise.As shown in Il6 and Srnx1 (Il6 is significantly increased at 12 h (p < 0.05), picking at 24 h  and then decreasing to control levels. Sxrn1 up-regulation  is maintained along the experimental period 48 and 96 h: both p < 0.05). The data suggest that, after the first hint, sustained inflammation and oxidative stress push TH+ DOPAn loss. At 12 h, the morphometric analysis of DOPAn presented a picture comparable with diagnosis . At thisnd Srnx1 , 12 h. IBdnf is significantly up-regulated (p < 0.001), falling below control levels at 48 and 96 h . At 24 h, we notice also a significant glutamate release (p < 0.05), continuing all long the 96 h .As shown in Anxa5, p < 0.05) starts, increasing even more later on .After 48 h of challenging, apoptosis  is increased, while Uchl1  and Il1b  are significantly down-regulated.Only at the end (96 h), microgliosis ; oxidative stress (glutathione reductase and glutathione peroxidase precursor), glutamate excitotoxicity (N-methyl-d-aspartate 2a receptor); and growth factors  were already altered. The immunohistochemistry also revealed a dramatic (compatible with diagnosis) decrease of TH+ DOPAn density. Altogether, these findings suggest that the experimental scheme applied , was too strong to create a slowly degenerative model, and indicating the need to inducing a more gradual damage, allowing to gain access to the pre-clinical phase and better unravel the temporal sequence of the molecular events in respect to the earliest DOPAn sufferance, before the starting of DOPAn loss. To the best of our knowledge, only one original study tried to compare the higher number of potential causative mechanisms by a time-course approach . In thisAs illustrated by The most important finding is that we obtained a temporal window , 3 h corHmox1) and inflammation markers . Redox imbalance is usually considered the first hint in PD [At the 3 h time (pre-diagnosis phase), we detected an increased expression of redox stress markers  [One of the adaptive mechanisms induced by ur model . In modend Il1β) , a featund Il1β) ,46, sleend Il1β) ,46,47, and Il1β) . The obsCox2 (and inflammation) is a first hit, already significantly up-regulated at 3 h , and picking at 24 h (Braak 4). The up-regulation of Tnfα at the same earlier (3 h) timing supports this interpretation.Persistent inflammation is a recognized player in PD ,53,79,80Il6 is upregulated at 12 h  and apoptosis (Anxa5) markers are also increased. Microgliosis is always detected in autopsy of PD brains [Lately on our model , in line with the clinical scenario [Snca) is one of the targets of Uchl1. Thus, the temporal windows we described in our model, may agree with the concept that dopamine loss precede synucleinopathy, as previously described [Among the final events of dopamine demise, we notice also the alteration of the ubiquitination mediated clearance of unfolded proteins  occurring in case of excessive oxidative stress [Uchl1 may be another  potential consequence of the inefficient adaptation to stress.Of notice, e stress . Thus, tBdnf , followed by a trend to decreasing under the level in controls thereafter (48/96 h). Altogether, the data may support the idea of the failure of the potential adaptive to stress mechanisms. Oxidative stress, and specifically Hmox1 modulation, has been linked also to the induction of growth factors  in animal models of PD , with coWe notice also an increase of glutamate release starting from 24 h, when DOPAn neurites loss starts , and perThe links between the damaging mechanisms reported in literature and fitting with our data, stress two critical aspects of the pathology: (1) The importance of blocking the early, inducing steps, to avoid the priming of the self-promoting and self-amplifying cascade of toxic events ongoing DOPAn degeneration, before it became uncontrollable.(2) Might explain the inefficacy of the current treatment schemes focused in facing a single molecular target. For this reason, and based on our results, we suggest a cocktail approach with a wide spectrum anti-inflammatory and an anti-oxidant.Admittedly, this study lacks of the assessment of the protein(s) content related to the genes variations. However, the selection of the genes we investigated was based on previous data showing a correlation between gene(s) and protein(s) variation ,61,64,65d-Glucose 10 mg/mL) until use. A McIlwain tissue chopper  was used to cut transversely 300 μm slices. Healthy slices were selected for structural integrity under stereomicroscope inspection. Slices were then transferred to sterile, semi-porous Millicell-CM inserts , fed by 1 mL of media and maintained at 37 °C, 5% CO2, 95% humidity in a humidified incubator. The media was changed the day after cutting and every two days thereafter. Slices were maintained in culture 10 days before starting treatment. The whole