Vibrio cholerae O1/O139 by culture, and characterized antibiotic susceptibility in a subset of culture-positive isolates. We estimated the odds of being culture positive among suspected cholera cases according to different potential risk factors. From May 4, 2014 through November 30, 2021, we enrolled 51,414 suspected cases from our sentinel surveillance sites. We confirmed V. cholerae O1 in 5.2% of suspected cases through microbiological culture. The highest proportion of confirmed cholera cases was from Chittagong (9.7%) and the lowest was from Rangpur Division (0.9%). Age, number of purges, duration of diarrhea, occupation, and season were the most relevant factors in distinguishing cholera-positive suspected cases from cholera-negative suspected cases. Nationwide surveillance data show that cholera is circulating in Bangladesh and the southern region is more affected than the northern region. Antimicrobial resistance patterns indicate that multidrug resistance (resistance to three or more classes of antibiotics) of V. cholerae O1 could be a major threat in the future. Alignment of these results with Bangladesh’s cholera-control program will be the foundation for future research into the efficacy of cholera-control initiatives.Despite focusing on cholera burden, epidemiologic studies in Bangladesh tend to be limited in geographic scope. National-level cholera surveillance data can help inform cholera control strategies and assess the effectiveness of preventive measures. Hospital-based sentinel surveillance among patients with suspected diarrhea in different sites across Bangladesh has been conducted since 2014. We selected an age-stratified sample of 20 suspected cholera cases each week from each sentinel site, tested stool for the presence of  With the commitment of cholera-prone countries, technical partners, and donors, as many as 20 countries including Bangladesh plan to eliminate cholera as a major public health threat by 2030.V. cholerae phenotypes , and monitoring the impact of the interventions planned for the cholera endgame.Standardized cholera surveillance is important for estimating incidence and allocating scarce cholera control resources, tracking changes in V. cholerae O1 or O139 confirmed by microbiological culture. Diarrhea can be fatal if it is not treated immediately and adequate fluid replacement is not administered. Early recovery may be possible when immediate rehydration can be achieved.We defined a suspected cholera case as an individual of any age presenting with acute watery diarrhea, although diarrhea is defined differently for those less than 2 months old and those 2 months and older. For those less than 2 months old, acute watery diarrhea was defined as stool habits that differed from the norm in terms of intensity  or type of stool . For those older than 2 months, acute watery diarrhea was defined as having three or more loose or watery stools in the preceding 24 hours or three or fewer loose/liquid stools causing dehydration.The nationwide sentinel surveillance network included both government health facilities and private medical college health facilities that provide care at a minimum cost. Initially, the surveillance included 10 sentinel sites in 2014; this was extended to 22 sites in 2016. In all, 13 districts, six subdistricts, two tertiary-level hospitals, and the Bangladesh Institute of Tropical and Infectious Diseases were included, as previously described.Supplemental Figure 1). In addition, when there were too few suspected cases in one age group, we overenrolled in the other age group.Given our funding, we aimed to enroll 20 suspected cholera cases each week from the diarrheal inpatient ward, outpatient care, and emergency ward of each sentinel site. We enrolled suspected cholera cases 5 days a week, with a goal of enrolling the first two who were younger than 5 years and the first two older than 5 years each day. When we were unable to enroll 20 suspected cases in a given week, we extended surveillance 1 additional day to try to meet this target . Questions on the participant survey included sociodemographic factors , clinical factors , potential exposure histories, and history of antibiotic use for the current diarrheal episode. The samples were then transferred to the icddr,b and IEDCR laboratories in Dhaka using Cary-Blair  transport medium twice monthly. We used Cary-Blair transport medium because it maintains the viability of V. cholerae. For enrichment, samples were inoculated in alkaline peptone water and incubated for a further 18–24 hours.V. cholerae O1 (Ogawa and Inaba) as well as O139 serogroups were used to categorize suspected colonies.,Samples were plated over taurocholate-tellurite gelatin agar and incubated overnight at 37 °C to identify Escherichia coli American Type Culture Collection 25922 susceptible to all antimicrobials was used as a control strain for susceptibility studies.A randomly selected subsample (about 20% of the positive cases) underwent antibiotic susceptibility testing. For the entire study period, we tested the following antibiotic classes: tetracyclines (doxycycline), macrolides , and fluoroquinolones (ciprofloxacin). At the end of 2017, we added beta-lactams including penicillin (ampicillin) and cephalosporins . Bacterial susceptibility to antimicrobial agents was determined with the disc diffusion method by measuring zone size (in millimeters) following the guidelines of the Clinical and Laboratory Standards Institute,,We described the frequency and percentage of different sociodemographic factors and clinical symptoms among all patients with suspected cholera and the proportion of cholera cases by month, division (the largest administrative unit), and age group. We examined the surveillance data by specific age strata  to evaluate the risk of cholera positivity among different groups; given our enrollment strategy, we investigated risk in the < 5-year-old group versus other age groups. We set the present age strata (< 5 and > 5 years) of the patients because diarrheal diseases are one of the leading causes of illness and death among children < 5 years of age.2 statistic. Given our sampling scheme, we estimated the odds of being a confirmed cholera case on the basis of different potential risk factors, with a binomial distribution controlling for individual clustering by sentinel site and stratified by age group . We assumed an independent correlation structure, and 95% CIs were calculated using robust standard errors. We conducted univariate analyses to estimate the crude odds ratio of cholera given each factor, which included sociodemographic factors, clinical factors, exposure history, history of antibiotic use prior to enrollment, and season . Given the large number of statistical tests, we corrected P values for multiple comparisons by using the Bonferroni correction method; we considered associations as significant only after the correction.To measure the difference between proportions of suspected and confirmed cholera cases, a bivariate analysis of various risk factors is presented as the frequency, percentage, and χWe used a random forest classification model to understand the predictive value of season, sociodemographic factors, clinical features, and exposure measures to predict whether a suspected cholera case would be culture confirmed. We excluded antibiotic use history because antibiotic use in the 24 hours prior to presentation could lead to false-negative results by culture.gee package to perform the risk factor analysis and the randomForest package version 4.7–1.1 for the random forest analysis.We conducted a descriptive analysis of the serotypes identified over the course of the surveillance period and the AMR patterns over time by geography and serotype. We examined resistance to different classes of antibiotics and multidrug resistance  and described the distinct AMR phenotypes by serotype. All analyses were carried out with R version 4.1.0 using the Supplemental Figures 2 and 3). Of the suspected cases, 28% were < 5 years old (per sampling design), and 97% of suspected cases were from inpatient departments. Twenty-two percent of suspected cholera cases were housewives and 6.3% were service holders (who used in the organization). For the suspected cholera cases, the median number of purges in the last 24 hours before presentation was 15 (interquartile range [IQR]: 10–20), and the median history of diarrhea was 2 days (IQR: 2–3). Overall, 44% of suspected cases reported taking antibiotics for their symptoms prior to attending the hospital, with the highest proportion (72%) recorded in the Mymensingh Division and the lowest in Barisal (23%) .From May 4, 2014 through November 30, 2021, we enrolled 51,414 suspected cases from our sentinel surveillance sites. There were slightly more male participants enrolled (54%) than female participants . Among tV. cholerae O1 in 5.2% of suspected cases , and one sample was positive for V. cholerae O139 through microbiological culture, with significant differences in positivity between age groups . Among confirmed cholera cases, 58% were 18–45 years of age, 29% were housewives, and 17% were service holders . The highest proportions of confirmed cholera cases were from Chittagong (9.7%), Dhaka (7.4%), and Barisal (5.6%), and the lowest proportions were recorded in Khulna (2.8%) and Rangpur Division (0.9%) . The southern sentinel sites had higher cholera positivity on average across all months and seasons than the northern sentinel sites .We confirmed e groups . In aggr holders . The medV. cholerae O1 was dominant across the country: From May 2014 to May 2016, nearly all samples were Ogawa (90%); from May 2016 to May 2018, nearly all were Inaba (96%); and from May 2018 to March 2020, nearly all were Ogawa (90%). After the pandemic period, there was co-circulation of the serotypes with 39% Ogawa and 61% Inaba .We observed three distinct periods when a single serotype of V. cholerae O1/O139 isolated in stool during pre-monsoon season (March–June) was 2.4 (95% CI: 1.50–3.93) times higher than the other season (November–February) in children under 5 years old. Although the pre-monsoon season was associated with greater odds of being a confirmed cholera case for both those aged 5–17 years and those > 18 years of age, this was not statistically significant. For children < 5 years old, the odds of being confirmed with cholera during the monsoon season (July–August) was 2.9 (95% CI: 1.57–5.51) times higher and during the post-monsoon season (September–October) was 2.3 (95% CI: 1.37–3.85) times higher than the other season, though these were not statistically significant after accounting for multiple tests. The odds of being a confirmed cholera case was 1.4 (95% CI: 1.12–1.80) times higher among children < 5 years old who presented with vomiting .We explored clinical, demographic, and ecological factors that helped to distinguish culture-positive suspected cases from culture-negative suspected cases. In age-stratified univariate analyses, we found that the odds of a suspected case having Among adults, the odds of testing positive for cholera was significantly higher among males than among females . Service holders  and day laborers, workers, or drivers  were also significantly more likely than housewives to be a confirmed cholera case. Although not significant after the Bonferroni correction, having tap water as the primary source of water in the household was associated with elevated odds of being a confirmed cholera case for children < 5 years old and adults.When combining all 19 measured attributes for each suspected case in a random forest classification model to predict who would be a confirmed cases, we found a cvAUC of 77% (95% CI: 72.3–81.5%). The most influential attributes