High-grade serous ovarian cancer (HGSOC) is a fatal form of ovarian cancer. Previous studies indicated some potential biomarkers for clinical evaluation of HGSOC prognosis. However, there is a lack of systematic analysis of different expression genes (DEGs) to screen and detect significant biomarkers of HGSOC.TCGA database was conducted to analyze relevant genes expression in HGSOC. Outcomes of candidate genes expression, including overall survival (OS) and progression-free survival (PFS), were calculated by Cox regression analysis for hazard rates (HR). Histopathological investigation of the identified genes was carried out in 151 Chinese HGSOC patients to validate gene expression in different stages of HGSOC.FAP was identified as the only novel gene that significantly contributed to both OS and PFS of HGSOC. In addition, FAP had a consistent expression profile between carcinoma-paracarcinoma and early-advanced stages of HGSOC. Immunological tests in paraffin section also confirmed that up-regulation of FAP was present in advanced stage HGSOC patients. Prediction of FAP network association suggested that FN1 could be a potential downstream gene which further influenced HGSOC survival.Of all 57,331 genes that were analyzed, High-level expression of FAP was associated with poor prognosis of HGSOC via FN1 pathway. Ovarian cancer is one of the major causes of death in females globally. According to 2018 global cancer statistics, 295,414 new cases and 184,799 deaths were reported , which rBased on the immunohistological variation, serous ovarian cancer is the most common subtype of ovarian cancer, which could be further categorized as high-grade and low-grade neoplasm according to tumor Federation International of Gynecology and Obstetrics (FIGO) grade , 6. HighFibroblast activation protein (FAP), a cell-surface serine protease, emerges as an imperative factor in cancer-associated fibroblasts (CAFs), especially relevant to tumor occurrence and progression. Structurally, FAP consists of a cytoplasmic tail, a single transmembrane domain, and an extracellular domain . FAP is In this study, we aim to identify potential biomarkers of HGSOC survival from TCGA ovarian cancer cohort bioinformatics data. We analyzed gene expression, clinical and/or demographic information, and targeted strategies for potential HGSOC biomarkers. In addition, we validated our findings by immunohistological investigation of tissues in a group of Chinese HGSOC patients. Results suggest that FAP expression could be an effective biomarker for HGSOC survival, which warrants further investigation as potential intervention of HGSOC.TCGAbiolink package in R 3.6.0 (https://www.R-project.org/) from the official TCGA website. After screening the clinical database, 320 patients were included for G3 histologic grade indicating HGSOC. Then, 2 of these patients were excluded because of lack of relevant information. Finally, a total of 318 participants were recruited in our study.Gene expression data  and clinical information were downloaded by the For gene expression analysis, we downloaded the entire 57,331-gene data in serous cystadenocarcinoma from the TCGA RNAseq database. After data cleaning, 19 of 57,331 genes were excluded because of their insufficient expression in total gene expression . Then RNA expression data were transformed to z-scores in survival analysis.This study followed the Declaration of Helsinki and was approved by the Institution of Research Ethics Committee of the First Hospital affiliated to Soochow University. All participants were fully aware of all protocols of this study, and signed up written consent forms to authorize the utilization of their tissues and relevant information.For the validation set of the findings in HGSOC TCGA database, 151 Chinese Han patients were diagnosed and recruited by the First Hospital affiliated to Soochow University, the Nanjing Maternity and Child Health Care Hospital, and the First Hospital affiliated to the Nanjing Medical University, from January 2013 to May 2019. After surgery, patients’ tumor tissues were prepared into the paraffin sections. Their demographic and clinical characteristics were collected as well.IHC was performed on paraffin sections of ovarian cancer tissues to characterize target gene (FAP) expression profile. Detailed steps can be found in our previous study . FAP antImmunohistochemical staining was performed by using the Boster SABC (rabbit IgG)-POD kit  with the recommendation of manufacturer. The above-mentioned FAP antibody was used to incubate the preparing sections overnight at 4 °C, and 3, 3′- diaminobenzidine was taken to dye for scoring, which was evaluated by two independent and qualified