Plasmodium falciparum erythrocyte-membrane-protein-1 (PF3D7_1150400/PF11_0521) contains both domain cassette DC13 and DBLβ3 domain binding to EPCR and ICAM-1 receptors, respectively. This type of PfEMP1 proteins with dual binding specificity mediate specific interactions with brain micro-vessels endothelium leading to the development of cerebral malaria (CM). Using plasma collected from children at time of hospital admission and after 30 days, we study an acquisition of IgG response to PF3D7_1150400/PF11_0521 DC13 and DBLβ3_D4 recombinant constructs, and five peptides located within these constructs, specifically in DBLα1.7_D2 and DBLβ3_D4 domains. We found significant IgG responses against the entire DC13, PF11_0521_DBLβ3_D4 domain, and peptides. The responses varied against different peptides and depended on the clinical status of children. The response was stronger at day 30, and mostly did not differ between CM and uncomplicated malaria (UM) groups. Specifically, the DBLβ3 B3-34 peptide that contains essential residues involved in the interaction between PF11_0521 DBLβ3_D4 domain and ICAM-1 receptor demonstrated significant increase in reactivity to IgG1 and IgG3 antibodies at convalescence. Further, IgG reactivity in CM group at time of admission against functionally active (ICAM-1-binding) PF11_0521 DBLβ3_D4 domain was associated with protection against severe anemia. These results support development of vaccine based on the PF3D7_1150400/PF11_0521 structures to prevent CM.The  It has been shown that iEs adhesion to endothelium is mediated by PfEMP1 protein family, the major clonally-expressed variant surface molecules involved in the malaria pathogenesis and antigenic variation7. The high polymorphism of these proteins enable iEs to modify their antigenic and binding properties allowing them to escape the host immune system8. Nevertheless, in endemic areas, individuals gradually acquire a protective immunity to various parasite-blood stage antigens including PfEMP-1 after limited number of malaria episodes, preventing them from severe and cerebral malaria complications11.Cerebral malaria (CM) is a major cause of mortality in endemic countries particularly among children in Africa where 91% of the 445,000 annual deaths occurs . Several studies, including recent Magnetic Resonance Imaging (MRI) observations, assumed that the cytoadhesion and accumulation of infected erythrocytes (iEs) to the brain micro-vessel endothelium are associated to the clogging and obstruction of blood circulation. This results in the disruption of blood–brain barrier (BBB) tight junctions, vascular permeability, severe edema, and micro-hemorrhages and swellingvar gene family, which undergo transcriptional switch allowing the parasites to express a single clonal variant protein at a time14. These proteins are composed of multiple Duffy binding like (DBL) and cysteine-rich interdomain region (CIDR) domains, which are classified according to their sequence into several types  and their numbered subtypes7. Sequence analysis of var genes from seven genomes led to the identification of twenty novel conserved structural units, so called domain cassettes (DC), each composed of several specific domain types15. Recent studies showed that parasite isolates causing severe and cerebral malaria transcribed preferentially var genes with DC 08, 13 and 0419. These DC have been demonstrated to bind to the endothelial protein C receptor (EPCR) through their CIDRα1.1/CIDRα1.4 domains20. The presence of parasites expressing PfEMP1s with dual binding capacity to ICAM-1 and EPCR was shown in CM isolates21. In addition, antibodies against recombinant ICAM-1-binding DBLβ3 domain, which are common in adults22, have been associated with protection against severe and moderately severe malaria hospitalizations23 and with a reduced risk of high-density clinical and severe malaria24. Therefore, these findings emphasized the importance of these specific sequence motifs/domains in the pathogenesis of severe and cerebral malaria and raised the question on their involvement in triggering the acquisition of protective antibodies in CM. In this study, we report on specificity of antibody immune response to PF3D7_1150400/PF11_0521 DC13 recombinant protein, to full-length functional (ICAM-1 binding) domain DBLβ3_D4, and to peptide motifs that aligned to the DBL α 1.7_D2 and DBLβ3_D4 domains of this protein in children with CM.PfEMP1 proteins are encoded by ~ 60 members of the Plasmas samples were collected from children consulting at Centre hospitalier universitaire Mère-enfant de la Lagune (CHUMEL), Centre National Hospitalier Universitaire Hubert Koutoucou Mega (CNHU-HKM), or to the Centre Hospitalier Universitaire of Suru-Léré CHU Suru-Léré, Cotonou, Benin, for malaria during the period of high malaria transmission . Informed consent was obtained from a parent and/or legal guardian for all children enrolled in the study.P. falciparum infection and a Blantyre coma score ≤ 2, with the exclusion of any other causes of coma. Uncomplicated malaria group (UM) have P. falciparum parasitemia infection along with fever, headache, or myalgia without signs of life-threatening malaria and evidence of vital organ dysfunction, as defined by the World Health Organization . Both ELISA experiments using PF11_0521-DC13 recombinant protein and peptides as well as IgG reactivity to the DBLβ3_D4 immobilized on BioPlex beads were performed on children with CM from the same cohort.Children of 6 years old or less were recruited in the study if they presented positive rapid diagnostic test for malaria  confirmed microscopically by Giemsa-stained thick blood smears, and meet the WHO clinical malaria definition criteria. CM group was defined as a microscopically confirmed Samples of 2–4 ml of blood were collected into citrate phosphate dextrose adenine-containing tubes from all children whose parents signed the informed consent. All patients were treated according to the guidelines established by the Beninese Ministry of Health. Children with severe cases were treated with 120 mg quinine perfusion.P. falciparum mono infection.Positive controls: the first positive control, included in triplicate to each ELISA plate, consisted of a pool of 20 plasmas from highly responsive individuals to MSP3 and GLURP  antigens. These plasmas were collected from adult males in Senegal. The second positive control were plasmas from individual children aged between 4 and 5 years old living in endemic malaria transmission area of the suburb of Yaoundé, Cameroun, tested individually in triplicate. These children were asymptomatic carriers of Negative controls consisted of a pool of 20 plasma samples from European adult blood donors who had never traveled to malaria endemic areas.The study protocol was reviewed and approved by the Research Institute of Applied Biomedical Sciences ethics committee (ISBA-CER), Cotonou, Benin (No 006/CER/ISBA/12 and No 21/CER/ISBA/13) and the Cameroonian National Ethics Committee as well as the Cameroonian Ministry of Public Health (authorization No 028/CNE/DNM/07). All the methods used in this study were performed according to the relevant guidelines and regulations of the ISBA-CER and the Cameroonian Ministry of Public Health.http://www.cbs.dtu.dk/services/VarDom/. Each DBL or CIDR sequence was isolated and homologous or paralogous sequences were found by searching for the nearest matching sequences at the site: http://blast.ncbi.nlm.nih.gov/ Blast.cgi. The DBL and CIDR sequences with the highest score were selected and aligned using the site: http://multalin.toulouse.inra.fr/multalin/. The solved (or predicted using https://toolkit.tuebingen.mpg.de/tools/hhpred) structures of the DBLs and CIDRs of interest were analyzed. The sequences of each of the corresponding variable regions for each DBL and CIDR domain were aligned. These analyses allowed us to determine constant and variable regions where only a few consensus sequences were observed, to be considered as sequences subjected to a selection pressure due to the host immune system escapement, as already described for the DBL6ε domain26. The absence of local homology to other existing proteins other than PfEMP1 was verified. DBLs alignments highlighting conserved and variable sequences are shown in supplementary Fig. 27.The number, type and boundaries of each DBL and CIDR domains of PF11_0521/PF3D7 1,150,400 protein were determined by the site E. coli expression. A sequence coding for a 6His-tag was added to the 3′-terminus of this construct. Encoded 727 amino acid residues (85.03 kDa) DC13-6His construct was expressed in E coli and purified in denaturing conditions in 8 M urea, and then refolded