in this model were age, number of purges, duration of diarrhea, occupation, and season . At the V. cholerae O1 Ogawa cases that were resistant to all the macrolides, penicillin, and cephalosporins tested  .We tested 581 isolates for AMR, and 35 (6%) were resistant to at least one of the antibiotics tested. There were nine unique AMR phenotypes . Among tefixime) . These cSupplemental Figure 7). The cephalosporins, ceftriaxone and cefixime, and azithromycin were largely sensitive over the years.Different AMR patterns were detected over the surveillance period. In 2018, intermediate resistance was 24.3% to doxycycline and 9.5% to ciprofloxacin, which was reduced to 0% in the following years  than in other seasons (November–February). A previously published paper from Bangladesh revealed that cholera had two distinct peaks in different geographical locations.,With 7 years of data, our study indicates that cholera cases occurred throughout the year in Bangladesh, although with different levels of frequency. This included those younger than 5 years old  and those 5–17 years old (except during the monsoon). Children younger than 5 years were more likely to have As indicated previously, the duration of diarrhea, number of diarrheal episodes, stool type, and dehydration were important variables for determining whether a patient with acute watery diarrhea had culture-confirmed cholera.V. cholerae O1 suggests that the inappropriate use of antibiotics likely occurs prior to seeking care as well, which was also found in another study.V. cholerae O1 may become more prevalent as a result of self-guided treatment regimens in the absence of accessible healthcare. Of note, resistance to cefixime was observed only among MDR cases that were also resistant to ceftriaxone, the other cephalosporin tested, and ampicillin. Further, there appear to be differential resistance patterns by serotype, which suggests that active monitoring of V. cholerae O1 may be critical for determining which first-line antibiotics should be administered to cholera patients in the hospital to reduce both morbidity and mortality. These data support the premise that antimicrobial-resistant V cholerae O1 may become a major public health concern.Distinct antimicrobial-resistant patterns were observed for ampicillin, ciprofloxacin, ceftriaxone, erythromycin, azithromycin, and doxycycline, and the fluctuation of antimicrobial-resistant patterns that were detected over time but were consistent across space in this study was similar to other findings from Bangladesh.,The surveillance system has several limitations. First, the surveillance system was confined to 22 sentinel sites, and later on, these were reduced to 16 sites because of a funding shortage. Setting up a higher-density surveillance network at varied levels of the healthcare system, including pharmacies and traditional healers, could improve our estimates of cholera incidence to determine both low- and high-incidence areas and to aid future implementation of interventions to reach the 2030 roadmap goal of ending cholera. Pharmacies and other nontraditional drug sellers are commonly visited by individuals with suspected cases of cholera,Because Bangladesh is a cholera endemic country, the establishment and continuation of surveillance activities give a key visual on any cholera outbreak in different areas of the country. They also help local and national governments track trends in cholera cases and initiate preparedness plans and public health response and interventions. Moreover, the data generated from this surveillance provided evidence of high-risk priority areas and helped the Government of Bangladesh decide to conduct mass cholera vaccination campaigns in the affected areas. In 2020, Bangladesh launched an oral cholera vaccination program in the high-risk areas of Dhaka city as a preventive campaign, and in 2022 another reactive campaign was conducted in affected areas of urban Dhaka. Since the introduction of the oral cholera vaccine program in Bangladesh, approximately 10.5 million vaccine doses have been delivered to different areas of Dhaka city that were highly affected, as well as in Cox Bazar, including the host community and Rohingya people. In addition, the surveillance system is geographically widespread across all eight divisions of Bangladesh, which has allowed us to closely monitor the changing cholera burden over time with the help of well-trained field and laboratory staff and to foster a collaborative partnership between the icddr,b and IEDCR (government partners). The insights obtained from this surveillance are valuable for designing and monitoring the progress of evolving prevention strategies to achieve the target of the GTFCC’s global roadmap to end cholera.Providing oral cholera vaccine (OCV) and even a safe water supply are the most important strategies to protect communities from cholera over the long term in endemic settings such as Bangladesh and elsewhere. Results from this study and our ongoing surveillance system will aid public health experts and government agencies in analyzing the effects of initiatives such as the OCV campaigns by evaluating how the risk of cholera and the cholera burden change over time. The GTFCC should prioritize adequate surveillance systems in targeted countries with systematic confirmatory testing schemes, particularly as the COVID-19 pandemic persists and healthcare-seeking behaviors have changed, resulting in fewer medically attended cases, higher antibiotic use, and likely increased AMR. Although an increase in handwashing and hygiene in response to the COVID-19 pandemic is expected to decrease food and waterborne infections such as cholera, evidence generated from surveillance systems is necessary to monitor such claims and help achieve long-term cholera control.We were able to identify priority areas for vaccination and awareness campaigns and were also able to focus our endeavors on areas where the cholera outbreak was most severe. However, more work still needs to be done to achieve the target of ending cholera by 2030 through an integrated, multi-sectorial approach.Supplemental materials


The impact of the polyunsaturated fatty acids (PUFAs) at physiological concentrations on the composition of eicosanoids transported within the extracellular vesicles (EVs) of rat bone marrow mesenchymal stem cells and cardiomyoblasts was reported by our group in 2020. The aim of this article was to extend this observation to cells from the cardiac microenvironment involved in the processes of inflammation, namely mouse J774 macrophages and rat heart mesenchymal stem cells cMSCs. Moreover, to enhance our capacity to understand the paracrine exchange between these orchestrators of cardiac inflammation, we investigated some machinery involved in the eicosanoid’s synthesis transported by the EVs produced by these cells . We analyzed the oxylipin and the enzymatic content of the EVs collected from cell cultures supplemented (or not) with PUFAs. We prove that large eicosanoid profiles are exported in the EVs by the cardiac microenvironment cells, but also that these EVs carry some critical and functional biosynthetic enzymes, allowing them to synthesize inflammation bioactive compounds by sensing their environment. Moreover, we demonstrate that these are functional. This observation reinforces the hypothesis that EVs are key factors in paracrine signaling, even in the absence of the parent cell. We also reveal a macrophage-specific behavior, as we observed a radical change in the lipid mediator profile when small EVs derived from J774 cells were exposed to PUFAs. To summarize, we prove that the EVs, due to the carried functional enzymes, can alone produce bioactive compounds, in the absence of the parent cell, by sensing their environment. This makes them potential circulating monitoring entities. Inflammation is a natural immune process, primarily involved in removing death tissues or pathogens. Cardiac repair upon myocardial infarction injury relies on coordinated immune cell infiltration to clean the infarct lesion, and foster angiogenesis and tissue remodeling ,2. AfterPreliminary publications reported that the surrounding medium from stem cells acts in a process of paracrine signaling, increasing cardiomyocytes’ in vivo and in vitro survival ,11. LateRecently, review articles reported about EVs obtained from different sources and considered their therapeutic potentials . The themicroRNAs, carried by EVs, have especially been investigated as they can fine-tune cell function. Linked with these RNA, some proteins transported by these nanoparticles were also reported ,27,28. OOne work recently reported on an analysis of resolvins (Rv) and prostaglandins (PG) in MSC EVs . RecentlIn the present work, we extend this study to mouse macrophages (J774) and to rat cardiac MSCs, opening up the landscape of the cardiac microenvironment to stromal cells described as orchestrators of inflammation and resolution processes. The aim remains to enhance our capacity to understand the paracrine exchange between the cardiac inflammation/resolution orchestrators. As it was only reported a few times that some enzymes encoding for eicosanoid synthesis could be transported by the EVs, we prove here that these are present in our models and that they are functional during their EV transportation. Moreover, we report the singular behavior of the EVs when secreted by J774 cells.Following the MISEV2018 , we perf2. When sub-confluence was reached, the cells were deprived, each over 24 h, to induce the same stress as those described in the literature. Then, the supernatants were collected for 24 h more. The apoptotic bodies (AB) were removed via a 10,000× g centrifugation. The EVs were collected after ultra-centrifugation at 100,000× g (1:30 h), suspended in PBS and submitted to NTA analysis.The cells  were plated at 5000 to 10,000 cells/cmWe also performed size exclusion chromatography on an aqEV35 500 µL column. In accordance with the recommended protocol, we collected ten 0.5 mL fractions c and meaFirst, the crude fraction of cMSCs, suspended in PBS, was analyzed using transmission electron microscopy (TEM). A large population of spherical particles ranging from 30 to 100 nm was observed , correspThe sample was then purified using the qEV35 size exclusion cartridge (Izon).To determine the size distribution of the fractions supposed to enclose EVs, NTA was used as it provides accurate measurements even for polydisperse samples. Thus, no complementary measurements via dynamic light scattering were needed.6 and 2.8 × 106 particles/mL. To confirm that the signals observed in NTA around 70 and 110 nm could be attributed to extracellular vesicles, TEM experiments were performed. Again, two main populations of particles were detected around 100 nm and a smaller one around 70 nm. These populations were composed of spherical vesicles, which can be attributed to EVs. As already observed by Sahlen [The size distribution obtained via the granulometric analysis performed with NTA showed that the sample was relatively polydisperse, with approximately 90% of the particles smaller than 170 nm. The size of the two main populations of particles were measured with NTA, centered on 72 nm and 114 nm a. They cy Sahlen  and Aalby Sahlen , these tIn order to validate that the particles obtained from deprived J774 cell cultures could also be attributed to EVs, the same experiments were performed.6 particles/mL , resulting in low eicosanoid concentrations, a problem that is amplified by the heavy analysis protocol, as indicated by the error bars. In particular, significant amounts of resolvins (RvE1) could be systematically characterized, but are very close to the quantification limit. However, we