pathologists who were blinded to actual clinical outcomes. IHC scoring was then established as follows. Percentage of positive cells and intensity of staining of FAP antibody were first calculated and then divided into these three major categories: ≤3, negative or weak; > 3 and ≤ 6, Moderate; > 6, strong.https://string-db.org/) and Genecard version 4.13 (www.genecards.org). In order to increase prediction accuracy, only common predictions present in both websites were included.Prediction of functional protein association networks of candidate genes was performed by STRING version 11.0 (The gene ontology (GO) functional annotation and KEGG pathway analysis of host genes of polymorphisms were carried out by using the package ‘clusterProfiler’ in R (version 4.0.1).R 3.6.0. Overall survival (OS) and progression-free survival (PFS) analyses in SOC patients were conducted by Cox regression and the Kaplan-Meier method. Multivariate Cox analysis was applied to identify potential influence of FAP expression on OS and PFS at different clinical stages of HGSOC. Other relevant demographic and clinical characteristics were compared by Student’s t test or Wilcoxon test whenever appropriate between the two groups. For immunohistochemical testing in HGSOC patients, the FAP expression score between Stage I + II and Stage III + IV were compared by Student’s t. P value < 0.05 was considered as statistical significance in this study.All statistical analyses were calculated in FAP and SSC5D were still present. And, FAP was finally included because of its characteristics of typical enzyme-catalyzed activity in uniprot database (www.uniprot.org/) and its potential role in HGSOC patient’s survival . Longitudinally, low FAP expression group had 91.1% survival rate in a period of 12 months, compared with 84.4% in high FAP expression group. Survival rate in 50 months decreased to 31.9% in low FAP group and 21.4% in high group, respectively . Additionally, negative FAP was observed in 33 (21.85%) patients.Sections of 151 HGSOC patients’ tumor tissues in different stages were stained with FAP-antibody. We identified an increasing trend of FAP expression with respect to severity of cancer stages Fig. . In addiBased on STRING and Genecard, prediction of co-influence genes with FAP and their potential regulating effects was shown in Fig. P = 0.018, as shown in Table FN1 was the only significant gene based on Cox regression with its hazardous influence on HGSOC (P = 0.001 for Cox regression), COL5A1 , COL8A1 , and COL4A1  (Table Considering the similar function of collagen (COL) family, we extended the search of collagen encoding genes. Additional COL genes that could influence HGSOC included COL16A1  analysis of FAP, the top three enriched GO annotations were listed as “regulation of fibrinolysis”, “negative regulation of extracellular matrix organization”, and positive regulation of execution phase of apoptosis” three in Biological Process, while as “dipeptidyl-peptidase activity”, “aminopeptidase activity”, and “metalloendopeptidase activity” in Molecular Function Fig. . On the In this study we extensively searched the TCGA database for HGSOC patients to identify significant biomarkers for HGSOC survival. We constructed a comprehensive summary of differentially expressed genes (DEGs) of HGSOC, especially with regard to patients’ survival, in order to evaluate the effects of gene expression on HGSOC survival through data mining.FAP is a typical plasma membrane-bound serine protease, which is implicated in matrix digestion and invasion , 27. OveBased on the location of FAP in HGSOC tissue, we suggest that FAP could be a strong positive cell-surface receptor. FAP has high expression in HGSOC patients in both Chinese population (over 60% in our study) and other ethnics (over 50%) . In addiCancer-associated fibroblasts (CAFs), including FAP, usually participate in extracellular matrix structure remodeling and tumor microarray reconstruction. In this study, we also retrospectively explored the TCGA database and investigated the effects of other CAF members on HGSOC prognosis, such as ACTA2, PDGFRα/β, S100A4, and αSMA. These CAF members are widely adopted as biomarkers of HGSOC. Nevertheless, none of these genes showed significant association with HGSOC survival. This result may be partly due to negative expression of αSMA .FN1 as the only potential gene regulated by FAP in HGSOC. Currently, there is no direct evidence to establish the causal relationship between FAP and FN1. Nevertheless, in this study, we demonstrated similar eventual clinical outcomes induced by FAP and FN1, positive correlation between their expression, significance of their role in overall survival of