experimental procedure agreed with the Italian Law (Decree 87–848) and European Community Directive (86-606-ECC), and was authorized by the competent OPBA  of the University of Trieste .Organotypic cultures were prepared as described before, with few modifications . Wistar l-Glutamine (Life Technologies Corporation), 2% Penicillin/Streptomycin (Life Technologies Corporation), and 10 mg/mL d-Glucose (Sigma-Aldrich).To create an ex vivo chronic (96 h) model reproducing all the features of human PD (needing dozens of years), slices were challenged with low doses of rotenone. The medium used to culturing the slices was composed of 65% Basal Medium Eagle , 10% heat-inactivated Fetal Bovine Serum , 25% Hank’s Balanced Salt Solution (Sigma-Aldrich), 1% Rotenone (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) and diluted to the final concentration of 10 nM in complete medium. At each time point, a well was challenged with rotenone, and one well exposed to the same amount of DMSO required dissolving rotenone (controls).The amount of total extracellular LDH in media, indicative of membrane leakage, was determined using a CytoTox-ONE™ Homogeneous Membrane Integrity Assay , as previously described ). The suTo follow the morphological degeneration of DOPAn, the cells number, the number of dopaminergic neuron’s neurites, as well as their length were assessed at 3, 12, 24, 48 and 96 h after challenging with low doses of rotenone. Slices were immediately fixed in 4% neutral buffered formalin for 30 min at 4 °C, washed with PBS, and then glycine 0.1 M (Sigma-Aldrich) in PBS to remove residues of paraformaldehyde. After washing, slices were pre-incubated 1 h in 10% normal donkey serum (Sigma-Aldrich), 1% bovine serum albumin (Sigma-Aldrich) and 0.1% Triton-X100 (Sigma-Aldrich) in PBS (blocking solution). Then, slices were incubated three days at 4 °C with primary polyclonal antibody anti-tyrosine hydroxylase  in an incubation solution containing 1% normal donkey serum (Sigma-Aldrich), 1% bovine serum albumin (Sigma-Aldrich) and 0.1% Triton-X100 (Sigma-Aldrich). After rinsing three times in PBS for 5 min, slices were incubated with labeled donkey anti-rabbit Alexa fluor 546 secondary antibody  overnight at 4 °C in incubation solution. Slices were then washed twice with PBS, stained with Hoechst 33258 , washed with water and mounted . Fluorescence was detected by fluorescent microscopy Leica DM2000 .Tyrosine hydroxylase-positive (TH+) DOPAn, the number of neurites, and neurites length were counted using a Leica DM2000 fluorescence microscope (Leica Microsystems Srl) by three separate individuals. For DOPAn counting, all the TH positive cells in the 2/3 slices challenged with rotenone (or vehicle) at each experimental timing were counted under a 10× magnification.Fiji software (ImageJ) ,94 was uThe amount of extracellular glutamate, a marker of excitotoxicity, was quantified using a Glutamate Assay Kit . Briefly, after the rotenone exposure, the supernatant was collected, and the assay was performed according to the manufacturer’s instructions. The absorbance (450 nm), proportional to the glutamate released in the medium, was determined using an EnSpire Multimode Plate Reader (PerkinElmer). Glutamate release in media was expressed as fold change compared to the control slices.® RNA Isolation Reagent (Sigma-Aldrich), following the manufacturer’s instructions. Only mRNA samples passing the quality test  were retro-transcribed and used in the study. Complementary DNA (cDNA) was synthesized with the High Capacity cDNA Reverse Transcription Kit .Genes reported to be involved in the biomolecular pathways of neuronal degeneration were chosen as markers of these pathways. The mRNA expression of the genes of interest was analyzed by quantitative real-time PCR. Total RNA was extracted using TRI ReagentCox2 and Il1β, 500 and 750 nM, respectively) were diluted in the Sso Advance SYBER green supermix . The genes of interest were analyzed by performing the qPCR as follow: 95 °C for 3 min; a 40 times repetition of: 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. Finally, the reaction was stopped by an additional step at 72 °C for 5 min. For the quantitative real-time PCR (qPCR), primers were designed using the Beacon Designer 4.2 software  on rat sequences available in GenBank . The reaAt the end of the amplification, we performed a melting curve analysis to confirm the amplification of the desired targets. We never observed non-specific products, primer dimers, or contaminations.Ct method, taking into account the efficiencies of the individual genes and normalizing the results to the housekeeping genes  [The relative quantification was made using the iCycler iQ software, version 3.1  by the ΔΔ: Gapdh) ,96.p value ˂ 0.05. A p value < 0.05 was considered as statistically significant. The results