found that, unlike the MSC and H9c2, the content of lipid mediators transported within the small EVs is reduced (compared to the quantity of cellular proteins) by a factor of 10 and their profile is close to the one of the parent cells .Similar to the studies we conducted in 2020 , we inveIn order to understand if the amplification of the profiles in the case of the J774 macrophages or of the cardiac MSC’s is due to a specific enrichment piloted by the cell or by the small EVs themselves, we incubated the EVs collected from normal cell cultures within media supplemented with the n-6 and n-3 PUFAs. Thanks to the high EV production level by cMSC, the eicosanoid quantification was more robust, as indicated by the error bars.Cardiac heart MSCs were identified based on CD45−, CD31−, Thy1+, CD44+, CD29+ and CD73+ expression after extraction from adult rat hearts and selection via adhesion on culture dishes, as stated in the Materials and Methods section.The lipidomic analyses indicated that the small EVs produced by cMSC cells can synthesize identical amounts and compositions of lipid mediators to the small EVs released by the treated cells .The J774 behavior is very different from the ones of the cardiac stroma cells . The lipTo verify that the observed autonomous biosynthesis by the EVs is effective, we investigated the presence of the biosynthetic enzymes within the EVs. We chose to investigate the enzymes synthesized by the MSC from rat hearts and the J774 mouse monocytes. We also added H9c2 rat cardiomyoblasts (described in ) on the PGES2, 15-LO and 5-LO were present in the investigated cells , but only PGES2 and 5-LO were found in J774 cells. The 5-LO and 15-LO are the only enzymes we could demonstrate as being embedded within the small EVs .Even if the prostaglandin PGD2 is often enriched in the EVs and PGES2 was abundant in cells studied, no PGD2 synthase signal could be observed in small EVs. We were not able to characterize the PGD2 synthase expressed in cMSC and H9c2. Unfortunately, hematopoietic PGDS antibody could not be tested here.Our previously published work demonstrated that small EVs produced by cardiac fibroblasts (cF), MSCs and H9c2 were enriched in lipoxin B4 (LXB4), PGD2 and HETEs compared to their parent cells . After tThese observations raised several questions we wished to address: Are the small EVs specific lipid-mediator-signaling vehicles? Are the small EVs simple carriers, or active signaling bodies? Is it mesenchymal cell specific, or are other circulating stroma cells as efficient?Formerly, we showed that, even if the oxylipin profiles of cardiac MSCs obtained from adult rat hearts are not exactly like the ones observed in bone marrow, the differences reported between the cell content and the small EV content remain comparable. Surprisingly, in the present work, the lipid mediator profile embedded in the small EVs released by J774 cultured in standard conditions  resembles those produced by the cells, even if their amount was reduced. Moreover, when cultured under PUFA supplementation, J774 cells do not transferring the lipid mediator profiles into the released small EVs. This observation would indicate that in the J774 background, small EVs do not embed specific eicosanoid signals, but carry the lipids present in the parent cytosol.However, when the small EVs produced by the J774 cells cultured under standard conditions are exposed to PUFAs , the lipid mediator profile embedded changes radically. Huge amounts of eicosanoids and docosanoids are produced, especially HETEs and lipoxins, but also some resolvins. For the latter, even if they are close to the detection limit, they are systematically observed. This observation upheld, this time, the hypothesis that small EVs carry functional oxylipin biosynthetic machinery.To assess this theory, we generated enough small EVs produced by cMSC, H9c2 and J774 cells under normal conditions to perform immunoblots on their cargo. Deduced from the lipidomic studies, we supposed that at least lipoxygenases might be globally present in the small EV cargo and possibly also PGD synthase in the H9c2 background. We assessed that the protein amounts were sufficient using Ponceau red coloration. Moreover, the efficiency of the antibody bindings was evaluated using specific tissues. Considering all these precautions, we could demonstrate that 5-LO and 15-LO were present in the small EVs secreted by the here-studied stromal cell. Contrarily to the suggestion made in the literature concerning the existence of a selective mechanism to load specific proteins onto EVs , the enzOther reports have shown that exosomecargoes are loaded with enzymes known to synthesize bioactive lipids. For example, exosomes secreted by hypoxic pulmonary artery endothelial cells are enriched in 15-LO2, whereas exosomes isolated from lung cancer pleural exudates or cancer cells contain enzymes capable of avidly converting LTC4 to the pro-tumorigenic cysteinyl leukotrien D4 ,42. HoweDue to its complexity, the protocol was schematized in ® Bright FITC, clone REA396, CD44H APC clone REA505) from Miltenyi biotech, Paris, France. Cells were incubated for 30 min with antibody diluted at 0.5 to 0.6 µg/mL in the dark on ice, and then the cells were washed with FACS buffer . Then, dead cells were determined using a LIVE/DEAD Viability kit   supplemented with 10% HIFCS and 1% Penicillin/Streptomycin culture medium. To collect the small EV fraction, the dishes were rinsed with 10 mL of PBS, and the medium was replaced with the same medium but deprived in 1% of ultracentrifuged fetal serum  for 24 h. The supernatant was collected over an additional 24 h period after the renewal of the medium.The experiments were carried out on three cell types: a commercial H9c2 cardiomyoblast line , J774 monocyte/macrophage cell line and mesenchymal stem cells from adult Lewis rat hearts, which were obtained using a protocol previously described . Briefly France) . The celProtein extraction of the samples was carried out using the RIPA lysis buffer containing phosphatase and protease inhibitors . The lysis was carried out in ice; 5 μL of the buffer was added to the samples, shaken with a vortex and finally incubated for 30 min at 4 °C. Total proteins were determined via the BCA method  in a 96-well plate. A standard range was produced from a protein solution of BSA  at 1 mg/mL . The samples were diluted 1/10 in PBS, and then 10 μL was deposited in the wells in the presence of 200 μL of the BCA reagent. After incubation at 37 °C for 30 min, the absorbance was measured at 570 nm using a spectrophotometer (TECAN).Immunodetection was realized using Western blotting with 25 to 40 μg of purified extracellular vesicles fraction or cell suspension. Detection of oxylipin-synthesizing enzymes was performed using antibodies from Santa Cruz biotechnologies (5-LO (sc-136195), 15-LO2 (sc-271290), PGES2 (sc-514224); L-PGDS (sc-390717) and HPGDS (Genetex)). Hsp60 (sc-13115) and Alix  were used according to the manufacturer’s instructions. The positive signals were detected via chemiluminescence using Biorad clarity ECL substrate and analyzed using Image lab .g to remove the apoptotic bodies. The supernatants were then subjected to precipitation via ultracentrifugation  (Beckman and Coulter).The supernatants of the cell culture were centrifuged for 20 min at 10,000× 6 cMSC and 6–8 × 106 J774/Petri dish) were collected using a scraper in 1 mL PBS and transferred to hemolysis tubes. An aliquot of 50 μL was taken to carry out an assay of the cellular proteins.Cells (around 4–5 × 102 and MgCl2) and then diluted ×100 in ultrapure Milli-Q water, filtered at 1.2 µm. The measurement of each sample was performed in triplicate, each for 60 s. The concentration of particles was presented as a function of the mean size of the particles.NTA analyses were performed at 25 °C using a NanoSight LM10  instrument equipped with an sCMOS camera. The camera level was fixed at 13 with a detection threshold of 5. Samples were prepared via EV dispersion in 200 µL of PBS  uranyl acetate in water. Aliquots (~100 μL) of EV solution prepared in PBS were put onto copper glow discharged TEM grids for 1 min. Then, the grids were placed in the solution of uranyl acetate for another minute. Then, the grids were dried at room temperature before the analysis.The presence of EVs was observed through the transmission electron microscopy (TEM) technique using a HT7700 Hitachi  microscope operated at an accelerating voltage of 80 kV. Samples for TEM were prepared using the negative-staining method with 2%  were mixed at a 400 ng/mL concentration in methanol and used as an internal standard (IS) solution. The 1 mL cell and extracellular vesicle suspensions were placed in the presence of 10 μL of IS, and the lipid extraction was carried out twice in glass hemolysis tubes with 500 μL of chloroform. The samples were vortexed and left to settle. The organic phases were collected, mixed and evaporated to dryness under nitrogen flow at 45 °C. The samples were first stored in pill boxes with glass inserts in 20 μL of methanol at −80 °C, and then subjected to spectrometric analysis by LC/MS-MS.v/v/v) (A) or  (B). The linear gradient was as follows: 0% B at 0 min, 85% B at 8.5 min, 100% B at 9.5 min, 100% B at 10.5 min, and 0% B at 12 min. The flow rate was 0.35 mL/min and the injection volume was 5 μL. The HPLC system was coupled on-line to an Agilent 6460 triple quadrupole MS (Agilent Technologies). Electrospray ionization was performed in negative ion mode. Analyses were performed in selected reaction monitoring detection mode. Peak detection, integration, and quantitative analysis were conducted using Mass Hunter Quantitative analysis software (Agilent Technologies) via correlation with the IS response.High-performance liquid chromatography was performed using an Agilent 1290 Infinity. The analytical column was a ZorbaxSB-C18 column  (Agilent Technologies) maintained at 40 °C. The mobile phases consisted of water, acetonitrile, and formic acid  to polyunsaturated fatty acid does not drastically impact the eicosanoid content of secreted small EVs. Secondly, we show that stromal cells’ small EVs (including for J774) carry the enzymatic machinery, allowing them to synthesize inflammation bioactive compounds. Finally, we report about the fact that this machinery is fully functional, as the small EVs, alone, produce high eicosanoid amounts by sensing the PUFAs in their environment.The macrophage-specific behavior that we reveal indicates that small EVs can synthesize bioactive compounds independently, even when the EVs produced by the parent cells exposed to the same environment do not. Therefore, although the molecules found in EV cargo are constrained by their parental cells, these small vesicles carry the molecular machinery allowing the modulated expression of the desired molecular profile after secretion. This observation upheld the hypothesis that they are key factors for the paracrine signaling, even in absence of the parent cell. Moreover, it opens their potential circulating monitoring role. This hypothesis now must be explored, integrating the ability of these nanovesicles to deliver their cargo in cells or even in the surroundings, as was proposed recently for tongue cancer, where many deregulated EV proteins are found in the plasma from patients .Indeed, it has yet to be discovered whether circulating or tissue EVs may have such autonomous synthesis function in vivo, or in interstitial fluid, or whether EVs are only used to transfer enzymatic material from cell to cell. Moreover, further investigations will be required to establish whether small-EV-derived cargos have functional significance in recipient cells as demonstrated by the delivery of EV-encapsulated RNA.