HGSOC, and direct regulating relationship predicted by STRING and Genecard. All these novel findings suggested FAP as a novel trigger for FN1, at least for HGSOC survival. For THY1 gene, though it was reported as a putative tumor suppressor of ovarian cancer, our study did not produce the same results as previous ones [THY1’s presence in our prediction of potential FAP association networks. It is possible that THY1 cooperates with FAP during HGSOC occurrence but not in prognostic period.We identified ous ones , despiteFN1 expression is potentially down regulated by FAP and further influences HGSOC survival.After extensive data mining of TCGA database, we identified FAP as a significant biomarker for HGSOC survival. FAP overexpression led to worse outcome of HGSOC patients, especially in the advanced clinical stage.


The public dental care sector is striving to fulfill the preventive and restorative needs of Egyptians, including pregnant women, who may not receive timely care due to misconceptions about dental treatment during pregnancy. Because of this, they are likely to suffer dental pain, with higher risk of infection affecting their offsprings.To compare the effectiveness of chemo-mechanical caries removal using Papacarie-Duo and Atraumatic Restorative Treatment (ART) in reducing dental pain among pregnant women.A randomized controlled clinical trial was conducted, in 2019, and included 162 pregnant women visiting family health centers in Alexandria, Egypt, with dental pain due to dental caries not extending to pulp. Patients were randomly assigned to Papacarie-Duo group (n = 82) and ART group (n = 80) after stratification by number of treated surfaces. The outcome variables were reduction in pain assessed using Visual Analogue Scale (VAS), satisfaction with treatment, and time taken for dental caries removal. T test/ Mann Whitney U test were used to compare groups and Freidman test was used to compare change across time.P = 0.001). Patients in the Papacarie-Duo group were significantly more satisfied with treatment than those in the ART, immediately after treatment  and after 6 months . Significantly less excavation time was recorded in the Papacarie-Duo group than in the ART group .Pain reduction was significantly greater in the Papacarie-Duo than the ART group ; 5/10/2020, retrospective registration. Physical and hormonal changes affect pregnant women in many ways and the oral cavity is no exception. There are many common oral problems during this stage such as pregnancy gingivitis, benign gingival lesions, tooth mobility, tooth erosion, periodontitis and dental caries . Dental Despite these consequences, pregnant women, sometimes, do not seek dental treatment, not only due to misconceptions about the safety of dental procedures during pregnancy but also because some dentists may be reluctant to provide dental treatment .In Rio de Janeiro, Brazil, 39% of the participating pregnant women experienced dental pain, which affected their daily normal activities . Unlike There has been an increased interest in the use of minimally invasive restorative procedures as an alternative to traditional rotary techniques of dental caries removal to overcome its various disadvantages such as vibration, pressure and heat generation. These minimally invasive methods include chemo-mechanical agents and atraumatic restorative treatment (ART) .The ART technique involves dental caries removal using hand instruments, followed by cavity filling using glass ionomer. There is no need for anesthesia and pain is reduced to minimum. This approach was originally developed for use outside the dental clinic, in remote areas with no electricity , 14. It Chemo-mechanical caries removal, on the other hand, involves the use of chemical solutions which soften dental caries and facilitate its removal using hand instruments. There are two categories of chemo-mechanical caries removal agents: sodium hypochlorite (NaOCl) such as Carisolv, and enzyme-based agents where Papacarie is considered its most common agent . PapacarMost studies involving oral health of pregnant women focused on periodontal conditions –21, whilThe aim of the study was, thus, to compare the effectiveness of two minimally invasive caries removal techniques: chemo-mechanical (Papacarie-Duo) and ART, in pregnant women, regarding pain reduction after one month and six months, their satisfaction with the treatment procedure and time taken for dental caries removal. The null hypothesis was that there would be no statistically significant difference between the two modalities in pain reduction.A randomized, two parallel arms, controlled clinical trial was conducted among pregnant women attending family