are expressed as the mean ± standard deviation (S.D.). Details on the number of independent biological repetitions for each experiment are given in figure and table legends.Data were analyzed with GraphPad Prism version 5.00 for Windows . Statistical significance was evaluated by one-way analysis of variance (ANOVA), followed by a Tukey-Kramer multiple comparisons test when In this work, we describe the development of a slowly degenerative ex-vivo model of Parkinson disease, used to identify the early causative mechanisms of dopaminergic neurons sufferance. Based on our results, we suggest the simultaneous activation of inflammation and oxidative stress as triggers of DOPAn demise, possibly sustained by glutamate neurotoxicity. Thus, we suggest a cocktail approach with a wide spectrum anti-inflammatory, an anti-oxidant, and possibly a glutamate blocker.


In our paper , an erro


Neisseria gonorrhoeae. However, generating full scaffolds of N. gonorrhoeae genomes from short reads, and the assignment of molecular epidemiological information  to multiple assembled samples, is challenging due to required manual tasks such as annotating antimicrobial resistance determinants with standard nomenclature for a large number of genomes.Recent adva1nces in whole genome sequencing (WGS) based technologies have facilitated multi-step applications for predicting antimicrobial resistance (AMR) and investigating the molecular epidemiology of N. gonorrhoeae. For its evaluation, the Gen2Epi pipeline successfully assembled the WGS short reads from 1484 N. gonorrhoeae samples into full-length genomes for both chromosomes and plasmids and was able to assign in silico molecular determinant information to each dataset automatically. The assemblies were generated using raw as well as trimmed short reads. The median genome coverage of full-length scaffolds and “N” statistics  were higher than, or comparable to, previously published results and the scaffolding process improved the quality of the draft genome assemblies. Molecular antimicrobial resistant (AMR) determinants identified by Gen2Epi reproduced information for the 1484 samples as previously reported, including NG-MLST, NG-MAST, and NG-STAR molecular sequence types.We present Gen2Epi, a pipeline that assembles short reads into full scaffolds and automatically assigns molecular epidemiological and AMR information to the assembled genomes. Gen2Epi is a command-line tool integrating third-party software and tailored specifically for ftp://www.cs.usask.ca/pub/combi or ftp://ftp.cs.usask.ca/pub/combiGen2Epi can be used to assemble short reads into full-length genomes and assign accurate molecular marker and AMR information automatically from NG-STAR, NG-MAST, and NG-MLST. Gen2Epi is publicly available under “CC BY-NC 2.0 CA” Creative Commons licensing as a VirtualBox image containing the constituent software components running on the LINUX operating system (CentOS 7). The image and associated documentation are available via anonymous FTP at The online version of this article (10.1186/s12864-019-5542-3) contains supplementary material, which is available to authorized users. Neisseria gonorrhoeae (Ng), is a global public health problem, magnified by high levels of resistance to antimicrobial agents , and a second study recently published by Harris et al. [Several experiments describe the use of WGS assemblies to understand the molecular epidemiology of gonococcal isolates –18, incls et al.  along wis et al.  to assems et al. . The webs et al.  is the lThere have been a number of methods and pipelines developed to assemble bacterial contigs into scaffolds; for instance, Ragout , SSPACE N. gonorrhoeae, different assemblers, pipelines, and web applications have been utilized to analyze the genomes [Neisseria community is the Nullarbor pipeline, which performs complete analysis, from read cleaning to variant calling [In published studies for  genomes , 26–28.  genomes , Velvet  genomes , and CLC calling . HoweverN. gonorrhoeae genomes from assembled short reads, and the assignment of in silico molecular marker information to multiple samples, is challenging due to required, typically manual steps, such as annotating antimicrobial resistance determinants with the standard nomenclature for a large number of genomes. Here, we present a one-step computational WGS pipeline, named Gen2Epi, to 1) assemble short read datasets into full-length genomes, and 2) assign molecular epidemiological and AMR designations to the assembled genomes automatically. We assessed the performance of the Gen2Epi pipeline by using Illumina short reads from 1484 whole genome sequencing samples from previously published studies [Despite the advances in WGS techniques, generating full scaffolds of  studies , 30, 31  studies , 30, 31.N. gonorrhoeae sequencing-read datasets from