Lipid profiles comprised 13 lipid classes and up to 755 lipid species. We found significant inter- and intrapatient alterations in lipid profiles for tumor and non-tumor samples . Thus, NIRL tissue sampling with consecutive MS lipidomic analysis is a feasible and promising approach for the differentiation of OPSCC and non-tumorous oropharyngeal tissue and may provide new insights into lipid composition alterations in OPSCC.Ultrashort pulse infrared lasers can simultaneously sample and homogenize biological tissue using desorption by impulsive vibrational excitation (DIVE). With growing attention on alterations in lipid metabolism in malignant disease, mass spectrometry (MS)-based lipidomic analysis has become an emerging topic in cancer research. In this pilot study, we investigated the feasibility of tissue sampling with a nanosecond infrared laser (NIRL) for the subsequent lipidomic analysis of oropharyngeal tissues, and its potential to discriminate oropharyngeal squamous cell carcinoma (OPSCC) from non-tumorous oropharyngeal tissue. Eleven fresh frozen oropharyngeal tissue samples were ablated. The produced aerosols were collected by a glass fiber filter, and the lipidomes were analyzed with mass spectrometry. Data was evaluated by principal component analysis and Welch’s  Oropharyngeal squamous cell carcinomas (OPSCCs) are a subgroup of head and neck squamous cell carcinomas (HNSCCs) and were estimated to be responsible for 98,412 new cases  and 48,143 deaths  worldwide in 2020 .Significant progress has been made in understanding the impact of lipid metabolism on cancer formation and progression. The dysregulation of lipid metabolism is known to contribute to the pathogenesis and progression of cancer. Alterations in lipid metabolism, including synthesis, uptake and storage, have been shown to support the growth, survival, and proliferation of cancer cells, as well as their ability to invade and metastasize. Therefore, the analysis of the lipid composition of tumor cells can provide insights into these mechanisms, thus indicating the high potential value of lipid-based molecular analysis of malignant diseases ,9,10,11.Mass spectrometry (MS)-based approaches are an essential tool for gaining comprehensive insights into cellular lipid profiles and their pathologic alterations ,16. FurtIn the last two decades, great efforts have been made to improve surgical guidance by enhancing tissue characterization on a molecular level through MS-based approaches. In these techniques, MS is used to analyze the molecular information of a tissue sample and, in particular, to identify tissue-specific alterations in lipids and proteins. However, the techniques vary in the way the samples are obtained. The application of an electrosurgical knife (iKnife) , desorptUltrashort pulse mid-infrared lasers (IRL) with pulse widths in the picosecond or nanosecond regime are an alternative emerging technology for tissue sampling, whereby the ablation occurs via desorption by impulsive excitation (DIVE) ,34,35,36In a recent study, we demonstrated tissue sampling and homogenization using a nanosecond infrared laser (NIRL) for MS proteomics with high spatial resolution ,45. HereIn this pilot study, we combined NIRL ablation-based tissue sampling with a shotgun lipidomics approach. By using lipid extraction prior to mass spectrometric measurement and differential mobility separation for targeted lipid analysis, isobaric molecular species can be separated and quantified. With this in-depth lipidome analysis, we achieved the quantification of 755 lipid species across 13 lipid classes, which enabled us to analyze the lipid profiles of OPSCCs and compare them to non-tumorous oropharyngeal tissue samples.n = 7 OPSCC and n = 4 healthy mucosa samples). Three technical replicates were obtained from each of the OPSCC and adjacent healthy tissue samples. The demographic and clinical data for all patients are presented in In total, 11 oropharyngeal tissue samples from four patients with HPV-positive OPSCC were sampled by NIRL ablation. For differential quantitative lipidome analysis, OPSCC samples and non-tumorous oropharyngeal tissue from the adjacent areas were taken from each patient  are shown in Overall, there was an increase in the proportion of PE and PC in OPSCC samples regardless of the tumor location. However, a characteristic specific to the BOT samples was a significant decrease in the proportion of TAG, from about 70% in the non-tumorous oropharyngeal tissue to 5% in the OPSCC samples.In the non-tumorous samples from patients A and B, we measured an exceptionally high concentration of TAG, which even exceeded the log2 concentration of FFA, PC, and PE. This result is consistent with the lipid composition results described above, where a very high proportion of TAG was found to be characteristic of the healthy BOT samples. However, for all patient samples, the TAG concentration was lower in the OPSCC samples than in the healthy samples. Our findings show that, for all patients, the PC and PE lipid concentrations were higher in the OPSCC samples than in the respective non-tumorous oropharyngeal tissue.To verify whether the tumor tissue could be clearly distinguished from the adjacent healthy tissue on the basis of the quantified concentrations of the 13 lipid classes, 122 lipid species (summarized by same fatty acyl chain length and degree of unsaturation), and 327 individual lipid species, we performed nonlinear iterative partial least squares (NIPALS) principal component analysis (PCA). Scatter plot visualizations of the PCA results are depicted in For the BOT samples (from patients A and B), there was a clear differentiation of tumorous and healthy tissue along the PC1, regardless of whether the NIPALS PCA was based on quantified lipid classes, lipid species with the same fatty acyl chain length and extent of unsaturation, or individual lipid species a,c,e. Fop < 0.05) for lipid classes are highlighted.Welch’s test was used to identify significant differences between the lipidomes of the OPSCC and healthy samples for each patient. As differences in the biological replicates are already shown in For samples that originated from the BOT  and non-tumor regions in the oropharynx and revealed significantly different lipid class compositions in healthy and cancerous tissue obtained from patients with differing gender, age, and habits .We focused in particular on the exact determination of the lipid composition of the analyzed tissue to provide valuable information for our collective understanding of the alterations in lipid composition in OPSCCs. Therefore, we chose to use fresh frozen tissue samples rather than formalin-fixed paraffin-embedded (FFPE) samples, as some lipids are not preserved during FFPE processing ,47,48. AIn the second step, we analyzed the specific lipid classes and species for tissue discriminability. We found significant changes in the concentrations of lipid classes when comparing tumorous and non-tumorous tissue samples from all given patients. The TAG, CER, PC, and PE lipid classes were the decisive classes for the discrimination of tumorous and non-tumorous samples in our analysis. In particular, the 60-fold decrease of TAG in tumorous samples from the base of the tongue and the 2–4-fold decrease of TAG in some of the tonsillar OPSCC samples make this lipid class potentially important and worth investigating in a larger patient population.It is noteworthy that previous studies on lipidome changes of other neoplastic entities do not follow a general principal pattern for tumor genesis or progression. Li et al. were able to discriminate prostatic tumor samples from non-tumor samples via changes in CE, CER, nonesterified fatty acids, and TAG . Anothern = 4), our findings show the method’s potential to differentiate between OPSCC and non-tumorous oropharyngeal tissues based on significant alterations in lipid profiles. However, the above-mentioned (epi)genetic factors should be considered for future studies in a larger patient cohort, as the lipidome analysis of OPSCCs and the understanding of these factors and their influence on individual lipidomic profiles remains limited [Similar to the study by Ogrinc et al., we were also confronted with high interpatient variability in lipidomic profiles . Even th limited .TM Platform is high when compared with approaches that use proteomics [Overall, this study demonstrates the feasibility of lipidomic analysis of oropharyngeal tissue using a NIRL-based ablation setup, which, in principal, allows three-dimensional tissue sampling. The ablated tissue volume needed for the quantitative analysis of lipid classes with the Lipidyzeroteomics ,53,54,55oteomics .Improvements in the efficiency of aerosol capture and post-ablation sample processing, and modification of the MS instrument may help exploit this potential and further improve the spatial resolution of tissue sampling and differentiation. With a further increase in resolution, it may be possible to resolve the heterogeneity of the tumor inside the samples.This study shows that IRL-based sampling in general—and NIRL-based sampling in particular—combined with tissue homogenization and an aerosol collection system is a very promising concept for future biopsies, as also demonstrated in our previous work . When imA total of 11 oropharyngeal tissue samples from four patients were included in this study. The tissue samples were collected from July 2020 to December 2020 under general anesthesia at our institution. Following collection, the tissue samples were immediately rinsed with sodium chloride solution (NaCl 0.9%), transferred to 15 mL centrifuge tubes and frozen in liquid nitrogen. Samples were then stored at –80 °C until further processing. The samples were collected and processed in accordance with the World Medical Association Declaration of Helsinki and the guidelines for experimentation with humans by the Chambers of Physicians of the State of Hamburg. All patients gave written informed consent for their excised tissue to be used for research purposes. The Hamburg Commissioner for Data Protection and Freedom of Information (HmbBfDI) was notified of the collection of head and neck tumor tissue in the context of a biobank, in accordance with local laws (§12 HmbKHG) and the local ethics committee (Ethics commission Hamburg WF-049/09). For histopathologic reconfirmation, representative parts of the tissue samples were H&E stained following the standard operating procedures of the Institute of Pathology. The stained slides were evaluated blind by an expert pathologist to confirm the histological diagnosis.The ablation setup is depicted in The tunable wavelength (2.70–3.10 µm) of the NIRL was set to 2.94 µm to match the O-H vibrational stretching band of water. The pulse energy was set to 1500 µJ at the sample position. In the custom-made control software, the scanning area was set to a square of 1.6 mm × 1.6 mm containing a pattern of 15 × 15 laser shots with a lateral spacing of 114 µm for each layer. For each sample technical replicate, six layers were ablated, resulting in a total of 1350 applied laser