health centers for their routine prenatal care in Alexandria, Egypt, from January to October 2019.The study was conducted in accordance with the CONSORT guidelines and HelsPregnant women were eligible to participate if they were in the first or second trimester, had at least mild dental pain as identified by a score of at least 5 mm on a Visual Analogue Scale (VAS) 100-mm-long , and witSample size was based on assuming a 5% alpha error, 20% beta error and reported percentage of patients with pain after using chemo-mechanical caries removal = 68% and 35% after ART . The minA computer-generated list of random numbers was used to randomly assign patients into one of the two study groups in a ratio 1:1 in blocks of four . The allPapacarie was introduced into the cavity using the applicator and left for 40 s and a blunt excavator was used to remove the softened dentin. The remaining gel was removed using a cotton pellet. When there was no change in gel color, the cavity was considered caries free . The cavThe tooth was cleaned with a wet cotton pellet to remove debris and plaque, dental caries was then removed using sharp spoon excavators , followed by cleaning the cavity using a small wet cotton pellet and finally dried with a dry cotton pellet. The cavity was considered caries-free when a leather-hard texture was reached and the excavator did not stick anymore . The GICDifference in pain was measured using Visual Analogue Scale (VAS) at baseline, after one month and a six months period. The scale is represented by a 100-mm-long horizontal line labeled “no pain” at one end and “worst pain” at the other end. Participants were asked to mark the place on the line representing their level of pain .Satisfaction with treatment was assessed using two questions: the first question was immediately following treatment: “Was the treatment carried out according to your expectations?” The second question was after 6 months” “Has the treatment solved the problem of your teeth?” Each question was answered on a 10-point scale, with lower values indicating a negative perspective and higher values indicating a positive experience .Time to remove dental caries was recorded using a stop watch from the start of excavation up to complete dental caries removal To control for potential confounders, information about age, education level, pregnancy stage, brushing frequency and last dental visit as well as clinical oral health indicators like number of decayed teeth, gingival index and plaque index were collected using the Arabic version of the WHO oral health assessment questionnaire for adults which was previously validated . ClinicaP < 0.05.SPSS  was used for data analysis. Quantitative variables were checked for normality using Shapiro Wilks tests, histograms and QQ plots. Descriptive statistics were displayed as frequencies and percentages for categorical variables and means and standard deviations for quantitative variables. Independent t test or Mann Whitney U test  was used to assess differences in quantitative variables: age, number of decayed teeth, gingival index, plaque index, pain and satisfaction scores. Chi square was used for categorical variables: education level, brushing frequency and last dental visit. Friedman test used to assess change in VAS across time in each group. An intention-to-treat analysis was performed by assigning the worst pain and satisfaction scores to those lost to follow up using worst case scenario analysis. Significance level was set at Among 250 pregnant women assessed for eligibility, 162 participants fulfilled the inclusion criteria and were randomly allocated to Papacarie-Duo group (n = 82) or ART group (n = 80) . Consumption of fresh fruits at least once daily was less common: (36.6% and 36.3% respectively).There were no differences between pregnant women in the Papacarie-Duo and ART groups regarding number of decayed teeth: mean (SD) = 2.91 (0.86) and 2.71 (0.92), plaque accumulation: mean (SD) = 1.69 (0.34) and 1.65 (0.29), or extent of dental caries: single surface = 54.9% and 51.3%. Women in the Papacarie-Duo had more gingivitis than those in the ART group: mean (SD) = 1.54 (0.32) and 1.43 (0.29).P = 0.454, including those with single surface cavities: 18.33 (11.28) versus 18.29 (10.22), P = 0.987 and those with multiple surfaces cavities: 27.3 (9.9) versus 29.49 (10.5), P = 0.353.Table P = 0.003 and in multiple surfaces cavities: mean (SD) = 8.11 (7.11) and 13.46 (8.75), P = 0.008. No significant difference was noted between groups among women with single surface cavities: 3.67 (6.16) and 5.85 (7.15), P = 0.100.At one month, women in the Papacarie-duo group had significantly less pain than women in the ART group