by rapid dilution to 2 M urea. Wells of ELISA 96-well, high protein-binding plates (Nunc MaxiSorp Thermofisher Courtaboeuf Cedex (Villebon-sur-Yvette) were coated with these proteins at 5 µg/mL concentration. All the peptides and the DC13 recombinant protein were synthetized by ProteoGenix SAS .The DNA sequences coding for PfEMP1 PF11_0521 DC13 of Pf3D7_1150400/PF11_0521 gene composed of DBLα1.7_D2 – CIDRα 1.4_D3 domain tandem  were chemically synthesized with codon optimization for An enzyme-linked immunosorbent assay (ELISA) to measure DC13- and peptide-specific IgG in plasma from Beninese children with cerebral malaria (CM) or uncomplicated malaria (UM) was performed in a blinded manner. Nunc MaxiSorp microtiter plates were coated with 50 µl of each peptide or recombinant protein at a concentration of 5 µg/ml in PBS pH 7,4  and incubated overnight at room temperature. The wells were blocked for one hour with 100 µl of blocking solution PBS-Bovine Serum Albumin (BSA) 4%, Sigma. The plates were washed three times with 200 µl of PBS-0.05% Tween 20 (PBST). Fifty µl of Plasma samples diluted 1:100 in PBST were incubated in triplicate at room temperature for 1 h. After washing, bound antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-human IgG  followed by washing and addition of 50 µl of 3,3′,5,5′-Tétraméthylbenzidine . The reaction was stopped by addition of 50 µl sulfuric acid to 0.2 M. IgG1 and IgG3 isotypes were also assessed against the entire DC13 from PF11_0521 protein (composed of DBLα1.7_D2-CIDRα1.4_D3 domains) in children with CM at admission (day 0) and at convalescence (day 30) and against the peptide DBLβ3 B3-34 located in the PF11_0521 DBLβ3_D4 domain in children with CM and those with UM collected at day 0 and day 30 of admission.The optical density (OD) values were measured at 450 nm using a Biochrom Asys UVM 340 reader and corrected for background by subtraction of OD values obtained in BSA-blocked wells. Antibody reactivity was expressed in Optical Density ratio (ODr) units calculated by the mean OD of samples divided by the mean OD of the pooled negative control. All means were calculated from the triplicate measurements. Each ELISA plate included a pool of 20 plasmas from malaria non-immune people in triplicate (Negative Control), which was used to determine the precise cut-off for positive and non-reactive samples.Median values of ODr were plotted together with CI95 using Prism v6 software . The Median values and the 25% and 75% interquartile values are presented in supplementary Table The same pool of positive and negative plasma controls was systematically included in each ELISA plate throughout the study.28. This domain expressed in mammalian system and immobilized on BioPlex beads is correctly folded, which was assumed from its strong binding activity toward ICAM-1, also confirmed for this work  were tested in these experiments. Normal distribution in all sets of data was tested by three tests  using GraphPad Prizm software. Statistical significance (P < 0.05) was determined by t-tests for normally distributed data and by Mann–Whitney tests for non-parametric distributions.The PF11_0521 DBLβ3_D4 domain and the control construct HAE  were expressed in COS7 cells and coupled to BioRad BioPlex beads as described inThe clinical and biological features of children included in the study are summarized in Table Our results show differential profiles of antibody response of children against the peptides and the whole Pf11_0521 DC13 (DBLα1.7_D2-CIDRα1.4_D3) recombinant protein. This response varies according to the tested molecules and to the clinical and immunological status of children. The median reactivity and 25%-75% interquartile ranges for each the DC13 and peptides are presented in Supplementary Materials Table The antibody response against PF11_0521 DC13 recombinant protein was induced in 56%, 70%, 30%, 63%, 93% of children with CM day 0, CM day 30, UM day 0, UM day 30, and children with asymptomatic parasitemia (AM group), respectively, based on ODr median values. Medians and their 25–75% interquartile ranges (in parentheses) were 1.21 (1.07–1.35); 1.34 (1.11–1.46); 1.06 (0.89–1.39); 1.36 (1.10–1.68); 1.48 (1.36–1.63), respectively. There was no statistically significant difference in the response for the plasma collected at days 0 and 30 in the CM group. However, this difference was significant in the UM group (P = 0.001). Similarly, there was no statistically significant difference either between CM and UM groups at day 0 or CM and UM groups at day 30. The IgG level of AM group was higher than that of CM at day 30 and the difference was statistically significant (P = 0.0035)  and 2.289 (2.047–2.768), respectively. At the same time, 75% of CM day 0 and 96% of CM day 30 had an IgG3 response with ODr median values of 1.63 (1.143–2.121) and 5.914 (2.98–7.33) CM day 30, respectively. Both IgG1 and IgG3 responses to the PF11_0521 DC13 in CM group at day 30 were higher than that at day 0 with statistical significance (P < 0.0001)  of 1.64 (1.44–2.19), 3.074 (2.467–3.71), and 2.21 (1.81–2.42) in CM Day 0, CM Day 30, and AM groups, respectively. The level of this response was higher in the CM day 30 group compared to CM day 0 with a statistically significant difference (P < 0.0001). Unexpectedly, the response of the AM group was lower than that of CM group at day 30 with statistically significant difference (P < 0.0001) ; 1.10 (0.98–1.30); 0.97 (0.83–1.04); 1.17 (0.88–1.34); 1.29 (1.12–1.58), respectively. There was no noticeable difference in the IgG response in the CM group between day 0 and day 30. However, IgG response in children with UM was statistically significant for samples collected at days 0 and 30 (P = 0.05). As expected, the IgG level was higher in the AM group compared to CM group (P = 0.0061) ; 1.25 (1.07–1.55); 1.22 (1.17–1.27); 1.32 (1.20–1.77); and 1.59 (1.26–1.98) for CM day 0; CM Day 30; UM day 0, UM 30 and AM group, respectively, with 64%, 51%, 62%, 80% and 92% of responders among these groups. This response seems to be similar within and between the CM and UM groups. However, the AM group IgG response was higher than that of CM group at day 30 and this difference was significant (P = 0.0098). ; 1.45 (1.37–1.56); 1.10 (1.01–1.17); 1.14 (1.03–1.56); 1.37 (1.12–1.65), respectively, in these groups. The response of CM day 30 was higher than that of CM day 0 with statistically significant difference (P = 0.007). However, there was no difference in the IgG level between AM group and that of children with CM day 30 ; 2.88 (2.43–3.34); 2.17 (1.76–2.71); 2.61 (2.23–3.22) and 2.74 (2.47–3.09) in children with CM day 0 CM day 30, children with UM day 0 UM day 30, and AM group, respectively). The difference in the antibody response was higher at day 30 vs. day 0 with statistical significance in both UM (P = 0.0069) and CM clinical groups (P = 0.0092) , 2.27 (1.89–2.89), 2.70 2.27–3.32), 2.74 (2.23–3.43) and 3.21 (2.58–3.63) in CM day 0, CM day 30, UM day 0, UM day30, and AM groups, respectively. It is clear that IgG1 response was higher at day 30 than at day 0 in CM group with statistically significant difference (P < 0.0001). This response was higher in UM day 30 group compared to CM day 30 group with statistical significance (P = 0.0135) and was not significantly different from that of the AM group. However, the IgG1 response of CM group was lower than that of AM group with statistically significant difference (P = 0.0008) , 2.29 (1.54–2.94), 1.66 (1.44–2.13), 1.86 (1.76–2.72), and 3.05 (2.51–3.37) for CM day 0, CM day 30, UM day 0, UM day 30, and AM control, respectively. The response was higher at day 30 than at day 0 for both CM and UM groups, but the difference was statistically significant only in CM group (P = 0.0012). The AM group IgG3 response was higher than that of both UM and CM groups at day 30 and the difference was statistically significant (P = 0.0005) and (P = 0.0145), respectively , 1.34 (1.27–1.71), 2.69(1.61–3.09), respectively. There was no difference in the level of the response between CM at admission and convalescence. Similarly, 66%, 62%, 94% of CM at admission, CM at day 30, and AM individuals responded to peptide MSA3 B-42 with ODr median values of 1.44 (1.18–2.09), 1.37 (1.14–2.05), 2.15 (1.68–2.99), respectively. Another peptide, MSA3 C-27, reacted with only 44% of children with CM at admission, while 96% of children with CM at convalescence and children with AM were reactive. Antibody reactivity ODr median values were 0.94 (0.76–1.81) for children with CM at admission, 2.1 (1.77–2.57) at convalescence, and 3.28 (2.68–3.86) for AM group, with statistically significant difference in the groups of children with CM