shots and the removal of a volume of about 1750 µm × 1750 µm × 160 µm, which corresponded to about 500 nL. The volume was determined by reference measurements with optical coherence tomography, as in our previous studies, where we demonstrated that the NIRL ablation volume is reproducible for technical replicates in a specific tissue type ,45. DuriTM Internal Standards Kit  was prepared according to the manufacturer’s instructions and resuspended in methyl tert-butyl ether (MTBE). The glass fiber filter was mixed with 50 µL internal standard, 500 µL MTBE, and 160 µL methanol and then extracted for 30 min at 20 °C at a rotation speed of 400 rpm on a shaker . After adding 200 µL water (LC-MS grade), the sample was centrifuged at 16,000× g for 5 min. The upper MTBE phase was removed and transferred to a new tube. After adding 300 µL MTBE, 100 µL methanol, and 100 µL water (LC-MS grade), the sample was mixed and centrifuged at 16,000× g  for 5 min. The upper phase was removed and combined with the previous upper phase. The combined phases were dried in a vacuum concentrator centrifuge  and stored at −20 °C until further use. Prior to MS analyses, lipids were resuspended in 250 µL of 10 mM ammonium acetate in dichloromethane : methanol (50:50 (v/v)).A LipidyzerTM) was operated with lipidomics algorithms . The LipidyzerTM Platform was tuned with a SelexION Tuning Kit , and a system suitability test was performed with a System Suitability Kit , both according to the manufacturer’s instructions. The LipidyzerTM Platform used 10 mM ammonium acetate in dichloromethane : methanol (50:50 (v/v)) as the running buffer, dichloromethane : methanol (50:50 (v/v)) as rinses 0 and 1, 2-propanol as rinses 2 and 3, and 1-propanol as a DMS modifier. 50 µL samples were injected for both multiple reaction monitoring (MRM) methods: one with DMS on and one with DMS off. A detailed description of this shotgun approach has been reported previously [TM lipidomics workflow manager; lipid concentrations are given in nmol/mL.Lipidomic analysis was carried out with a triple quadrupole mass spectrometer  equipped with a differential mobility spectrometer (DMS) interface operating with SelexION technology . This deeviously . Data prp-value and fold-change significance . Significant lipid species are listed for each patient in Quantified lipid species concentrations were loaded into Perseus  and log2 transformed. Nonlinear iterative partial least squares (NIPALS) principal component analysis (PCA) was performed in RStudio , with 70% valid values in at least one patient. That corresponded to 13 lipid classes, 122 lipid species with the same fatty acyl chain length and extent of unsaturation, and 327 individual lipid species . ResultsIn this pilot study, we have demonstrated that tissue sampling and homogenization utilizing a modified nanosecond infrared laser (NIRL) setup and consecutive mass spectrometric lipidome analysis is a promising approach for tissue classification and differentiation, which also has the potential to provide new insights into lipid composition alterations in OSPCC. To our knowledge, this study is the first to provide MS insights into the lipidome of this specific anatomic location. Subsequent studies with a larger patient population and with optimizations to reduce the minimum sample amount are necessary to fully explore the capacity of the NIRL MS platform and to prospectively enable in vivo tissue sampling with improved spatial resolution.


In parallel, high, and comparable separation rates (>92%) were achieved by all membranes regardless of the MXene content. In addition, promising antifouling features were observed with the nanocomposite membranes, disclosing that these nanocomposite membranes could offer a promising potential to treat heavy metals-containing wastewater for various applications.In this work, MXene as a hydrophilic 2D nanosheet has been suggested to tailor the polyphenylsulfone (PPSU) flat sheet membrane characteristics via bulk modification. The amount of MXene varied in the PPSU casting solution from 0–1.5 wt.%, while a series of characterization tools have been employed to detect the surface characteristics changes. This included atomic force microscopy (AFM), scanning electron microscopy (SEM), contact angle, pore size and porosity, and Fourier-transform infrared spectroscopy (FTIR). Results disclosed that the MXene content could significantly influence some of the membranes’ surface characteristics while no effect was seen on others. The optimal MXene content was found to be 0.6 wt.%, as revealed by the experimental work. The roughness parameters of the 0.6 wt.% nanocomposite membrane were notably enhanced, while greater hydrophilicity has been imparted compared to the nascent PPSU membrane. This witnessed enhancement in the surface characteristics of the nanocomposite was indeed reflected in their performance. A triple enhancement in the pure water flux was witnessed without compromising the retention of the membranes against the Cu The massive wastewater amounts discharged into the aquatic system are becoming an increasingly significant concern threatening the ecosystem. The toxic nature of some contaminants in the wastewater, such as heavy metal ions, produced by some industrial activities can adversely affect marine life, plants and human health ,2. ThereAmong the realistic treatment methods, membrane separation endows limitless opportunities to separate these hazardous heavy metal ions at minimal economic and environmental costs ,14,15. I2 electrospun hybrid membranes was carried out by Moradi et al. (2016) [3C2Tx) nanosheets are promising materials with exceptional potential for the future of material science [2 and NaCl) was minimized during the forward osmosis process. Wang et al. (2021) employed a laminar hydroxylated MXene membrane for separating heavy metal cations  and co-existing anions (Cl− and/or NO3−) from wastewater [3N4 photocatalyst-modified PVDF mixed-matrix membrane [6+).With the rapid growth in nanotechnology and its outstanding applications, various novel nanoscale materials  have simply found their way into water treatment applications. The nanoscale materials were extensively harnessed for versatile wastewater treatment applications as a standalone or nanocomposite adsorbents . Their l. (2016) . Having  science . This st science ,33. Depe science . MXene a science , medical science , lithium science , energy  science , antimic science , superca science . Paralle science . Many at science . The memstewater . Comparemembrane . ExperimHerein, MXene has been suggested for fabricating a nanocomposite membrane comprising MXene and PPSU for nanofiltration of heavy metal ions removal using a facile, cost-effective and rapid method. So far, in the literature, there is a limited amount of research considering the applications of the novel MXene for polymeric membrane modification compared to conventional nanoscale materials. Additionally, most research focuses on fabricating a standalone MXene membrane (thin layer), while only a few papers reported the application of MXene for mixed-matrix membrane fabrication. In addition, no work has considered using PPSU as a host polymer for MXene mixed-matrix membrane fabrication or MXene-modified NF membranes for heavy metals removal.® R-5000) with a molecular weight of 52,000–55,000 g/mol was purchased from Solvay Advanced Polymer, Belgium. Dimethyl acetamide (DMAC) , used as an organic solvent for polymer dissociation, was purchased from the local market. Polyvinylpyrrolidone K30 (PVP-K30) was used as a pore former and purchased from Sigma-Aldrich, St. Louis, MO, USA. MXene  was obtained from the department of materials, University of Technology, Iraq. Cu (NO3)2·3H2O, Cd (NO3)2·4H2O and Pb(NO3)2 salts for filtration tests of the Cu2+, Cd2+ and Pb2+ heavy metal ions were purchased from Sigma-Aldrich, USA. All chemicals were in analytical grades and used without further purification.Polyphenylsulfone (PPSU) polymer (RadelThe neat and MXene-modified NF membranes have been fabricated via the non-induced phase inversion (NIPS) process. For preparing the membranes, a fixed amount of the PPSU polymer and PVP were dissolved in DMAC with continuous stirring at 50 °C overnight. The required composition of MXene was added to the solution and sonicated for an hour in an ultrasonication bath to achieve a homogenous dispersion and avoid agglomeration of MXene. Following that, the casting solution was left aside to get rid of air bubbles (degassing) and an appropriate amount of the solution was poured on a clean and dry glass substrate which was then cast using an automated casting machine. Finally, the film was directly placed in a tab water bath for coagulation. After the phase inversion process completion, the formed membrane was taken out, rinsed with deionized (DI) water and stored wet in a sealed container, ready for characterization. The composition of all control and modified NF membranes is given in wetW = wet membrane weight (g), dryW = dry membrane weight (g), wD = density of water (0.998 g/cm3) and pD = density of PPSU polymer (1.3 g/cm3).To investigate the influence of the MXene content on the PPSU membrane porosity, a 2 cm × 2 cm sample of each membrane was cut precisely and soaked in water. The samples were then wiped with a tissue to remove excessive drops at the surface and the weight is recorded. Following that, the samples were dried naturally at room temperature overnight. The dry weight of the samples was measured, and the porosity of each membrane is calculated according to the following equation :(1)ρ%=Wwmr) of the membranes was determined according to Guerout–Elford–Ferry equation, as given below [ε = porosity of membrane, μ = water viscosity (0.00089 Pa·s), h = membrane thickness (m), wJ = water flux (m3/m2·s) and ∆P = differential pressure.The mean pore radius rm =Scanning electron microscopy (SEM)  has been utilized to visualize the morphology of the surface and cross-section of the membranes before and after modification with various MXene contents. Before SEM imaging, membrane samples were cut into strips and frozen in liquid nitrogen. The membrane strips were then directly fractured and coated with a 5-nm thick gold layer using a sputtering coat machine. The sample was then attached to an appropriate sample holder and placed inside the SEM chamber ready for imaging.q) and the average roughness (Ra).The surface topography of the membrane was scanned using atomic force microscopy (MultiMode 8 AFM) with NanoScope V Controller. The measurements were conducted via a tapping mode in the air at room temperature using a silicon tip and were analyzed using special AFM software to determine the surface roughness parameters, including the root mean square roughness  has been employed. FTIR is a powerful tool for determining the molecular structure and existence of various hydrophilic functional groups. A background spectrum was initially taken as a baseline and then the MXene, PPSU membrane and MXene-modified membrane final spectrum is recorded. All transmittance spectra were taken within a range of 4000 cmHydrophilicity