previously published studies  [N. gonorrhoeae datasets from New Zealand [N. gonorrhoeae reference genomes were downloaded from the NCBI [N. gonorrhoeae plasmids  . Full re, and Z)   , 31. The Zealand  download Zealand . Six samthe NCBI . SequencN. gonorrhoeae NCCP11945 for New Zealand [N. gonorrhoeae FA1090 N. gonorrhoeae reference strain genomes for Saskatchewan [During Gen2Epi testing, a number of reference genomes were utilized for the scaffolding process. For instance, available WHO reference genomes were used in the case of WHO datasets , N. gono Zealand  isolatesatchewan , 31 and atchewan  isolatesatchewan .ftp://www.cs.usask.ca/pub/combi or ftp://ftp.cs.usask.ca/pub/combi. Each sequential step of the pipeline are automatic, linked with each other and described below.Gen2Epi is a Linux-based command-line pipeline providing fully integrated analysis to assemble reads to scaffolds and link the assembled genomes to strain typing (NG-MLST and NG-MAST) and AMR determinant (NG-STAR) information in one place. The pipeline design integrates third-party software  using Trimmomatic  after ang Kraken  and Bowtg Kraken . The pipIn the second stage, the output from step one is used to generate contigs using separate de novo assemblies of chromosomes and plasmids. Raw or trimmed reads are assembled into contigs using SPAdes   from step two are joined into full scaffolds by using the reference-based scaffolding approach implemented in Ragout . The resin Mauve .N. gonorrhoeae plasmids   using BN. gonorrhoeae loci associated with AMR are assigned to each allelic combination by searching against AMR gene profiles and metadata , along with alignment and parsing, are carried out using a single custom script. The WHO sequencing read datasets and reference genomes, along with locally created plasmid databases used in step 4, are included within the Gen2Epi distribution.N. gonorrhoeae WGS samples into full-length genomes. Using the recommended hardware configuration (see Availability and Requirements below), full analysis required approximately 12 h of elapsed time to complete on a laptop with a 3.60 GHz Intel(R) processor. The detailed results of the full genome assembly evaluation in terms of measures such as median misassemblies, alignment length, and different “N” statistics  are shond NGA50 , full gend NGA50 , the assnd NGA50  was highnd NGA50 , 31 (93.nd NGA50  (92.62%)N. gonorrhoeae NCCP11945 genome, whereas Gen2Epi aligned scaffolds.To compare the completeness of the genomes generated by Gen2Epi with published results, optimally fully assembled genomes from these previously published studies should be used , 30, 31.Even though full genomes generated by the proposed pipeline were accurate and overcame the limitation of gene fragmentation over multiple contigs, there were still some irregularities  and obtaining identical results.The pipeline was validated by one of the authors (Demczuk) at a separate site  by analyzing  the WHO  N. gonorrhoeae research community. The detailed comparison is shown in Table We compared the Gen2Epi pipeline with existing tools developed using different techniques and exhibiting various features, and which have been widely used by the Firstly, in terms of functional features, both the Gen2Epi and Nullarbor pipelines can perform data cleaning and contamination checks on the raw reads. For assembly, four tools  require AMR genes identified from the assembled contigs in FASTA format. Gen2Epi requires input data in FASTQ (raw or trimmed readsets) and FASTA formats.Finally, the biggest limitation with both NG-MAST and NG-STAR is that these tools do not use assembled contigs or scaffolds to determine strain typing and AMR determinants. Gen2Epi overcomes this limitation by allowing users to determine strain typing directly from assembled scaffolds and predicted genes. Therefore, Gen2Epi is superior in comparison because it enables users to assemble multiple samples efficiently into scaffolds and assign molecular epidemiological results automatically and simultaneously.Functions for variant calling, mismatched read elimination, novel AMR determinant prediction, assembly improvement using long reads, and scripts that can take advantage of API (Application Programming Interface) facilities implemented in pubMLST, will be included in the next version. Modules for phylogeny based on core genome analysis  will alsChlamydia trachomatis, N. meningitidis, and Mycobacterium tuberculosis. However, functions for the complete WGS analysis (including novel scaffolding and typing scripts) of other bacteria will be implemented in the future GUI version of Gen2Epi.The first two steps  of the current Gen2Epi version are universal for the analysis of other pathogenic bacteria such as N. gonorrhoeae isolates from four previously published studies [We have developed a novel WGS pipeline