at days 0 and 30 (P = 0.0001). As expected, the level of the AM group IgG response against the three MSA3 peptides was higher than that of CM group at day 30 with statistically significant difference of P = 0.001 for MSA3 A-27 and MSA3 C27 peptides and P = 0.0004 for MSA3 B-42  IgG reactivity against PF11_0521 DBLβ3_D4 domain had higher parasitemia (PE)  and lower Hb  compared to group with high (above median) IgG. Median levels for Hb concentrations in these two groups were 4.65 (low IgG) and 6.5 g/dL (high IgG), respectively  domain cassette DC13, which is composed of tandem of domains DBLα1.7 and CIDRα1.4, and binds EPCR receptor through CIDRα1.435, and (b) neighboring downstream domain DBLβ3_D4, which binds ICAM-1 receptor36. It has been shown previously that the presence of these two features together in one protein plays a pivotal role in the pathogenesis of CM19. Therefore, we focused our efforts to measure IgG response against the entire DC13, functionally active (ICAM-1 binding) full-length domain DBLβ 3_D4, and smaller peptides located on the surface of these two constructs.The feasibility of a protecting vaccine against malaria is supported by the naturally acquired immunity in people living in endemic areas. It has been shown that as the rate of exposure increase so does the level of protection against severe life-threatening malaria. This observation is underpinned by several immuno-epidemiological studies17 thus indicating the acquisition of specific antibody response against this cassette. However, this response was also found in children with UM, which may indicate previous exposure of these children to epitopes from DBLα1.7 and CIDRα1.4, or sufficiently homologous domains. This difference between the response at admission and at day 30 was statistically significant (P = 0.001)  and was higher in semi-immune children  further contribute to understanding protective immune response to PfEMP1 proteins with dual receptor binding, important for CM.With the exception of DBLβ3 B1-17 peptide, IgG response against other DBLβ3 peptides located in the DBLβ3-D4 domain was substantially different at admission and convalescence, with statistical significance for DBLβ3 B2-37 and DBLβ3 B3-34 in children with CM  compared to admission (day 0), we tested for reactivity of these plasma samples against peptides from MSA antigen  PF11_0521 DBLβ3_D4 domain (immobilized on BioPlex beads) with plasma from children with CM at admission indicate that children with high IgG levels have higher Hb (P = 0.0159) and lower parasitemia . This suggests that high levels of IgG against this domain are associated with protection of patients, which develop CM, against low Hb levels and severe anemia. As association was found at the admission, it indicates that IgG reactivity against this domain either persists after previous experience or quickly raised from immunological memory upon current infection, which leads to CM and hospitalization. If level is high enough, it may protect against severe anemia in these patients with CM (conjectured from the observed association). This observation is of high interest since severe malaria anemia is highly prevalent among children with severe malaria including CM and, in our cohort, it reaches 40%.Interestingly, IgG response in children at admission and at day 30 is strong against the entire DC13 cassette, as well as against peptides DBLα1 A1-31 and DBLβ3 B3-34. For the DBLα1 A1-31 and DBLβ3 B3-34 peptides the level of this response was similar to that obtained in AM group, which consisted of semi-immune children from Cameroon, and in positive controls obtained from adults living in Senegal, suggesting the acquisition of IgG response to these antigens and potential role in protection.24. Definitely, the non-statistically significant observation about difference in parasitemia in children with high and low IgG levels to PF11_0521 DBLβ3_D4, and peptides DBLβ3 B1-17, DBLβ3 B2-37 needs to be further confirmed. It can be done, for example, by using HRP2 ELISA-based parasite load quantification, which also accounts for the sequestered parasites. Besides, the heterogeneous response between individuals might also originate from the genetic polymorphism of genes associated with immune response, such as FcγRIIa and TNFα37. These hypotheses should be tested using larger set of samples.These data also indicate that these peptides most likely represent conserved epitopes. Our results also indicate that IgG response is highly heterogeneous within patients in the same group, particularly in children with CM at day 30. This was even more noticeable for the IgG3 response against PF11_0521 DC13 (Fig. 40. Our results demonstrate an acquisition of IgG1 and IgG3 antibodies against entire PF11_0521 DC13 and against peptides from DBLβ3_D4 domain in a group of children with CM with statistically significant difference between admission and convalescence. However, for IgG1 and IgG3 responses in children with UM there was no difference between admission and convalescence, suggesting an existing immunity, which might be acquired due to previous exposure to these or homologous antigens. The IgG1 and IgG3 response was significantly higher in AM group than in CM and UM groups at day 30 highlighting the importance of these IgG isotypes in the immune protection.The IgG1 and IgG3 isotypes are the cytophilic antibodies shown to be dominant in clinically protected compared to non-protected individuals, thus associated with acquired protective immunityOur results further support the idea of development of a vaccine based on PfEMP1 DBLβ domains that would elicit immune response against this domain and potentially prevent interactions with ICAM-1. However, other experiments to investigate the antibody-dependent cell-mediated cytotoxicity initiated by antibodies against purified DC13 as well as against DBLα1 A1-31, and DBLβ3 B3-34 peptides are needed to better comprehend relevance of these antigens as vaccine candidates. Also, additional studies of ICAM-1 binding inhibition activity using IgG from these plasmas will allow better understanding the development of immune protection against CM. It is also essential to assess the levels of IgG responses to these antigens in cohorts from other epidemiological settings.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.


The PAH toxicities oscillated from 20.0–2670.8 μg TEQ·kg-1 dw in the O horizon and from 1.73–694.7 μg TEQ·kg-1 dw in the A horizon. The enrichment factor values point to a more intensive accumulation of PAHs with relatively high molecular weights along the pollution transect. The PAH diagnostic ratio values indicated that the main PAH emission sources were from coal and wood combustion.Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that are released into soils primarily from the air, with wet and dry deposition. To assess the contamination of the forest soil environment, soil samples were collected from organic and mineral horizons from three study areas representing a gradient of pollution across Poland (the ‘pollution transect’). The soils examined varied in PAH contents, generally from 124.3 μg·kg Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic pollutants, and many of them belong to the group of persistent organic pollutants (POPs). Their chains are composed of condensed benzenoid rings. With the increase in the number of rings in the molecule (increase in the molar mass), the lyophilic characteristic of PAHs increases, while their volatility and solubility in water decrease. Therefore, they do not degrade sufficiently in the environment. PAHs occur in the environment in the form of complex mixtures and can react with other matrix components, producing derivatives with higher toxicity to the environment , 2. Due 10-2.5), which is involved in smog formation pyrene. The use of TEF allows the concentration of PAH compounds to be converted into an equivalent toxic concentration of benzo[a]pyrene (TEQ). The TEQ method is used to assess the ecotoxicological risk at a specific site , 29. TheSoil samples were air-dried, homogenized in a mortar grinder and screened through a 1 mm sieve. Extraction was carried out in a Soxhlet apparatus in dichloromethane at 120 °C for 5 h . The extracts were concentrated to less than 1 ml in a vacuum evaporator with a water bath at 45 °C. The residue was dissolved in 5 ml of n-hexane and quantitated on top of glass columns filled with 5 cm silica gel (bottom of the column) and with basic aluminium oxide, which was previously washed with 30 ml of n-hexane. The samples were then eluted with 20 ml of n-hexane and dichloromethane at a ratio of 3:1 and 20 ml of n-hexane and dichloromethane at a ratio