regarding all cavities: mean (SD) = 5.67 (6.93) and 9.56 (8.79), P = 0.002, in those with single surface cavities: 2.33 (4.88) and 4.88 (6.66), P = 0.044 or in those with multiple surface cavities: 7.43 (7.32) and 12.82 (9.23), P = 0.012.After 6 months, women in the Papacarie-duo group had significantly less pain than those in the ART group whether in the whole sample: mean (SD) = 4.63 (6.56) and 8.75 (8.9), P =  < 0.001). The difference in the percent reduction from baseline to 6 months between the groups was statistically significant for the whole sample: 81.55 (28.84) and 69.43 (30.35), P = 0.01, for women with multiple surface cavities: 72.07 (31.67) and 58.93 (27.9), P = 0.04 but not among women with single surface cavities: 89.53 (23.78) and 79.41 (29.49), P = 0.084.The reduction in pain from baseline to one and 6 months was statistically significant in both groups  and 9.84 (0.37) versus ART: 8.32 (0.93) and 8.27 (0.89), P < 0.001) or with multiple surface cavities: Papacarie-Duo: 9.3 (0.81) and 9.38 (0.72) versus ART: 7.67 (0.66) and 8.05 (0.89), P < 0.001. The change in satisfaction across time in the two groups was not statistically significant for the whole sample  and for women with single surface cavities . In women with multiple surfaces cavities, there was no significant difference in the Papacarie-Duo (P = 0.183), while in the ART group, the satisfaction after 6 months increased significantly (P = 0.004), Table The mean (SD) satisfaction scores immediately after treatment and after 6 months were significantly higher in the Papacarie-Duo: 9.6 (0.68) and 9.63 (0.59) than in the ART group: 8 (0.87) and 8.16 (0.89), P =  < 0.001) indicating that patients who reported less pain were more satisfied with the treatment provided.A significant negative moderate correlation was found between pain and satisfaction at 6 months  time in minutes taken for dental caries removal was significantly less in the Papacarie-Duo-duo: 10.38 1.94) for all cavities, 9.36 (1.8) for one surface cavities and 11.62 (1.28) for multiple surfaces cavities than in the ART group: 11.56 (1.62) for all cavities, 10.71 (1.37) for one surface cavities and 12.46 (1.37) for multiple surfaces cavities,  for all Papacarie-Duo was associated with significantly greater pain reduction, higher satisfaction and less time of dental caries excavation than ART in pregnant women. The greater effectiveness of Papacarie-Duo was more observed in multi-surface cavities when pain and excavation time were considered whereas greater satisfaction compared to ART was reported after 6 months in one surface cavities. The null hypothesis of the study can, therefore, be rejected. The current findings have implications for the dental care of women who suffer from dental pain during pregnancy and are reluctant to seek conventional treatment for fear that it may harm their babies. Even if dental caries extends to more than one surface, it can still be restored with Papacarie-Duo if adequate access for the instruments can be ensured. The duration of the trial shows that this treatment can adequately last throughout the period of pregnancy after which other types of treatment can be provided if needed. Further studies with longer follow up and with economic evaluation are needed to inform policy decision makers about the use of Papacarie-Duo as an efficient restorative modality in low-resource settings.Pain reduction was greater in multi-surfaces cavities treated with Papacarie-Duo than ART whereas there was no difference in one surface cavities. This may be attributed to the components of the gel that contains papain enzyme which has an anti-inflammatory, bactericidal and bacteriostatic action. Papacarie-Duo removes carious tissues without affecting the sound collagen fibers in the affected normal dentin, and as a result, less instrumentation is required with less pain generated during excavation . This fiThe present study is the first to evaluate the effect of two minimally invasive restorative techniques (Papacarie and ART) on pain reduction and satisfaction among adults. Only one study by Barata et al.  comparedIn the present study, there was a significant increase in satisfaction among patients with multiple surfaces cavities treated by ART, after 6 months, but not among those treated with Papacarie-Duo. This may be attributed to the fact that some patients felt relatively mild pain during excavation in larger cavities and thus longer time was taken to completely remove dental caries than in single surface cavities. This led to initially lower levels of satisfaction with subsequent potential for improved satisfaction as time passed and dental pain resolved. Although direct comparison