named Gen2Epi to assemble Illumina short reads to full genomes and assign strain typing (NG-MAST and NG-MLST) and AMR determinant information (NG-STAR) automatically to the assembled genomes. The accuracy of the pipeline was validated by testing it on 1484  studies , 30, 31.Project name: Gen2Epi.Project homepage: Anonymous ftp from URL ftp://www.cs.usask.ca/pub/combi. Any standard web browser can be used to access this site.Operating system(s): Platform Independent.Programming language: Perl.Other requirements: VirtualBox 5.2.14 or higher. The Gen2Epi virtual machine requires a minimum of 29 GB of free hard drive space, 2 GB of RAM, and 2 processors to efficiently run the pipeline on the test dataset included with the software distribution. With this virtual machine configuration on a computer with a 3.6GHz CPU, approximately 12 h of elapsed time are required to complete the full analysis of the test data.License: CC BY-NC 2.0 CA.Additional file 1:Table S1. Datasets used for pipeline testing and evaluation. Table S2. Names and accession numbers of input data used by the Gen2Epi pipeline. Table S3. List of third-party software incorporated into Gen2Epi. Table S4. Sample output generated from Gen2Epi’s AMR & Molecular Epidemiological Analysis step shown in tabular form. (DOCX 25 kb)Additional file 2:Technical details. (DOCX 20 kb)Additional file 3:Figure S1. Genome alignment of the Gen2Epi-produced WHO G scaffold against the corresponding Neisseria gonorrhoeae reference genome using Mauve. The extent of the colored bar indicates the strong similarity between the scaffold and the reference genome. (DOCX 48 kb)


They came to the conclusion that one component of the latter, mGPD, is responsible and a direct molecular target for metformin.+/NADH as co-factor [a Km value around 30 μM for glycerophosphate (GP)] and mGPD with FAD as co-factor. The mGPD has a high Km for GP (2 to 8 mM), is an important reactive oxygen species (ROS) producer  stimulation of succinate-driven OCR by metformin that has never been reported before. Third, high throughput screening for mGPD inhibitors with several thousands of chemicals yielded membrane permeable, high-affinity blockers such as iGP  but not metformin or related structures (Orr et al., Thus, the data by Calza et al.  and the After submission of the manuscript two publications came to our attention, which support the notion that mGPD must be removed from the list of direct molecular metformin targets. Alshawi and Agius  report tHG and OL discussed the topic and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The implementation of an integrated molecular surveillance (IMS) of tuberculosis (TB) is of high priority for TB control. IMS is defined as the systematic inclusion of molecular typing results in the national TB surveillance system. Although not standardized, an IMS of TB is already implemented in several low TB incidence countries. Germany is in the process of implementing a nationwide IMS of TB. This requires close collaboration between national and local health authorities. We conducted an online survey to understand the current use of molecular typing results for TB surveillance among the local public health offices (PHO)s in Germany, and to collect their perception and expectations towards the implementation of a nationwide IMS of TB.The online survey was developed using the software Voxco and included 31 questions. The survey was sent to all the 377 local PHOs in Germany in April 2017. Responses were collected until June 2017.did not use molecular typing results (n = 86) vs. PHOs that did use molecular typing results (n = 88): the latter perceived the use of molecular typing results as more beneficial for their work compared to the former . Moreover, the PHOs using molecular typing results expect for the future more support and coordination from regional and national public health institutes, especially regarding the identification and analysis of molecular clusters.A total of 174/377 (46.2%) local PHOs participated in our survey, and 88/377 (23.3%) used molecular typing results in their routine TB surveillance work. The PHOs used molecular typing results especially as support for epidemiological contact tracing . We found statistically significant differences between answers of PHOs that Our study is a step forward in the broader goal of implementing an IMS of TB in Germany. The local PHOs currently using the molecular typing results highlighted their positive attitude towards the implementation of an IMS, but also their needs of more support. Similar assessments might serve as an example for other countries which are on the way to implement a nationwide IMS of TB.The online version of this article (10.1186/s12889-019-6631-6) contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis (MTB) isolate (such as genetic lineage or