of 1:1. The collected eluent was concentrated in a nitrogen atmosphere. The residue was dissolved in 1 ml of acetonitrile and analysed using the HPLC-FLD method.The analysis was performed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) using a 2695 Waters Alliance HPLC system  with Waters 2475 Multi-Wavelength Fluorescence (FLR) Detector  operating within 250–400 nm. The separation of the PAH compounds was carried out using a C18 capillary column . The identification of PAHs was based on retention times. The mobile phase was 50% acetonitrile and 50% water. The flow rate was 0.5 ml/min. The injection volume was 10 μl. The elution was in gradient mode for 22 min to 100% acetonitrile and then isocratic mode to 36 min. The excitation and emission wavelengths for the FLD are presented in -1 (0.1–1 μg·kg-1 dw). Each calibration curve was analysed twice, and each calibration level was analysed three times . If the PAH concentration in a sample exceeded the range of the calibration curve, the sample was diluted. The LOQ of the method was established at the lowest point of the calibration curve (0.1 μg·kg-1) and confirmed by a six-fold replicate of the LOQ-fortified sample . To ensure the quality of the results, blank and reference materials were analysed with each series of samples, and the precision (RSD <20%) and recovery (80–110%) of the method were determined based on this. The results of the analysis were corrected for recovery values. Information on standards and reagents is presented in the supplementary materials. The detailed data on method validation are presented in The quantitative analysis was conducted using the calibration curve method. The linearity was established using five calibration levels of 13 PAH mixed standard solutions in the range of 0.01–0.1 μg·mlThe statistical analysis was performed with STATISTICA 12 software. The statistical parameters  were calculated for all soil types in accordance with the three sampling areas  located along the pollution transect. The Pearson correlation analysis was set to be significant at p < 0.05. The sum of PAH content in soil was defined as ∑PAHs.The enrichment factor (EF) was calculated for each compound and sum of PAHs as the content of PAHs in the A horizon divided by the content of the same PAHs in the O horizon within the same soil profile. An enrichment factor > 1 indicates the possibility of accumulating PAH compounds in the soil profile , 31.To determine potential sources of PAH emissions that could affect PAH accumulation in forest soils, PAH diagnostic ratios were used . Based o-1 dw in the O horizon and 85.62 μg·kg-1 dw in the A horizon. The ΣPAH TEQ content in the NE did not exceed the value of 214.96 μg TEQ·kg-1 dw in the O horizon and 32.23 μg TEQ·kg-1 dw in the A horizon. In central Poland (C), the mean ΣPAH content was 1946.26 μg·kg-1 dw in the O horizon and 334.71 μg·kg-1 dw in the A horizon. The ΣPAH TEQ contents in the C area did not exceed 374.47 μg TEQ·kg-1 dw in the O horizon and 140.33 μg TEQ·kg-1 dw in the A horizon. The highest ΣPAH contents were determined in the S area, with a mean value of 3761.55 μg·kg-1 dw in the O horizon and 1050.38 μg·kg-1 dw in the A horizon. The ΣPAH TEQ contents in the S area did not exceed 2670.84 μg TEQ·kg-1 dw in the O horizon and 694.68 μg TEQ·kg-1 dw in the A horizon.The results of ΣPAHs and ΣPAHs TEQ content are presented in Although the maximum ΣPAH contents in the O horizon of the NE and C areas are of similar values, the maximum content in the A horizon is much higher in the C area. However, the mean ΣPAHs contents in the O and A horizons of the NE area were significantly lower than the mean ΣPAH contents in the C and S areas, which justifies the recognition of this region as a background zone. Although the mean ∑PAH contents in the O and A horizons of the C area were three times higher than that in the NE area, the ∑PAH TEQ content in the C area is only twice as high as that in the NE area. The differences between the ∑PAHs in the C and S areas are also smaller compared to the differences between the ∑PAHs TEQ content in the C and S areas. Therefore, the toxicity of the PAH compounds did not increase in parallel with the PAH contents.In the research conducted in agricultural soils of the same regions of Poland , 3, the In the NE area, the ΣPAH contents in the O and A horizons were higher in comparison with the study results obtained in the forest soils of areas considered to be uncontaminated in other parts of the world , 10. In -1 dw), IcdP (11.8–490.9 μg·kg-1 dw), CHR (11.5–427.7 μg·kg-1 dw) and PYR (10.0–420.1 μg·kg-1 dw) in the O horizon and for FLT (0.17–31.4 μg·kg-1 dw), BbF (1.47–26.0 μg·kg-1 dw), FLU (2.5–20.4 μg·kg-1 dw) and CHR (0.74–22.33 μg·kg-1 dw) in the A horizon. In the C area, the highest contents were recorded for FLT (95.08–1145.4 μg·kg-1 dw), CHR (120.7–515.1 μg·kg-1 dw), BbF (89.9–467.4 μg·kg-1 dw), PHE (41.54–449.6 μg·kg-1 dw) and IcdP (40.1–410.2 μg·kg-1 dw) in the O horizon and for IcdP (2.0–322.5 μg·kg-1 dw), BbF (4.6–184.3 μg·kg-1 dw), FLT (3.4–150.4 μg·kg-1 dw), CHR (3.5–124.6 μg·kg-1 dw) and BkF (0.75–123.3 μg·kg-1 dw) in the A horizon. In the S area, the highest values were recorded for BaP (98.1–2128.6 μg·kg-1 dw), BbF (114.1–1601.1 μg·kg-1 dw), FLT (18.7–1311.6 μg·kg-1 dw) and IcdP (93.9–1119.3 μg·kg-1 dw) in the O horizon and for BbF (5.1–667.8 μg·kg-1 dw), CHR (5.5–609.7 μg·kg-1 dw), BaP (3.2–550.6 μg·kg-1 dw), PYR (3.0–546.9 μg·kg-1 dw) and IcdP (3.7–482.8 μg·kg-1 dw) in the A horizon. The detailed results for the individual PAHs are presented in The content of individual PAHs increased along the pollution transect. In the NE area, the highest levels were reported for PHE . The detailed results are presented in The enrichment factor was calculated for individual PAHs and ∑PAHs for all sampling points of the research areas: NE, C and S . The enrThe mean enrichment factor (EF) values for the individual PAHs were calculated for the three research areas . Subsequently, the mean EF values of the individual PAHs were correlated with their logKow values using Pearson’s coefficient at 13 degrees of freedom. A negative correlation (-0.58) was obtained in the NE area, while a strong positive correlation was obtained in the C (0.93) and S (0.80) areas. Similar results of the positive correlation between the EF values and logKow were reported in forests of the industrial zone in northern Germany , while a10 dust collected by monitoring stations in the research area Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Both the 7th and 8th editions of the American Joint Committee on Cancer (AJCC) staging system for perihilar cholangiocarcinoma (pCCA) had the same definition for T2a and T2b. But the value of this classification as prognostic factor remains unclear.178 patients with stage T2a or T2b who underwent curative intent resection for pCCA between Jan 2010 and Dec 2018 were enrolled. Relationships between survival and clinicopathological factors including patient demographics and tumor characteristics were evaluated using univariate and multivariate Cox regression analysis. The overall survival (OS) were calculated by Kaplan-Meier method.P = 0.354). Both the 7th and 8th edition of the AJCC TNM staging demonstrated a poor prognostic predictive performance. High level of preoperative AST (≥85.0 IU/L) and CA19–9 (≥1000 U/mL), vascular resection and lower pathological differentiation of the tumor were the independent predictors for poor survival after resection.There was no significant difference in OS between T2a and T2b groups, and the median OS duration were 37 and 31 months (The newly released 8th edition of AJCC staging system demonstrated a poor ability to discriminate the prognosis of patients with stage T2a and T2b pCCA after resection. Cholangiocarcinoma (CCA) is one of the most challenging diseases in hepatobiliary surgery field , 2. CCA Currently, the AJCC staging system remains as the most popular method in predicting survival. However, prognostic accuracy of the AJCC staging scheme is controversial. Some authors have reported that AJCC system did not predict survival accurately , 17. WhiThus, the objective of the current study is to validate the rationality and prognostic value of the 8th edition of AJCC staging system for stage T2a and T2b pCCA using data from a high-volume center in China.This study was performed as a retrospective observational study. The ethics committee of West China Hospital, Sichuan University approved this retrospective analysis and waived the requirement for informed consent. All patients who underwent radical surgical treatment for pCCA in our institution during January 2010 and December 2018 were identified. Routine histopathological workup was conducted for all resected pCCA by the Department of Pathology. The T stage for every patient was determined according to ‘perihilar cholangiocarcinoma’ chapter in the 8th edition of AJCC staging system. Patient selection criteria: pathologically confirmed pCCA with stage T2a and T2b. Exclusion criteria: pCCA with Tis, T1, T3, T4 tumors; patients who underwent palliative surgery or had R2 margins; primary liver tumors involved the hilum, such as iCCA, hepatocellular carcinoma (HCC) or combined HCC-cholangiocarcinoma; distal bile duct cancers; benign tumors; metastasis tumors. By these criteria, 202 patients were identified consecutively. 