is not possible, these findings were in line with Anegundi et al.  who repoThe present study revealed significantly shorter time of excavation in the Papacarie-duo group than in the ART, in both single and multiple surface cavities. This is because Papacarie-Duo softens dental caries so that it is easily removed. This is comparable to other studies of permanent dentition, in adults, where the time for excavation in the present study was slightly less in the Papacarie-Duo group as compared to that reported by Kumar et al.  and lessOne of the limitations of the study is that blinding could not be applied due to the differences between the two techniques. Also, patients who received Papacarie-Duo may have over-reported their satisfaction because of their attraction to the new material and some patients had difficulty translating pain to a linear scale. There was a slight difference between groups in the severity of gingivitis. However, both groups were in the mild to moderate level of gingival inflammation and thus, this minor difference is unlikely to affect the study conclusions. Also, in the Papacarie-Duo group, the cavity was considered caries free when the gel stayed clear. On the other hand, in the ART group, excavating till hard dentin was reached might have been responsible for greater pain felt with less pain reduction reported , 43. FutPapacarie-Duo is available at low cost (73$ per tube which may be used for 25 applications) providing a cost reduction of 42% compared to traditional drilling method . AlthougThis study was performed in public healthcare centers which mainly serve the disadvantaged communities. Hence, this new low-cost line of treatment is suggested for use in remote areas, deprived from proper facilities and equipment. In addition, this study aids in filling the knowledge gap by providing evidence about the effectiveness of minimally invasive caries treatment in adults which makes possible the translation of the results of scientific research to clinical practice. Future studies, based on action research, are needed to evaluate the feasibility and cost-effectiveness of integrating minimally invasive caries removal into comprehensive dental care programs for pregnant women.Chemo-mechanical caries removal using Papacarie-Duo is more effective in reducing dental pain, in pregnant women, and is associated with more satisfaction and less excavation time than ART which offers an alternative low-cost method for treatment of dental caries among women who suffer from dental pain during pregnancy but and are reluctant to seek conventional treatment.


Plant diversity in the Arctic and at high altitudes strongly depends on and rebounds to climatic and environmental variability and is nowadays tremendously impacted by recent climate warming. Therefore, past changes in plant diversity in the high Arctic and high-altitude regions are used to infer climatic and environmental changes through time and allow future predictions. Sedimentary DNA (sedDNA) is an established proxy for the detection of local plant diversity in lake sediments, but still relationships between environmental conditions and preservation of the plant sedDNA proxy are far from being fully understood. Studying modern relationships between environmental conditions and plant sedDNA will improve our understanding under which conditions sedDNA is well-preserved helping to a.) evaluate suitable localities for sedDNA approaches, b.) provide analogues for preservation conditions and c.) conduct reconstruction of plant diversity and climate change. This study investigates modern plant diversity applying a plant-specific metabarcoding approach on sedimentary DNA of surface sediment samples from 262 lake localities covering a large geographical, climatic and ecological gradient. Latitude ranges between 25°N and 73°N and longitude between 81°E and 161°E, including lowland lakes and elevated lakes up to 5168 m a.s.l. Further, our sampling localities cover a climatic gradient ranging in mean annual temperature between -15°C and +18°C and in mean annual precipitation between 36 and 935 mm. The localities in Siberia span over a large vegetational gradient including tundra, open woodland and boreal forest. Lake localities in China include alpine meadow, shrub, forest and steppe and also cultivated areas. The assessment of plant diversity in the underlying dataset was conducted by a specific plant metabarcoding approach.We provide a large dataset of genetic plant diversity retrieved from surface sedimentary DNA from lakes in Siberia and China spanning over a large environmental gradient. Our dataset encompasses sedDNA sequence data of 259 surface lake sediments and three soil