drug-resistance determinants) with epidemiological data of TB patients (such as demographics or contacts) [According to public health institutes in Europe, the implementation of a nationwide integrated molecular surveillance (IMS) is of high priority to improve tuberculosis (TB) control . The IMSontacts) , 3. The ontacts) –6, distiontacts) , 8, distontacts) , evaluatontacts)  and undeontacts) .A nationwide IMS of TB, although not standardized, is implemented in few countries such as Denmark , EnglandSuch local studies conducted in Germany can be seen as “building blocks” for the preparation of a nationwide IMS of TB. The implementation of a nationwide IMS of TB is a public health intervention which requires a close collaboration between several players, such as the Robert Koch Institute  acting as coordinator, the regional and local public health offices (PHO)s which collect and transmit the epidemiological data, the laboratories performing the molecular typing and the clinical services which communicate with both health authorities and laboratories.Infektionsschutzgesetz, IfSG) on July 2017; available results of molecular typing now need to be included in the notification. In addition, we wanted to see if there was any difference in perception and expectations on the implementation of the IMS of TB between the PHOs belonging to the federal state of Baden-Württemberg–which participated in the pilot study on the implementation of the IMS of TB [In this study, we focussed on the perspective of the local PHOs in Germany. We conducted an online survey among the local PHOs in Germany to understand their current use of molecular typing results in their routine TB surveillance work, and also their perception and expectations regarding the implementation of a nationwide IMS of TB. At the time of this study, the use of the molecular typing result was not routinely used and could not be reported. This situation has changed with the amendment of the German infection protection law  and the Information Security Officer  of the RKI examined our study design and gave us their ethical approval, basis on the fact that there were no data protection concerns against the implementation of our study. As our study did not involve interventions no further ethical clearance was necessary.We developed an online survey using the software Voxco . The questionnaire was divided into four parts: 1) general information on each PHO; 2) information regarding the use of the molecular typing result up to 2016; 3) information on the performance of contact tracing and 4) expectations for the implementation of a nationwide IMS of TB. We developed a total of 31 questions. In 19 questions only one answer was possible, in 11 questions multiple answers were possible and one question was an open question. In 10 questions we collected quantitative information such as the number of TB cases per PHO .Pearson’s chi-squared and Fisher’s exact tests were used to determine significant differences in the answers to the survey between the different groups. Statistical significance was considered with a two-sided test if A total of 174/377 (46.2%) of PHOs participated in our online survey. All 16 German federal states were represented with at least one PHO  PHOs used the molecular typing results in their routine TB surveillance work in 2016 or prior to 2016. The molecular typing was performed in 76/88 (85.2%) cases at the German National Reference Centre for Mycobacteria , in 6/88 (6.8%) cases at other laboratories, in four cases at multiple places and in two cases this information was unknown. The most common molecular typing method analyzed was spoligotyping in 41/88 (46.6%), followed by MIRU in 26/88 (29.5%), WGS in 7/88 (7.9%) and multiple methods in 14/88 (15.9%). The costs for the molecular typing were covered by the local PHO in two-thirds of cases  or by the laboratory performing the typing . The estimated median cost for the PHO per typed isolate was 50 Euro (ranging from 0 to 360 Euro), considering all the typing methods. The median turnaround time per isolate, defined as the time taken from the request to the delivering of the molecular typing result, was 21 days . Among the PHOs that used molecular typing results, the majority  did not systematically document such results in their TB-surveillance reporting system.We asked the PHOs: “For which purpose did you use the molecular typing result?” The most common answer was “support for epidemiological contact tracing” 62/88 (70.4%), highlighting the added value of combining molecular typing and epidemiological data in the control of TB transmission. Among the other purposes, the PHOs reported: distinguish between reinfection-relapses , molecular detection of drug-resistance  or detection of false-positive laboratory results . We further elaborated on this question and