7 patients were excluded because N categories could not be determined. 12 patients were censored because of lost in follow-up since discharge. 5 patients died within 90 days post operation were excluded from further evaluation. Thus, a total of 178 patients were available for evaluation.n = 32).All patients were evaluated with systematic inspection and elaborative imaging examination prior to surgery. Surgical procedures were finally determined and conducted according to preoperative multidisciplinary team (MDT) discussion and intraoperative exploration. Selective preoperative biliary drainage is mandatory in cholangitis and when future residual liver (FRL) is small (< 50%) in patients requiring extended resection  infection; cholelithiasis; fluke; percutaneous transhepatic cholangial drainage (PTCD); maximum diameter of the tumor; Bismuth type; resection margin status; vascular resection; postoperative complication; histologic grade; T stage; presence of lymph node metastasis; presence of perineural invasion; invasion of hepatic parenchyma; adjuvant therapy; preoperative total bilirubin (TBIL); direct bilirubin (DBIL); indirect bilirubin (IBIL); alanine aminotransferase (ALT); aspartate amino transferase (AST); alkaline phosphatase (ALP); gamma-glutamyl transpeptidase (GGT); carcinoembryonic antigen (CEA); carbohydrate antigen 19–9 (CA19–9); survival status. Overall survival (OS) was computed as the interval between the date of surgery and the date of death or the last follow-up. R0 was defined as no macroscopic or microscopic residual tumor. The T staging of pCCA was mainly determined by operation and pathologic records. All laboratory indicators were examined within 1 week before surgery.t test or Mann-Whitney U test, when appropriately. Categorical variables were compared using the χ2 test or Fisher’s exact test, when appropriately. The cutoff value of TBIL, DBIL, IBIL, ALT, AST, ALP, GGT were 157.4 μmol/L, 145.3 μmol/L, 17.8 μmol/L, 105.5 IU/L, 85.0 IU/L, 320.0 IU/L and 343.5 IU/L, which were their median respectively. The cutoff value of CEA and CA19–9 were 3.4 ng/mL (lower limit of threshold level) and 1000 U/mL (upper limit of threshold level). Survival analysis was estimated using the Kaplan-Meier method and compared using the log-rank test. Additionally, 1-, 3-and 5- year survival rates were calculated. Univariate and multivariate Cox regression analyses were performed to determine predictors of OS. Variables that were significant in univariate analysis (P < 0.05) were involved into the multivariate analysis. AJCC stage was not used as a dependent variable in the multivariate survival analysis to avoid confounding effect. In addition, the survival analyses were conducted for the patients stratified by stage T2a and T2b pCCA, which was further followed by the subgroup survival analyses based on different N stages of 8th edition AJCC. Variates were presented as the hazard ratio (HR) with 95% confidence interval (95%CI). The concordance index (C-index) was used to assess the performance of the 7th and the 8th editions of the AJCC staging systems [https:www.r-project.org) and Medcalc . Two tailed P values< 0.05 was considered to indicate a statistical difference significantly. Threshold levels of significance were adjusted for multiple comparisons by Bonferroni’s correction.Continuous numerical data were presented as means with standard deviation or as medians with the range, and were compared by means of the student’s  systems . Statistn = 14, 7.9%), moderate-  or poor-  differentiated. Perineural invasion were present in 155 (87.1%) patients. Most patients had an R0 surgical margin , and 23 (12.9%) patients had an R1 margin. Postoperative main complication include infection (n = 15), hypohepatia (n = 2), biliary fistula (n = 8), postoperative bleeding (n = 6), deep venous thrombosis (n = 2). 14 patients (7.9%) accepted gemcitabine-based chemotherapy. Lymph node metastases were present in 57 (32.0%) patients, while 121 (68.0%) patients had not metastatic lymph node identified in the surgical specimen. The median number of harvested lymph node were 5 (range 1–20).The mean age of the 178 patients was 61(range 26–80) years. The population had a male dominance . The median blood loss for the resections was 400 mL (range 50–2000 mL). The median postoperative hospital stay was 17 days (range 8-50 days). Of the 178 patients, 80 were T2a and 98 were T2b. Preoperative biliary drainage was performed in 32 patients (18.0%). At the time of surgery, major hepatectomy was conducted in most patients : left hepatectomy in 60 (33.7%) patients, right hepatectomy in 35 (19.7%) patients, mesohepatectomy in 21 (11.8%) patients and a left and right trisectionectomy in 5 (2.8%) patients and 4 (2.2%) patients, respectively. The remaining 53 (29.8%) patients underwent out-hepatic bile duct resection. Caudate lobe resection was performed routinely . 10 (5.6%) patients had partial pancreatectomy. For final pathology of the resected tumor, tumor grade were classified as well- . The median survival of patients with high level of AST (≥85.0 IU/L) was 31 months, whereas that of patients with low level of AST (< 85.0 IU/L) was 41 months (P = 0.047). The median survival of patients with high level of CA19–9 (≥1000 U/mL) was 28 months, whereas that of patients with low level of CA19–9 (< 1000 U/mL) was 37 months (P = 0.015). In patients underwent vascular resection, the median survival was 10 months, while 35 months for those did not receive vascular resection (P = 0.008). The patients with postoperative complication had a median survival of 26 months, however, those without postoperative complication had a median survival of 36 months (P = 0.035). The median survival of patients without perineural invasion was 58 months, but that of patients had perineural invasion was 33 months (P = 0.003). The patients with positive resection margin had a median survival of 24 months, whereas those with negative resection margin had a median survival of 36 months (P = 0.030). The median survival for the patients with well-, moderate- and poor- differentiation tumors were 51, 37, 21 months, respectively (P = 0.001). The median survival of patients with stage N0, N1, N2 were 39, 27 and 28 months, respectively (P = 0.035).During a median follow-up of 51 (range 4–117) months, 110 (61.8%) patients died. The overall median survival were 35 months. In the univariate analysis , high level of CA19–9 (≥1000 U/mL), vascular resection and pathological differentiation of the tumor remained as independent predictors for poor survival Table . Figure P = 0.354). For group T2a, the 1-, 3- and 5-year OS rates were 88.4, 50.2 and 21.3%, respectively, with a median survival of 37 months. In T2b cohort, 1-, 3- and 5-year OS rates were 87.6, 45.0 and 23.9%, respectively, with a median survival of 31 months.In total, after curative intent resection of pCCA, there were no significant difference of survival between groups T2a and T2b . For group T2a, the 1-, 3- and 5-year OS rates were 91.5, 54.0 and 27.4%, respectively, with a median survival of 44 months. In T2b cohort, 1-, 3- and 5-year OS rates were 91.4, 49.3 and 27.4%, respectively, with a median survival of 36 months.In subgroup N0, 61 patients were included in group T2a and 60 patients were categorized into group T2b. There was no significant difference of survival between group T2a and T2b as well . For group T2a, the 1-, 3- and 5-year OS rates were 80.0, 34.3 and 0.0%, respectively, with a median survival of 33 months. In T2b cohort, the 1-, 3- and 5-year OS rates were 85.7, 37.3 and 8.2%, respectively, with a median survival of 24 months.In subgroup N1, 15 and 28 patients were respectively categorized into group T2a and T2b. No significant difference of survival existed between group T2a and T2b yet . For group T2a, the 1-, 3- and 5-year OS rates were 75.0, 50.0 and 0.0%, respectively, with a median survival of 19 months. In T2b cohort, 1-, 3- and 5-year OS rates were 70.0, 43.8 and 43.8%, respectively, with a median survival of 28 months.In subgroup N2, only 14 patients were included. 