samples originating from Siberian and Chinese lakes. We used the established chloroplastidal P6 loop trnL marker for plant diversity assessment. The merged, filtered and assigned dataset includes 15,692,944 read counts resulting in 623 unique plant DNA sequence types which have a 100% match to either the EMBL or to the specific Arctic plant reference database. The underlying dataset includes a taxonomic list of identified plants and results from PCR replicates, as well as extraction blanks (BLANKs) and PCR negative controls (NTCs), which were run along with the investigated lake samples. This collection of plant metabarcoding data from modern lake sediments is still ongoing and additional data will be released in the future. Arctic and high-elevation ecosystems are very sensitive to natural and anthropogenically-induced climate variability. Anthropogenic warming and changes in land-use have been considered to shift vegetation composition and plant richness in these areas during the last centuries and decades . Still, Plant diversity from sedimentary DNA in Siberian and Chinese lakesLakes are located in the Siberian Arctic and in low- to high-elevation lakes from Northern China and from the Tibetan Plateau. Lake sites include large lakes, which were formed during past glacial periods and smaller lakes formed by thermokarst.The lake sites in Siberia were accessed during field trips conducted by the Alfred Wegener Institute in the years 2005 to 2016. The lake sites in China were visited from 2003 to 2018. This data-set has been established to correlate modern genetic plant diversity with modern vegetation mappings and climate and environmental data. The modern sedDNA data will be used to a.) evaluate suitable localities for sedDNA approaches, b.) provide analogues for preservation conditions and c.) conduct reconstruction of plant diversity and climate change mainly across glacial/interglacial phases in the Late Pleistocene–Holocene and during recent environmental change in the Anthropocene.The CAS Strategic Priority Research Program supported the sample collection on the Tibetan Plateau in 2018 . Jian Ni, Xianyong Cao and Kai Li were also supported by the Grant No. XDA20090000 and the China Scholarship Council (CSC). Further, fieldwork on the Tibetan Plateau was supported by the Research program (STEP) of the Chinese Academy of Science , the Strategic Priority Research Program of Chinese Academy of Sciences, Pan-Third Pole Environment Study for a Green Silk Road (Pan-TPE) (XDA20040000) and the National Natural Science Foundation of China (41877459). The National Natural Science Foundation of China (NSFC) and the German Research Foundation  supported the project as well. The expedition to Sakha in 2007 (07-SA) was funded by the grant from the Ministry of Nature Protection of the Republic of Sakha (Yakutia) “Bioindication assessment of the quality of drinking water of the Anabar River" . The Kolyma expedition 2008 (08-KO) was partly supported by the contractual theme of the Ministry of Nature Protection of the Republic of Sakha (Yakutia). The Tiksi expedition in 2009 (09-Tik) was supported by the Russian-German Biological Monitoring (BioM) research network in the terrestrial Arctic, funded by the German Ministry of Education and Research (BMBF) and supported by the German Research Foundation (DFG). The expeditions to Chatanga in 2011 and 2013  were co-financed by a Project of the Ministry of Education of the Russian Federation “NEFU Development Program: Activity 2.8 Biomonitoring of the tundra ecosystems of the North-East of Russia under the conditions of global climate change and intensification of the anthropogenic process . The expeditions to the Omoloy region in 2014 (14-OM) and Chukotka in 2016 (16-KP) were funded by the Ministry for Education and Science of the Russian Federation (project 5.184.2014/K).Sampling localities comprise lakes from Northern , Eastern  and Central Siberia (05-Yak), Northern and Central China and Tibet  or from soil on a lake’s shoreline (three samples).The first centimetre of the surface sediment (259 samples) and shoreline soil (three samples) was carefully sampled by using gloves and single-use plastic spoons. Samples were transferred into sterile Whirl-Pak® or sterilised Nalgene tubes and were kept dark and cool (+4°C) until further treatment in the laboratories for environmental and ancient DNA at Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research.The lake localities cover a large geographical, climatic and ecological gradient, including elevated lakes from 0 up to 5168 m a.s.l. Latitude ranges between 25°N and 73°N and longitude between 81°E and 161°E. The climatic gradient ranges in mean annual