measurements of the membranes were evaluated via the sessile drop method. An optical contact angle instrument  connected to PC software was utilized to conduct the membranes’ CA measurement. The membrane sample was cut into 4 × 1 cm strips and attached to a glass microscope slide. Then, a 3 μL DI water drop was placed by the instrument’s syringe on the flattened membrane surface. The contact angle of the drop with the flat membrane surface was captured using the camera and processed using the instrument software.2+, Cd2+ and Pb2+ in the feed and the permeation was measured at a wavelength of 324.8 nm, 217.0 nm and 228.8 nm, respectively.An Atomic Absorption Spectrophotometer (AAS) instrument (iCE 3000 Series AA Spectrometers) has been harnessed to acquire the element concentration. The concentration of Cu2. More details with schematic diagrams of the testing rig were presented elsewhere [2·h), V: permeated water volume (L), Δt: the measurement period (h) and A: membrane area (m2).The water permeation and heavy metal ions retention of all NF membranes were determined using a custom-made crossflow testing rig. The system is composed of a feed tank, circulating pump, flowmeter, two pressure gauges, membrane cell and Teflon tubes. The membrane cell was constructed from Teflon and has an active filtration area of 14.4 cmlsewhere . For evap: solute concentration in the permeate and Cf: solute concentration in the feed.Following the pure water flux measurements, the membrane retention performance against three heavy metal ion synthetic solutions was conducted. Two concentrations (10 and 50 ppm) of copper, cadmium and lead-contaminated solutions have been utilized to assess the performance of the membrane. An amount of 250 mL of each concentration (10 and 50 mg/L) of the three heavy metal solutions was passed through the selected membrane at the 3-bar transmembrane pressure. About 20 mL of the permeate samples have been collected at 10-min intervals and the concentration of the heavy metals was measured utilizing Atomic Absorption Spectroscopy (AAS) after calibration. The rejection magnitude was calculated according to the following equation.Total) of the membrane induced by various heavy metal ions, the reversible fouling (Fr) and the irreversible fouling (Firr) were determined as given below.0 = the initial water flux of control membrane, J1 = the solute flux of heavy metal ions and J2 = the water flux of fouled membrane after cleaning. All measurements are replicates and an average value was presented [The fouled membrane was then backwashed with DI water for 30 min and the water flux is recorded. The total fouling  and the fouling behavior of these nanocomposite membranes under operation conditions. It should be noted that there was a trivial gradual difference in the color of the membrane’s top surface upon increasing the MXene content in the nanocomposite membranes. This change in the color was proportional as the PPSU control membrane surface was white while tending to turn into darker grey at the 1.5-MXene membrane. However, the bottom sides of all membranes were much lighter than the top surface. This indicates that MXene have migrated during the phase separation process and accumulated close to the surface of the membrane. Practically, this is a well-desired feature that could impart the hydrophilic characteristics of MXene close to the surface of the nanocomposite membrane.Scanning electron microscopy (SEM) has been employed to visualize the MXene content influence on both surface and cross-sectional morphologies of the fabricated NF nanocomposite membranes. As depicted in Meanwhile, the impact of 2D MXene nanosheets on the cross-section of the PPSU membrane was more distinguishable, as depicted in a) and root-mean-square height (Rq) of all membranes were given in a and Rq recorded 56.75 nm and 89.3 nm, respectively, which were almost double the rest of the samples. These high roughness values agreed with what was reported in the preceding literature for control membranes [a values ranging between 20.5 nm and 27.3 nm  reported a similar behavior when using MXene for PSU membrane modification [Topography parameters of the control and MXene-modified membrane surface were analyzed using atomic force microscopy (AFM). The 50 μm × 50 μm 3D images of the six membrane samples were illustrated in  27.3 nm . Comparefication .−1, 3066 cm−1 and 3092 cm−1 are the absorption bands of C–H bonds. The peaks observed at 1482 cm−1 and 1584 cm−1 are assigned to the C=C bond while the other two spectra at 1237 cm−1 and 1322 cm−1 are attributed to the S(=O)2 bond [The FTIR spectra of neat and 1.5 wt.% MXene-modified nanocomposite membranes were compared, as depicted in O)2 bond . In compO)2 bond .3AlC2 [3AlC2 were not recognizable in the nanocomposite membrane sample  between the water drop and the surface. A lower contact angle indicates higher hydrophilicity and vice versa. Higher hydrophilicity of the membrane is a well-desirable characteristic for enhancing the permeation, retention and antifouling performance of that membrane. The CA values of the control and MXene-modified membranes are illustrated in The influence of the MXene content on the mean pore size and porosity of the NF/PPSU nanocomposite membranes was investigated. Pore size and porosity are critical surface characteristics controlling the permeability and retention of any membrane. Smaller pore sizes give rise to a higher rejection rate, while a higher porosity results in higher permeability. In the meantime, all control and nanocomposite membranes revealed no change in their porosity values upon varying the MXene content in the casting solution. All fabricated membranes exhibited an average porosity of around 80%, regardless of the MXene wt.% see . This inThe pure water flux (PWF) of the MXene-modified membranes was measured and compared with that of the control PPSU membrane to investigate the influence of the MXene content on the nanocomposite membrane’s permeability. As illustrated in 2+ > Cd2+ > Pb2+, regardless of the membrane type. Compared to other metal ions, the slightly higher rejection of copper could stem from the precipitation of metal hydroxide at an alkaline or neutral pH [2+ ions’ retention, which exhibited lower values than Cu2+, despite Cu2+ having a lower hydrodynamic radius. The variation in the Cd2+ retention between the control and all nanocomposite membranes was trivial and ranged between 93.2% and 94.5%. Nonetheless, the retention of Pb2+ ions was slightly lower than the Cd2+ ions and recorded separation rates between 92–94%. These results enclosed that all control and modified membranes have comparable separation potential, despite a significant variation in their permeabilities and pore size.To investigate the influence of the MXene content on the retention potential of the nanocomposite membranes, a lab-scale crossflow filtration apparatus has been employed. All membrane samples were tested against three individual heavy metal ions  containing synthetic feed solutions. Experimental conditions were fixed during all measurements where feed concentration = 50 ppm, operating pressure = 3 bars, flowrate = 1 LMH, pH = 7 and temperature = 25 °C. The rejection measurements of the NF membranes against the copper, cadmium and lead ions are compared, as illustrated in utral pH . The conutral pH . Most prFouling is the major cause of membrane flux decline that diminishes membrane performance. Depending on the membrane surface characteristics, operating conditions and solute nature, disparate fouling scenarios could be seen, such as internal pore blocking and narrowing, deposition and cake formation . To eval2+ could be easily fixed by membrane surface charges, and this could disturb the solute passage through the membrane due to reduced pore sizes [2+ > Cd2+ > Cu2+. These results disagreed with several other reported studies that ascribed higher fouling to be associated with copper ions [A similar performance trend was also observed for the cadmium feed solutions, as shown in re sizes . The conper ions , while oper ions .To explain the results, the permeate flux reduction could occur due to many reasons, such as the concentration polarization, adsorption of solutes on the membrane surface, cake layer formation due to metal hydroxide precipitate and osmotic pressure .Despite the relatively reported promising potential of MXene in membrane field applications, there is still a long way to go before a comprehensive evaluation can be performed. Similar to all metal-based nanoscale materials, MXene should undergo thorough extensive investigation to evaluate its stability inside the polymeric membrane and final destination into water streams. In this context, a pilot plant and long-term experiment are essential to assess the real service life of the membrane. On the other hand, there are a number of reports that claimed MXene poses trivial toxicity, while others reported high toxicity to stem cells . HoweverThis study presented an endeavor to fabricate a novel nanocomposite NF membrane that comprises polyphenylsulfone (PPSU) polymer and 2D MXene nanosheets for heavy metals removal from wastewater. Results disclosed that playing with the MXene content could significantly influence some of the surface characteristics of the membranes, whereas no effect was seen on others. The roughness of the nanocomposite membranes was notably enhanced by obtaining a ~50% smoother surface compared to the control PPSU membrane (56.75 nm). In addition, the wettability of the nanocomposite membrane showcased a gradual improvement and reached its maximum value at a 0.6 wt.% MXene ratio (contact angle = 49.4°) compared to only 60.3° for the control membrane. Similarly, the mean pore size was doubled from 0.84 nm to 1.6 for the control and 0.6 wt.% MXene-modified membrane, respectively, while no observable change in the porosity was obtained for all membranes (around 79%).2+ compared to Cd2+ and Pd2+, which exhibited slightly lower rejections of 93.2–94.5% and 92–94%, respectively. In the meantime, the nanocomposite membranes manifested distinguished antifouling features and were proportional to the MXene content in the nanocomposite membrane. Even though the total reduction fouling reached up to 20% for the 0.1 wt.% MXene-modified membrane, the flux was mostly recoverable and still much higher than the control PPSU membrane.Alongside that, performance evaluations demonstrated that upon MXene incorporation, the pure water flux witnessed a 28% improvement and recorded 3.8 LMH when only 0.1 wt.% MXene was used. The uppermost flux (11.1 LMH) was revealed by the nanocomposite membrane prepared with 0.6 wt.% MXene, compared to only 2.74 LMH for the control PPSU membrane. Likewise, there was a trivial non palpable change in the retention potentials of all control and modified membranes against all metal ions solutions. In this context, the retention was higher (96–98%) for CuObtained results disclosed that MXene-functionalized membranes could provide superior potential for the functional nanofiltration of heavy metals contaminated wastewater effluents.