4 patients belonged to group T2a and 10 patients belonged to group T2b. There was still no significant difference of survival between group T2a and T2b .According to 7th edition of AJCC TNM staging system, 113 patients were categorized as stage II, while 26 patients as stage IIIB, 9 patients as stage IVA, and 30 patients as stage IVB, with a median survival of 36, 27, 49 and 24 months, respectively. The 1-, 3- and 5-year OS rates were 90.9, 50.0 and 27.0% for stage II; 73.1, 43.1 and 14.4% for stage IIIB; 100, 75.0 and 28.1% for stage IVA; 86.7, 34.6 and 13.8% for stage IVB. There was no significant difference in prognosis when patients were stratified by the 7th edition of AJCC TNM staging system . The 1-, 3- and 5-year OS rates were 71.4, 46.3 and 27.8% for stage IVA. Interestingly, there appeared overall significant difference between the outcome of groups categorized by the 8th edition of AJCC TNM staging system .Sorted by the 8th edition of AJCC TNM staging system, 121 patients were categorized as stage II, while 43 patients as stage IIIC and 14 patients as stage IVA, with a median survival of 39, 27 and 28 months, respectively. The 1-, 3- and 5-year OS rates of stage II were 91.4, 51.5 and 27.3%. However, those of stage IIIC were 83.7, 36.3 and 7.3%. In paired comparison, only patients with stage IIIC had a worse outcomes than those with stage II . The C-index for the 8th edition of the AJCC TNM staging system was 0.563 (95%CI 0.512–0.614). In total, both the 7th and 8th edition of the AJCC TNM staging demonstrated a poor prognostic predictive performance (C-index < 0.7).Both the 7th and 8th editions of the American Joint Committee on Cancer (AJCC) staging system for perihilar cholangiocarcinoma (pCCA) have recommended that tumor invades beyond wall of bile duct to surrounding adipose tissue could be graded as T2a and those with liver parenchyma involved into T2b. To date, there is no article focused on the prognosis of patients with stage T2a and T2b of pCCA uniquely and specifically. The current analysis represented the role of stage T2a and T2b exclusively on outcomes in resected pCCA. We found that the current AJCC T staging systems poorly stratified the prognosis of patients with T2a and T2b after curative intent resection and several clinicopathological factors of the tumor were the independent predictors for poor survival.P = 0.030). The 2-year and 3-year of survival rate were 46 and 28% for T2a, 84 and 18% for T2b in that study. Ruzzenente et al. [In comparison of basic clinicopathological characteristics between T2a and T2b groups, age, intraoperative blood loss, caudate lobe resection and Bismuth type were significantly different, whereas none of those factors were associated with poor survival, thus the baseline was balanced. There were no difference in overall survival in comparisons of group T2a and T2b, this result remained both in N0 subgroup and subgroups with lymph node metastasis (N1 and N2). This result was consistent with a few studies , 24. Howe et al.  found thDespite improvements in treatment, pCCA was associated with limited treatment options and poor prognosis . The oveThe lymph node status have been reported as one of the most important independent prognostic predictor for patients undergoing hepatectomy for pCCA , 31. In P = 0.031 for 8th AJCC vs. P = 0.055 for 7th AJCC). In a recent study, the 8th edition of AJCC staging system had a slightly better discriminatory ability with a C-index of 0.624 compared to 0.619 for the AJCC 7th edition [T4 category excluded Bismuth type IV in the 8th edition of the AJCC Staging Systems [ edition . HoweverThe present study had several limitations of note. Firstly, our study was limited by its retrospective nature, there may have been a selection bias in diagnosis and treatment. Secondly, only 14 patients underwent gemcitabine-based chemotherapy in this study and revealed no impact on OS. However, adjuvant therapy was demonstrated to association with improved survival, especially for those with node positive disease . This reIn summary, the newly released 8th edition of AJCC staging system failed to discriminate prognosis of patients with stage T2a and T2b pCCA. Both the 7th and 8th editions of the AJCC staging systems demonstrated a poor ability in predicting prognosis of patients undergoing curative intent resection for pCCA. In addition, high level of AST (≥85.0 IU/L), high level of CA19–9 (≥1000 U/mL), vascular resection and lower pathological differentiation of the tumor were the independent predictors for poor survival. At last, we proposed to merge stage T2a and T2b to simplify the AJCC staging system for pCCA in future amendments to the TNM classification.Additional file 1 Table S1. Univariate regression analyses of the prognostic factors.


Based on the experimental data, we demonstrated that under high pressure, the samples experienced two different high-pressure structural phase transitions up to 29.4 GPa: from monoclinic P21/n structure  to orthorhombic P212121 structure (phase II) at 8.9 GPa and then to monoclinic P21/n structure (phase III) at 12.4 GPa. The critical phase transition pressures for phase II and phase III are approximately 2–3 GPa higher than that of 30 nm Ag2S nanoparticles and bulk materials. Additionally, phase III was stable up to the highest pressure of 29.4 GPa. Bulk moduli of Ag2S nanosheets were obtained as 73(6) GPa for phase I and 141(4) GPa for phase III, which indicate that the samples are more difficult to compress than their bulk counterparts and some other reported Ag2S nanoparticles. Further analysis suggested that the nanosize effect arising from the smaller thickness of Ag2S nanosheets restricts the relative position slip of the interlayer atoms during the compression, which leads to the enhancing of phase stabilities and the elevating of bulk moduli.An in situ high-pressure X-ray diffraction study was performed on Ag Under ambient conditions, Ag2S is stable in a monoclinic structure with P21/n space group (α-Ag2S) [2S undergoes a thermo-induced phase transition and reforms into a body-centered cubic (bcc) structure  [−1 cm−1 [2S is considered as a fast ionic conductor (FIC), which has potential applications including in energy, analytical chemistry, biomedicine, solid-state ionic devices and so forth [2S further converse into a face-centered cubic (fcc) phase (γ-Ag2S), then keeping stable up to melting temperature [As a well-known metal sulfide, Agnductor ~.5 eV, wioperties . So far,icient 10 m−1, goo sensors . Recentl(α-Ag2S) . While hp Im3¯m) ,9. In thp Im3¯m) , leading[−1 cm−1 . Therebyso forth ,14,15. A2S, structural and optical behaviors under high pressure have been systematically studied by synchrotron X-ray diffraction and infrared spectroscopy measurements [2S bulk material experiences a series of phase transitions up to 40 GPa. At 5.1 GPa, bulk Ag2S transforms from the P21/n structure  to an orthorhombic P212121 structure (phase II). Elevating the pressure up to 8.8 GPa, Ag2S undergoes the second phase transition, from phase II to a monoclinic P21/n structure (phase III), which is isosymmetric to phase I. At 28.4 GPa, phase III further transforms into a Pnma structure (phase IV). Accompanied by structural transformations, the band gap of Ag2S is narrowed gradually by increasing the pressure. Beyond 22 GPa, the pressure effectively tunes the semiconducting Ag2S into a metal [Instead of temperature, high pressure is an effective approach for tuning both structural phase transitions and physical properties ,17. Undeurements ,18,19. I2S have been prepared, such as quantum dots [2S nanomaterials, where they expected to obtain novel properties under pressure. Thus far, there has been one report on the high-pressure behaviors of pure and Y-doped Ag2S nanoparticles [2S nanocrystals with random geometric shapes. It is still unclear what behaviors of Ag2S nanocrystals with special morphologies will actually be induced by high pressure. In particular, it is still unknown whether or not they will choose the known structural evolution laws as well as the nanoparticles. These require further studies.Up to now, with the rapid development of synthesis technology, many kinds of nanostructured Agtum dots , nanopartum dots ,22, nanotum dots , nanowirtum dots , nanorodtum dots , sheet-ltum dots  and so farticles . The par2S nanomaterials with sheet-like morphologies using the in situ high-pressure X-ray diffraction up to about 30 GPa. A set of phase transitions of Ag2S nanosheets were observed. Interestingly, the Ag2S nanosheets exhibit even higher structural stability and lower