asked: “Which were the benefits of using the molecular typing results for your work?” The answers of the PHOs reflected once again the advantages of combining molecular typing and epidemiological data: detection of unknown transmission , transmission detection over longer time period , exclusion of previously assumed transmission  early detection of outbreaks  or detection of transregional transmission . Among the PHOs participating in our survey, the major barriers to the implementation of the IMS of TB were: costs , lack of staff capacity  and complex logistics related to sample shipping .A total of 38 PHOs answered that they did not perceive the IMS of TB as beneficial  vs. local PHOs that did use molecular typing results (n = 88). Indeed, the group that used molecular typing results perceived it more often as beneficial for their work compared to the other group . Another difference between these two groups is that PHOs in Baden-Württemberg expect more support in the identification of molecular cluster when compared to all other PHOs .We analyzed the answers of the PHOs in the federal state of Baden-Württemberg since in this federal state a pilot study on the feasibility of the IMS was carried out and we wanted to see their perception about the IMS . The PHOin 2016 (n = 39) perceived it as more beneficial for their work compared to the PHOs that used molecular typing results prior to 2016 (n = 49)  compared to the WGS. This is true also for the other countries where the IMS of TB was implemented in the previous years: for example in the Netherlands, Slovenia, Sweden and the United States, the molecular typing method used was the MIRU –18, whilAmong the participating PHOs using molecular typing results, less than half included such results systematically in their TB surveillance system, which is a prerequisite to implementing a nationwide IMS of TB. This challenge can be solved by providing training about the benefits of the IMS and of the systematic inclusion of the molecular typing results in the TB surveillance system.We observed that a third of PHOs that did not use molecular typing results answered that they did not know if a nationwide implementation of the IMS of TB would be beneficial for their work. In addition, several PHOs answered that they did not perceive the IMS of TB as beneficial for their routine work. These answers might reflect the need for training and education on the benefits of the IMS of TB. The PHOs that used molecular tying results were indeed more aware of the benefits of IMS of TB compared to the PHOs that did not use molecular typing results. We observed a similar trend between the PHOs that used it in 2016, compared to those using it prior to 2016. Therefore there seems to be a growing perception of the benefits of the IMS over time, and this might be due to several factors: the growing experience in the interpretation of molecular typing results, the availability of more advanced and faster methodologies and the increased education and awareness about the IMS of TB. The major benefits identified by the responding PHOs in our study were the support in transmission detection, contact tracing and cluster investigations in outbreaks. Similar results were obtained also in a study in the Netherlands assessing the impact of molecular typing on contact tracing . Two stuOne of the limitations of our study is that the PHOs who used molecular typing results might have been more willing to participate in our survey and to provide answers, therefore leading to an overestimation of the use of the molecular typing results. Moreover, prior experience may have also influenced responses due to greater insight and education on the value of the IMS in practice. In addition, PHOs using molecular typing results in 2016 might also remember more answers and details compared to the ones that used them prior to 2016. Moreover, we did not ask the PHOs if they ever received the molecular typing results but they did not use them. This could have added further insights on the usefulness of the molecular typing results for the PHOs. Lastly, around half of the invited PHOs did not participate in our study, highlighting the need for more training and awareness on the importance of a national IMS of TB. Possible reasons to explain why certain PHOs did not participate in our survey might be the rarity of TB cases in certain PHOs and also the fact that the molecular typing result was not part of the official notification, thus making the PHO less motivated to participate in such type of survey.Our study is a step forward in the broader goal of implementing a nationwide IMS of TB in Germany. The answers of the local PHOs to our survey highlighted their positive attitude towards the implementation of an IMS, but also their needs of more support in data interpretation. Therefore, a