Pleurozium schreberi (Brid). Mitt in boreal landscapes near six gold mine sites at different stages of the mine lifecycle. We found that (1) both mining stage and ecosystem type are drivers of the phyllosphere microbial community structure in mine offsite landscapes; (2) Bacterial alpha diversity is more sensitive than fungal alpha diversity to mining stage, while beta diversity of both groups is impacted; (3) mixed and deciduous forests have a higher alpha diversity and a distinct microbial community structure when compared to coniferous and open canopy ecosystems; (4) the strongest effects are detectable within 0.2 km from operating mines. These results confirmed the presence of offsite effects of mine sites on the phyllosphere microbiota in boreal forests, as well as identified mining stage and ecosystem type as drivers of these effects. Furthermore, the footprint was quantified at 0.2 km, providing a reference distance within which mining companies and policy makers should pay more attention during ecological assessment and for the development of mitigation strategies. Further studies are needed to assess how these offsite effects of mines affect the functioning of boreal ecosystems.Plant-microbe interactions play a crucial role in maintaining biodiversity and ecological services in boreal forest biomes. Mining for minerals, and especially the emission of heavy metal-enriched dust from mine sites, is a potential threat to biodiversity in offsite landscapes. Understanding the impacts of mining on surrounding phyllosphere microbiota is especially lacking. To investigate this, we characterized bacterial and fungal communities in the phyllosphere of feather moss  Sphagnum–methanotrophs  alone, through the activity of its phyllosphere nitrogen-fixing bacteria , was shown to fix 1.5 to 2.0 kg nitrogen ha–1 yr–1 in boreal forests  generally produce more dust pollutants than non-operating sites . These results will contribute to understanding the impact of mine sites on microbial communities in moss and identifying potential indicators of offsite mining effects.This study aimed to characterize the impact of mining on the feather moss phyllosphere microbiota in boreal landscapes and sought to answer the following questions: (Q1) do mine sites affect the feather moss phyllosphere microbiota offsite? (Q2) what is the main driver of the phyllosphere microbiota in mine offsite landscapes: ecosystem type, mining stage, or their interaction? (Q3) at what distance are offsite effects of mines on the phyllosphere microbiota detectable? To answer these questions, three operating mines and three non-operating sites were selected in the boreal forest of Quebec, Canada  and 1-kmTo conduct the study, six mine sites were selected in the Abitibi-Témiscamingue and Nord-du-Québec regions of the province of Québec, Canada , includiP. schreberi were randomly selected around the center point of each plot. The distance between colonies was approximately 10 m. At least 20 shoots per colony were picked, and debris and other plants were removed in the field. Only the top green parts . In addition, forest environmental variables were also measured in the field as previously described  of escribed . Principg for 20 min at 4°C. The supernatant was discarded and the pellet was transferred to a PowerBead tube of the DNeasy Power Soil DNA Isolation Kit  for DNA extraction, following the manufacturer’s instructions. DNA extracts were quantified with the Qubit dsDNA HS assay kit using a Qubit Fluorometer 2.0 . Bacterial and fungal communities were characterized by amplifying and sequencing the V4-V5 regions of the 16s ribosomal RNA gene with the primer pair 515F-Y (5′-GTGYCAGCMGCCGCGGTAA−3′)/926R (5′-CCGYCAATTYMTTTRAGTTT−3′) , and 2.5 μl of DNA extract. Thermocycling conditions were as follows: initial denaturation step at 95°C for 5 min, 34 (bacteria) or 40 (fungi) cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and a final elongation step at 72°C for 10 min. PCR products were purified with magnetic beads . Unique codes were added to each sample using the Nextera XT Index Kit following the manufacturer’s instructions (Illumina). Indexed amplicons were purified with magnetic beads, quantified with the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific) and pooled at equimolar concentration. Sequencing was performed on an Illumina MiSeq platform with a MiSeq Reagent Kit v3 (600 cycles) at the Next Generation Sequencing Platform of the Centre hospitalier universitaire de Québec-Université Laval Research Centre. Sequence data from this study are available at the NCBI Sequence Read Archive (SRA) under the BioProject PRJNA800026.The DNA extraction process followed the protocol of GTTT−3′)  and the GTTT−3′) /ITS4 (5′GTTT−3′) , respectGTTT−3′) . The firThe DADA2 Pipeline  (decontam R package (The tables generated by the DADA2 Pipeline were imported into R (3.4) as a phyloseq object ( 1.12.3)  for furt package  was used package , whereas package .ggplot2 package version 2.0.0 (To answer the three specific questions identified in the sections “1. Introduction” (Q1–Q3) and “2.5. Statistical analyses” were divided in two sections: (1) examine offsite effects of mines on the diversity, community structure and composition of the bacterial and fungal communities of the phyllosphere and identify drivers (ecosystem type and mine stage) of these communities (Q1 and Q2); (2) determine the distance influenced by offsite effects (Q3). All analyses were performed with the statistical platform R 4.0.5 (2021-03-31) with R Studio software. Results were visualized with the on 2.0.0 .lme4 R package version 1.1-23, phyloseq package) were selected as the response variables for bacterial and fungal alpha diversity, separately. Based on the inherent statistical characteristics of response variables, Negative Binomial  and Gaussian  distribution were used in the models. Marginal R squared of GLMMs were calculated with the function “r.squaredGLMM” of the MuMIn R package R package 1.15.6  and identify potential drivers of microbial diversity, community structure and composition (Q2), alpha, beta diversity and relative abundance of individual taxa were analyzed. For alpha diversity analysis, generalized linear mixed models  with 999 permutations. PERMANOVA tests were performed for the bacterial and fungal ASV matrices as the response and ecosystem type, mining stage and their interaction as the explanatory variables and site factor as a “strata” term to reduce randomizations across all sites. Bray-Curtis distance matrices were further visualized using non-metric Multi-dimensional Scaling  with three dimensions. We also used post hoc pairwise PERMANOVAs  algorithm  was used as response variables to determine the effects of mining stage and ecosystem type . Microbiome relative abundances are compositional data that range between 0 and 1 and are also generally zero-inflated , therefolgorithm  method ion 2.4.0 . Furtherpost-hoc test) was similar to the models we used in Q1 and Q2, but here transects were nested into sites, and ecosystem type were used as random factors. Distance influenced by offsite effects of mines were determined based on the results of TukeyHSD tests and graphs . Considering the very large ranges of variation (orders of magnitude) between taxa and zeros in relative abundance datasets, row data were replaced by log (x + l) transformation to present the spatial pattern with the distance in graphs. Finally, the offsite footprint of mines was evaluated and estimated using the influenced distance for alpha and beta diversity of bacterial and fungal communities as well as the relative abundance of individual taxa.The effects of distance from the mines on alpha and beta diversity of bacterial and fungal communities (Q3) and on the relative abundance of individual taxa  were analyzed. Control transects were removed from these analyses given that the control transects were designed for landscape-level analysis (Q1 and Q2). Firstly, to assess variations in diversity and community structure  along the transects, alpha diversity indices  and Bray–Curtis dissimilarity matrices were used as response variables in GLMMs and Pairwise PERMANOVAs, respectively. The effect of distance and its interaction with mining stage (only operating and non-operating here) were used as predictors. The analysis process , 44 (6 phyla) and 22  and one fungal ASV  and six bacterial ASVs, as well as two fungal genera  and four fungal ASVs were negatively affected by either operating mines or all mine sites  based on the differences in alpha and beta diversity, relative abundance of individual taxa, and indicator species of mined sites (operating or non-operating) and controls. These findings extend the offsite effects of mines on surrounding ecosystems to microbiomes, as only vegetation and animals were used to determine the effects in previous studies . SurprisDifferences in phyllosphere fungal and bacterial beta diversity between mining stages confirmed that the mining stage is a driver of microbial community structure in mine offsite landscapes. This can be attributable to the fact that mine production directly determines dust production . PreviouSolibacter, Acidiphilium and Phenoliferia, respectively. The ability of members from the first two bacterial taxa to overcome stressful conditions  have been reported in mining environments of other minerals (Phenoliferia can degrade phenolic compounds (Phenoliferia) near mines. Nevertheless, fungal ASV 10, with its relatively high abundance  led to those effects being overlooked . If our Our results provide further evidences that feather moss phyllosphere diversity and composition differed between ecosystem types . HoweverSurprisingly, the effects of mining stage on the alpha, beta diversity and relative abundance of most phyla were not ecosystem type-dependent. It suggests that ecosystem types did not influence the ability of the moss phyllosphere to resist to the offsite effects, which is in contrast with a previous study indicating that bryophyte diversity was more affected by mining stage in deciduous forest than in coniferous forest . CompareWe also found that fungal communities were more resistant to the offsite effects than bacterial communities. According to previous