compressibility under high pressure, remarkably different from the Ag2S bulk materials and nanoparticles. Further analysis suggested that the stronger nanosize effect arising from the smaller thickness of Ag2S nanosheets effectively restricts the relative position slip of the interlayer atoms during the compression, which leads to the enhancement of phase stabilities and the elevation of bulk moduli. Our findings give a further insight into high-pressure behaviors of Ag2S nanomaterials.In this work, we investigate the high-pressure behaviors of Ag2S nanosheet was provided by Tongshun Wu, Ph. D. of State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University , which was prepared by solvothermal method. The initial sample was dispersed in ethanol, with no substrate or coating. A HITACHI H-8100  transmission electron microscope (TEM) with accelerating voltage of 200 kV was employed to analyze the typical product. X-ray powder diffraction (XRD) was used to characterize the product and the XRD patterns at ambient condition were collected by a Rigaku D/max-rA XRD  spectrometer .In this study, Agθ = 50°. A sheet of T301 stainless steel was selected as the gasket and pre-indented in the DAC to an initial thickness of about 40 μm. Then, a hole of 150 μm in diameter was drilled manually by a tungsten carbide needle in the middle of the dent, which served as a sample chamber. The Ag2S nanosheet samples along with the dispersant were loaded simultaneously into the sample chamber to keep the dispersity. A mixture of methanol and ethanol at a ratio of 4:1 was used as the pressure transmitting medium. A tiny ruby ball ~5 μm in diameter was loaded in the sample chamber, and pressure was determined by the frequency shift of the ruby 1R fluorescence line. High-pressure X-ray diffraction experiments were carried out up to 30 GPa using a synchrotron X-ray source (λ = 0.386 Å) of the beamline X17C of National Synchrotron Light Source (NSLS), Brookhaven National Lab (BNL). The monochromatic X-ray beam was focused down to 25 μm × 25 μm using Kirkpatrick-Baez mirrors. For filtering out the tail of the X-ray beam, a pinhole was placed before the sample position as a cleanup aperture. The X-ray diffraction images were collected by a MAR345 image plate, located ~350 mm behind the sample. Exposure times were typically 300~600 s. Geometric correction and radial integration was done using the Fit2D software . The observed intensities were integrated as a function of 2θ in order to give conventional, one-dimensional diffraction profiles. Rietveld refinement was performed by GSAS-EXPGUI package . A Birch-Murnaghan (BM) equation of state (EOS) was employed to fit the experimental P-V relationship, using Origin Pro software .A symmetric diamond anvil cell (DAC) with flat diamond culets of 400 μm in diameter was used to generate high pressure. The DAC allowed access to the X-ray diffraction angular range 42S samples, TEM and selected area electron diffraction (SAED) analysis was performed. 2S samples. It can be clearly seen that all Ag2S nanocrystals are well dispersed and possess sheet-like morphology; no other shapes can be observed. SAED pattern , which demonstrates that the thickness of samples is relatively thin, far below 29 nm. All the analyses show that Ag2S samples are well crystallized and have an almost-uniform morphology as nanosheets, providing ideal samples for high-pressure research.To investigate the morphology and structure of the Aginset in a determi2S nanosheets, XRD analysis was carried out. 2S phase (Joint Committee on Powder Diffraction Standards (JCPDS) card no. 14-0072) . Vertical markers indicate the Bragg reflections of the P21/n monoclinic structure. It can be seen that no other diffraction peaks exist, which means that no other phases or other impurities are mixing in the sample. The Rietveld refinement of the XRD pattern at ambient pressure yields a = 4.214 ± 0.001 Å, b = 6.912 ± 0.001 Å, c = 7.853 ± 0.001 Å, β = 99.580° ± 0.010°, 0V = 225.55 ± 0.034 Å3 for Ag2S nanosheets, slightly smaller than 30 nm Ag2S nanoparticles and bulk materials [2S nanosheets could be attributed to the stronger nanosize effect as a result of their smaller thickness.In order to further study the crystal structure of Agaterials ,28. The 2S nanosheets, we carried out an in situ high-pressure X-ray diffraction study. 2S nanosheets under different pressures. It is clear that under high pressures, two phase transitions for the samples are observed up to 29.4 GPa. With pressure increasing, all the peaks of α-Ag2S phase shift to smaller d-spacing, indicating a pressure-induced reduction of d-spacing and shrinkage of the unit cell. The initial phase can be maintained up to 8.9 GPa, much more stable than that in Ag2S nanoparticles and bulk materials [2S), the measured XRD pattern is consistent with the starting monoclinic P21/n structure (phase I). The mutual agreements encourage us to perform subsequent fine Rietveld analysis on our experimental data by using the P21/n structure. As shown in 2S nanosheets at 5.38 GPa are as follows: a = 4.107 ± 0.002 Å, b = 6.659 ± 0.003 Å, c = 7.616 ± 0.004 Å, β = 99.62° ± 0.03, 0V = 205.3 ± 0.1 Å3. According to the fitting data, all of the diffraction peaks can be indexed to the initial monoclinic P21/n structure. No new phases were observed at this pressure.To systematically explore the high-pressure behavior of Agaterials ,27. At 5α-Ag2S undergoes a phase transition. According to previous reports, these new peaks located at 8.96°, 9.18°, 9.39°, 10.90° and 11.05° can be indexed into the (120), (112), (121), (113) and (201) diffractions of orthorhombic P212121 structure (phase II). Both phase I and phase II of Ag2S coexist under this pressure. No pure phase II structure could be obtained in our work. With continuous compression, all the peaks of ambient phase I became weak gradually.While compressing up to 8.9 GPa, several new peaks (marked as asterisks) start to emerge obviously and their intensity grows with pressure increasing see , indicatθ = 9.39°, 9.78°, 10.87°, 11.30°) emerged (marked as arrows in 2S nanosheets undergo the second phase transition. These new diffraction peaks can be indexed into (022), (12-1), (200) and (014) diffractions of monoclinic P21/n structure (phase III), isosymmetric to the ambient phase. Both phase II and phase III coexist under this pressure. However, the peaks corresponding to phase II lost a lot of intensity and then completely disappeared at the pressure of 16.5 GPa. Under this pressure, all of the remaining peaks can be indexed to phase III, indicating Ag2S nanosheets crystallized in monoclinic P21/n structure completely. Typical Rietveld refinement for phase III is plotted in a = 4.079 ± 0.003 Å, b = 5.800 ± 0.006 Å, c = 7.948 ± 0.008 Å and β = 93.15° ± 0.07 (V = 187.7 ± 0.2 Å3). Compressing the samples up to 29.4 GPa, the highest pressure in our study, no new diffraction peaks were observed, Ag2S nanosheets are mainly still maintaining in phase III.Further compressed to 12.4 GPa, it can be clearly seen that a set of new diffraction peaks (22S undergoes the phase transition sequence P21/n (phase I) → P212121 (phase II) → P21/n (phase III) → Pnma (phase IV) at pressures of 5.1GPa, 8.8 GPa and 28.4 GPa, respectively [2S nanoparticles, the transition pressures elevate slightly compared to the bulk: from phase I to phase II at about 6.83 GPa and then to phase III at 9.3 GPa [2S nanosheets was about 2–3 GPa higher than that in 30 nm Ag2S nanoparticles and bulk crystals. Moreover, phase IV [2S nanosheets exhibit a different high-pressure property compared with previous reports. According to the analysis in the TEM image before, it was shown that the Ag2S nanosheets have a relatively thin thickness, far below 29 nm (the average size of the sample). Therefore, we suggest that the stronger nanosize effect arising from the smaller thickness of Ag2S nanosheets effectively restricts the relative position slip of the interlayer atoms during compression, which leads to the enhancing of phase stabilities.In previous studies, we noted that bulk Agectively . For pur 9.3 GPa . Comparephase IV , which f2S nanosheets. A third-order BM EOS was introduced to fit our pressure-volume data. For monoclinic P21/n structure (phase I), by fixing the first pressure derivative of isothermal bulk modulus (B0′) to 4, the bulk modulus (B0) was obtained as 73(6) GPa. The obtained bulk modulus 0B for phase I, the ambient phase, was much higher than that of the bulk Ag2S (~34–38(2) GPa), even higher than that of the 30 nm Ag2S nanoparticles (B0 = 57.3(6) GPa) [B0) and zero pressure cell volume (V0) were left to vary freely, and the first derivative of B0 with pressure (B0′) was fixed to 4. As shown in P-V curve yields a bulk modulus B0 of 141(4) GPa and unit cell volume V0 of 207(6) Å3. Compared to previous studies, the obtained bulk modulus B0 for phase III of Ag2S nanosheets was also larger than the bulk counterparts (102(4) GPa) and 30 nm nanoparticles (123.1(0) GPa) [2S nanosheets exhibit significant difference in compression properties under high pressure, a much lower volume compressibility than previous reports. A similar phenomenon was observed in some other nanomaterials, e.g., C60 nanosheets and ZnO nanocrystal [2S nanosheets have a very small size in thickness, much lower than their grain size of 29 nm. Thus, the stronger nanosize effect from our samples may cause the bulk modulus to largely elevate.