closer collaboration is needed between local and national public health authorities. For example, molecular typing data from different PHOs should be made more accessible, organized and centralized, or regular training workshops on molecular surveillance could be organized. Such closer collaboration might also encourage and convince those PHOs in Germany, who do currently not use molecular typing results in their work, of the benefits of the IMS. In Germany, the concept of implementing a nationwide IMS of TB started years ago with smaller and local molecular surveillance studies–the so-called building blocks in our introduction–which helped identify specific needs and room for improvements first at a smaller scale. After these studies, we are now in a good position to launch a nationwide IMS of TB. Such an approach could be used also by other countries, which are on the way to develop a nationwide IMS of TB.Additional file 1:Word format of the online survey. (DOCX 43 kb)Additional file 2:Qualitative answers to the survey between different groups of PHOs. (XLSX 17 kb)


This paper provides information related to the sensor measurements obtained from five different unreinforced masonry (URM) walls subjected to incremental dynamic shake-table tests at EUCENTRE, Pavia, Italy. This information has been made available to assist in the development and calibration of analytical and numerical models intended to simulate the out-of-plane (OOP) two-way bending response of URM walls. For further interpretation of the sensor recordings, and for a detailed discussion on the observed seismic performance of the specimens, the reader is referred to the article entitled “Experimental Response of URM Single Leaf and Cavity Walls in Out-Of-Plane Two-Way Bending Generated by Seismic Excitation” [1]. Videos documenting the failure of each specimen are also available on YouTube [2]. Each specimen was densely instrumented with various sensors measuring accelerations and displacements throughout the testing sequence. The locations of these sensors and their operating status throughout the testing sequence  is provi2e.g. Refs. e.g. Tomassetti et al. This article presents the experimental data obtained from incremental dynamic testing of five full-scale URM walls subjected to two-way bending OOP seismic excitation. Four full scale URM walls (single leaf as well as cavity) were also previously tested by the authors under one-way bending excitation The five tested walls represent the first full-scale URM walls tested up to collapse that have been reported in literature. Three of these specimens were constructed in calcium silicate masonry (CS), one in clay (CL) masonry and another one was a cavity wall consisting of an inner leaf in CS and an outer leaf in CL masonry connected to each other by metal ties. All dynamic tests were carried out at the uniaxial shake table of EUCENTRE Pavia. The main input motions used in this part of the campaign corresponded to second floor accelerograms recorded either from a building prototype tested by Graziotti et al. Every specimen was densely instrumented with sensors that recorded the dynamic response at various locations. The instrumentation adopted for each specimen consisted of accelerometers, potentiometers, wire potentiometers and a 3D optical acquisition system . The location of all the instrumentation adopted for each specimen was decided based on the boundary conditions envisaged and correspondingly expected deformed shapes. Accelerometers were installed on the OOP panel of the specimen in order to record acceleration-time histories. Additional accelerometers were also installed at the specimen foundation, top beam, rigid frame and the return walls. Potentiometers were used to measure relative displacements associated with various locations of the specimen. Wire potentiometers attached to the rigid frame in several locations were used to record horizontal displacements relative to the shake table. Potentiometers were also adopted to record the relative displacements between the main panel and the return walls.All data acquired was filtered from frequencies higher than 50 Hz. Accelerations and forces are provided in units of g and kN, respectively; displacements are given in mm. For each specimen, a folder is created named as the specimen: the folder containing data from all the tests corresponding to the second specimen is named as “CS-000-RF”. This folder contains .txt files for each test named as “TestT#” where “T#” refer to the same quantity provided in the testing sequence included in the reference article .txt files). This associated lumped mass distribution changed throughout the testing sequence with the development of cracks and the adopted distribution throughout the testing sequence can also be found in As an illustrative example, with reference to