studies, fungi appear to be more tolerant to heavy metals than bacteria , and expDetermining the offsite footprint of mines is challenging but necessary for policy maker and governments to assess ecological impacts of mining projects and, subsequently, develop mitigation strategies. Our findings indicated that phyllosphere alpha and beta diversity were affected by the distance from mine site, but the effect was only detectable up to a distance between 0 and 0.05 km from the edge of mine sites, which is much lower than what was detected using the relative abundance of individual taxa  for the three operating mines in our study. With more than 6,000 active mines across the globe (2 (about the land area of Cyprus). Considering non-operating sites still have effects on surrounding phyllosphere structure, the actual offsite effects of mines could be even larger. Although the mining method, size, shape and local environmental conditions could affect the range of offsite effects, our results still provide justification to include offsite footprints of mines when evaluating the total footprint of mining on landscapes.Based on all results of alpha, beta diversity and relative abundance of individual taxa, we found that significant effects are detectable within 0.2 km of mine sites for most taxa, especially for operating mines. Therefore, if the average width of the offsite footprint surrounding mines is set to 0.2 km, the total area for footprint calculation would be about 4.5 kmhe globe , the estPhenoliferia) have higher indicator roles than community-level diversity to determine the presence and footprint of offsite effects of mines. Though the average distance influenced by the offsite effects is small (about 0.2 km) near individual mines, the sum might be large and impactful across the globe. Moreover, as more mining projects will be established in the coming decades with the energy transition, more landscapes will be exposed to the offsite effects of mines. The global offsite footprint of mines is therefore expected to expand and become a problem over larger areas. Further studies are needed to assess how these changes in microbial communities of the phyllosphere in mine offsite landscapes affect ecosystem functioning, including carbon and nitrogen cycling in boreal ecosystems, and to develop appropriate mitigation strategies.Our findings highlight the presence of offsite effects of mines on microbiomes, as exemplified here in the feather moss phyllosphere. Although both mining lifecycle and forest composition were identified as factors affecting the offsite effects of mines, our study indicates that these effects are cryptic and can easily be ignored at the community level. Specific taxa such as Bacteroidota and fungal ASV10 .The data presented in this study are deposited in the NCBI-SRA (Sequence Read Archive) repository, accession number XY, CM, and NF contributed to conception and design of the study. XY organized the database and wrote the first draft of the manuscript. XY and AS performed the statistical analysis. All authors contributed to manuscript revision, read, and approved the submitted version.


Introduction: Monoclonal antibodies (mAbs) targeting immunoglobulin E (IgE) [omalizumab], type 2 (T2) cytokine interleukin (IL) 5 , IL-4 Receptor (R) α [dupilumab], and IL-5R [benralizumab]), improve quality of life in patients with T2-driven inflammatory diseases. However, there is a concern for an increased risk of helminth infections. The aim was to explore safety signals of parasitic infections for omalizumab, mepolizumab, reslizumab, dupilumab, and benralizumab.Methods: Spontaneous reports were used from the Food and Drug Administration’s Adverse Event Reporting System (FAERS) database from 2004 to 2021. Parasitic infections were defined as any type of parasitic infection term obtained from the Standardised Medical Dictionary for Regulatory Activities® (MedDRA®). Safety signal strength was assessed by the Reporting Odds Ratio (ROR).Results: 15,502,908 reports were eligible for analysis. Amongst 175,888 reports for omalizumab, mepolizumab, reslizumab, dupilumab, and benralizumab, there were 79 reports on parasitic infections. Median age was 55 years (interquartile range 24–63 years) and 59.5% were female. Indications were known in 26 (32.9%) reports; 14 (53.8%) biologicals were reportedly prescribed for asthma, 8 (30.7%) for various types of dermatitis, and 2 (7.6%) for urticaria. A safety signal was observed for each biological, except for reslizumab (due to lack of power), with the strongest signal attributed to benralizumab .Conclusion: Parasitic infections were disproportionately reported for mAbs targeting IgE, T2 cytokines, or T2 cytokine receptors. While the number of adverse event reports on parasitic infections in the database was relatively low, resulting safety signals were disproportionate and warrant further investigation. The discovery of human immunoglobulin E (IgE) in 1968  and an iThe availability of biologicals has aided patients with severe asthma in reducing exacerbations and oral corticosteroid use, while improving lung function and quality of life, especially in patients with T2 asthma . The IgEIn recent years, concerns were expressed for a hypothesized increased risk of parasitic infections among patients using biologicals affecting the T2 immune response . Such biIn current clinical studies and routine practice, reporting of parasitic infections is based on spontaneous reporting, rather than systematic anamnestic or biochemical screening. Especially in endemic regions, routine screening for parasitic infections may be useful. So far, no studies were performed on the Food and Drug Administration’s (FDA) Adverse Event Reporting System (FAERS) investigating the risk of parasitic infections among patients using biologicals affecting the T2 immune response. Therefore, the objective of this study was to determine if parasitic infections are disproportionately reported for biologicals affecting the T2 immune response.® (MedDRA®) are used as a standardized coding practice for the reported events in a received report.For this study, we used publicly available quarterly data files from FAERS covering the period 2004–2021 . FAERS dData processing of the quarterly files was performed by the Adverse Event Open Learning through Universal Standardization (AEOLUS) system . AEOLUS ® High Level Group Term (HLGT) “Helminthic Disorders” or a PT related to parasitic infections noted under High Level Term (HLT) “Infections—Not elsewhere classified (NEC)” according to MedDRA® version 24.1. All these PTs are listed in All the selected FAERS reports from the FAERS database as described previously were processed. The reported AE of interest was a parasitic infection, which was defined as any PT falling under the MedDRASignals of disproportionate reporting (SDRs) were produced by AEOLUS and expressed as Reporting Odds Ratio’s (RORs) with a 95% confidence interval (CI). The ROR and 95% CI were calculated according to Eqs A SDR was considered disproportionate if the lower boundary of the 95% CI was greater than 1 and the drug-event count was at least 3, in accordance to guidelines from the European Medicines Agency (EMA) . Three dThe FAERS database contains 16,757,507 AE reports from 2004 to 2021. The data extraction process is displayed with a flowchart . A totalHealth outcomes varied per mAb of interest . OmalizuParasitic infections were found in 34 (43%) reports for dupilumab, followed by 24 (30.4%) reports for omalizumab, 9 (11.4%) for benralizumab, and 12 (15.2%) for mepolizumab. No parasitic infections were reported for reslizumab. The specific parasitic infections for each biological can be found in In the 1st secondary analysis  , it coulFor reslizumab no AE reports were found on parasitic infections within FAERS, which could be attributed to a general low reporting rate for this biological and most probably due to lack of power. Indeed, spontaneous reports for the other biologicals were roughly a 12–16-fold more often received by the FDA than for reslizumab. The lower reporting rate for reslizumab might be due to a low number of patients receiving the drug because reslizumab has to be administered intravenously in the hospital. In contrast, other biologicals can be self-administered subcutaneously , making treatment with reslizumab less practical and potentially more costly within the healthcare system . ExcludiNotably, 50 out of 79 parasitic infections were unspecified, raising concerns on the validity of the submitted reports. The parasitic infections might have been reported by individuals, such as consumers or even healthcare workers, with no experience in the diagnosis of parasitic infections. On the other hand, sufficient information might have been provided by an experienced or treating healthcare professional to the reporter of the AE. The validity of the start date of treatment, end date of treatment, and event date  should be carefully interpreted, as in most cases these data elements were not reported, and in some cases the parasitic infection was reported before the start date of the mAb treatment. The latter would be unlikely as only primary drug suspects for the reported events were selected for analysis. Recently, a correspondence article by While theoretically it can be expected that patients using these biologicals would be at an increased risk for parasitic infections, literature is relatively scarce on the topic, and evidence on the increased risk is weak. A 2007 clinical trial performed in Brazil reported that 50% (34 of 68) of the omalizumab arm experienced at least 1 intestinal helminth infection, compared to 41% (28 of 69) of the placebo arm . The oddBesides the well-known shortcomings of randomized clinical trials (RCTs) compared to observational studies , RCTs inWhile the FAERS database contains a rich dataset which is publicly available, it is subject to underreporting and selective reporting of cases , potentiParasitic infections were disproportionately reported for mAbs targeting IgE, T2 cytokines, or T2 cytokine receptors. While the number of AE reports on parasitic infections in the FAERS database was relatively low, the resulting safety signals were disproportionate and warrant further investigation.