(6) GPa) ,27. For (0) GPa) ,27. Accoocrystal ,30, and 2S nanosheets quenched to the ambient conditions are shown in 2S samples could not maintain their uniform shape because of the high pressure and high shear stresses, it still keeps the sheet-like morphology in nanoscale. Based on this result, we can reasonably draw a conclusion that during the compressing process, Ag2S samples in our experiment remain nanomaterials, and no agglomeration took place. After decompressing to ambient pressure, the shapes of all the diffraction peaks for Ag2S nanosheets return to the initial monoclinic P21/n structure (phase I), indicating the reversibility of the pressure-induced structural transitions. Further analysis showed that the (-112), (-103) peak was enhanced after compression. The effect of non-hydrostatic pressure and high pressure gives nanosheet samples a certain orientation and texture.In our high-pressure research, the quench process is implemented. TEM images and XRD pattern of Ag2S nanomaterials with sheet-like morphologies using in situ high-pressure X-ray diffraction up to about 30 GPa. Under high pressure, Ag2S nanosheets undergo the phase transition sequence P21/n (phase I) → P212121 (phase II) → P21/n (phase III) at pressures of 8.9 GPa and 12.4 GPa. Interestingly, the Ag2S nanosheets exhibit a wide field of structural stability and much lower compressibility under high pressure, remarkably different from the corresponding bulk materials and 30 nm nanoparticles. Further analysis suggests that a stronger nanosize effect arising from smaller thickness in Ag2S nanosheets effectively restricts the relative position slip of the interlayer atoms during the compression, which leads to the enhancing of phase stabilities and the elevating of bulk moduli. Our findings give a further insight into high-pressure behaviors in Ag2S nanomaterials.In summary, we studied the high-pressure behaviors of Ag


Halophilic archaea have been proposed to exchange DNA and proteins using a fusion-based mating mechanism. Scanning electron microscopy previously suggested that mating involves an intermediate state, where cells are connected by an intercellular bridge. To better understand this process, we used electron cryo-tomography (cryoET) and fluorescence microscopy to visualize cells forming these intercellular bridges. CryoET showed that the observed bridges were enveloped by an surface layer (S-layer) and connected mating cells via a continuous cytoplasm. Macromolecular complexes like ribosomes and unknown thin filamentous helical structures were visualized in the cytoplasm inside the bridges, demonstrating that these bridges can facilitate exchange of cellular components. We followed formation of a cell–cell bridge by fluorescence time-lapse microscopy between cells at a distance of 1.5 μm. These results shed light on the process of haloarchaeal mating and highlight further mechanistic questions. Pyrococcus furiosus, and Thermococcus kodakaraensis are naturally competent taking up linear and circular DNA   . SSV1 st1 (SSV1) . Also ha1 (SSV1) .Methanococcus voltae PS produces viral particles named “voltae transfer agent” (VTA) which can carry chromosomal fragments instead of viral DNA  to preserve whole cells in a frozen hydrated state. We identified and imaged cell–cell bridges connecting the cytoplasms of pairs of cells grown in liquid media. Tomograms revealed that two mating cells shared a continuous membrane, a continuous S-layer and had continuous connected cytosols. Strikingly, macromolecular structures were detected in the cell–cell bridges likely to be ribosomes. Fluorescence time-lapse microscopy of H. volcanii cells with fluorescently stained S-layers showed how cells established an intercellular bridge as an intermediate state prior to cell fusion.To better characterize the mechanism of horizontal gene transfer by fusion in H. volcanii H26 and RE25 was performed as described previously  with 0.5% (wt/vol) yeast extract (Difco), 0.1% (wt/vol) peptone (Oxoid), and 0.5% (wt/vol) casamino acids (Bacto). Selective Casamino acid medium (CA medium) was prepared as Hv-YPC medium but yeast extract and peptone were omitted. CAB medium was prepared as CA medium and contained a 100× fold diluted expanded trace element solution, containing 5 g/L ethylenediaminetetraacetic acid (EDTA), 0.8 g/L FeCl3, 0.05 g/L ZnCl2, 0.01 g/L CuCl2, 0.01 g/L CoCl2, 0.01 g/L H3BO3, 1.6 g/L MnCl2, 0.01 g/L Ni2SO4, and 0.01 g/L H2MoO4. The pH was adjusted to pH 7.0 with NaOH and the solution was filter sterilized.Growth of eviously . The celHaloferax volcanii RE 25 was inoculated in 5 mL CA medium supplemented with 1 g/l thiamine and 0.1 μg/L biotin, 10 μg/mL uracil and 50 μg/mL tryptophan that was incubated at 42°C overnight. A total of 5 and 15 μL of the pre-culture was inoculated in 20 mL CA medium and incubated again at 42°C overnight. At OD600 of 0.05, the cells were harvested at 2,000 × g for 20 min at 40°C. The pellet was dissolved in 1 mL CA medium and again pelleted at 2,000 × g for 10 min at 40°C. The cell pellet was dissolved again in CA medium to a theoretical OD600 of 3 or 5. The cells were mixed with BSA-coated 10 nm gold fiducial markers and 2.5 μL of cells were applied to a freshly glow-discharged copper Quantifoil R2/2 grid (300 mesh). The vitrification of the grid was done using the Vitrobot Mark IV (FEI). The grid was blotted on the back using a repellent Teflon membrane and subsequently plunge-frozen in liquid ethane. Electron cryotomography was conducted with a 200 kV Twin FEI F20 (FEG) equipped with a Falcon II direct electron detector. The software package Leginon was used to record tilt series  was added. The cells were incubated at RT for 1 h while rotating. To remove excess dye, the cells were washed three times as above with 500 μL CAB medium.g for 10 min at RT. SDS was added to a final concentration of 0.01% and the mixture was centrifuged again at 3,400 × g for 10 min at RT. The supernatant was divided into 2 × 500 μL and centrifuged at 190,000 × g for 1 h at 4°C. The resulting pellets were resuspended in 50 μl 1× loading dye and in 5 μL 1× PBS, 0.1% Triton-X 100. Both pellets were incubated at 6°C, in a light-protected manner for 48 h and then mixed together. This was incubated for 2 1/2 h at 37°C and 10 μL was used for an SDS-PAGE and detection of the fluorescence signal.Staining was checked by sonicating the cells for 10 min in an ultrasonic bath. Afterwards, the cell debris was pelleted at 3,400 × 600 of 1.37. The cells were pelleted at 6,200 × g for 25 min at 4°C. The pellet was resuspended in 200 mL CA medium and 60 mL of 0.5 M EDTA (pH 6.7) was added. Subsequently, the pellet was incubated at 37°C while shaking for 30 min. The spheroplasts were removed via centrifugation in an iterative manner at 3,000 × g for 15 min, 7,000 × g for 5 min and 13,000 × g for 10 min. The supernatant was concentrated via Amicon  to 500 μL. A total of 16 μL were used for an SDS-PAGE and sent for Mass spectrometry.For S-layer isolation, 400 mL of H26 was grown at 45°C to an OD® software. Images were taken with the PCO Edge sCMOS Camera (PCO) with 50 ms exposure time for the phase contrast images and 500 ms for the GFP images. Images were analyzed using ImageJ, Fiji  have also been identified between members of bacterial species and also connect the cytoplasms of neighboring cells, but still little is known about how they are formed . In Euka unclear . Also thThe original contributions presented in the study are included in the article/SS, PN, MB, and S-VA designed the research. SS performed the cryoET with technical help from Florian Rossmann (FR). SS, S-VA, and MB analyzed the tomograms, designed the figures, and wrote the manuscript. HW and PN performed the fluorescence microscopy. HW, PN, SS, and S-VA analyzed the fluorescence microscopy data and designed the figures. All authors read and reviewed the manuscript.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.