temperature between -15°C and +18°C and in mean annual precipitation between 36 and 935 mm. The localities in Siberia span a large vegetational gradient including tundra, open woodland and boreal forest. Lake localities in China include alpine meadow, shrub, forest and steppe and also cultivated areas.25° and 73° Latitude; 81° and 161° Longitude.The retrieved DNA metabarcoding data provide 623 unique plant sequences which mainly include terrestrial and aquatic vascular plants and a few mosses. Plant DNA sequences are identified to different taxonomic levels. About 78% of sequences types are assigned to species level, 13% to genus-level and 9% to higher taxonomic levels .Sampling of lake surface-sediments was conducted in the years 2003–2018.Creative Commons Public Domain Waiver (CC-Zero)Environmental and plant diversity data from modern sedimentary DNA of lakes in Siberia and Chinahttps://doi.org/10.1594/PANGAEA.920866. DNA sequence data can be retrieved at Dryad https://doi.org/10.5061/dryad.k6djh9w4r.Environmental data can be retrieved at Pangaea 2Environmental data of lake localitiesTable25Compilation of environmental data for the 262 investigated localities, which include additional intra-lake localities taken within three large lakes, namely: 16-KP-01-L02 (nine samples), 16-KP-03-L10 (five samples), 16-KP-04-L19 (four samples)  and are based on the average climate data for the years 1970–2000 at a spatial resolution of 30 seconds (ca. 1 km2). The site-specific climate data was interpolated to the location area by using the R packages raster  and pH and electrical conductivity were measured with a WTW multi-340i device. Annual mean temperature, mean temperature in July and January and mean annual precipitation were downloaded from WorldClim 2  and PowerMax Soil DNA Isolation kit  following the manufacturer’s instructions with the following modifications: To each sediment sample in the Power Bead solution 2mg/ml proteinase K, 0.5ml dithiothreitol (DTT) and 1.2 ml C1 buffer were added, then vortexed for 10 min and incubated overnight in a rotating shaker at 56 °C. The final elution step was carried out with 1.6 ml C6 buffer. Each DNA extraction batch contained a maximum of nine samples and one extraction blank. Precautions were taken for all the experimental steps to avoid potential contaminations  by keeping the total count number for each sample in which the read was detected. Further, we used obiclean to remove probable PCR and sequencing errors. For taxonomic assignment, we used ecotag with two different databases. One database comprises a sequence reference database generated from the EMBL standard sequences (http://www.ebi.ac.uk/ena) using the release 138. To apply the EMBL database with ecotag, a transformation of the EMBL into an ecoPCR database was required. Therefore, we first ran a simulated in silico PCR , containing 1664 vascular plant taxa and 486 bryophyte species  are plotted in Fig. We analysed in total 262 lake samples, resulting in 688 PCRs, which divide into 553 sample PCRs and 135 PCRs of extraction blanks and NTCs. The analysis of the resulting sequence data and taxonomic assignments was done by using the OBITools package . Firstlylico PCR  with the species  already After merging raw paired-end reads and demultiplexing according to the internal barcode, two sample PCRs from lakes 16-KP-02-L08 and 16-KP-04-L22, as well as seven BLANKs and five NTCs yielded no read counts and were discarded from the dataset. After taxonomic assignment of the remaining 674 PCRs, we identified a total of 15,754,779 reads which had a 100% match to either the EMBL or arctborbryo database. For 99.6% of the reads , we identified 621 unique plant sequences types, while 0.39%  of the reads were assigned to non-plant taxa, including bacteria, algae and higher eukaryotic taxa . Further, we identified 340,346 reads  in extraction blanks and NTCs, whereof 38.7%  of reads in the BLANKs and NTCs were of non-plant origin. Amongst the samples, excluding BLANKs and NTCs, we found large differences in sample read counts which range between 1 and 718,279.DB66053C-AB5A-588A-B269-EA9795742CD910.3897/BDJ.8.e57089.suppl1Supplementary material 1Environmental data of lake localitiesData typeLake environmental dataBrief descriptionCompilation of environmental data for the 262 investigated localities, which include additional intra-lake localities taken within three large lakes namely: 16-KP-01-L02 (nine samples), 16-KP-03-L10 (five samples), 16-KP-04-L19 (four samples). The table includes information about the geographic coordinates, elevation, type of sample material, geographic region, water depth (at which samples were taken), pH, water conductivity, mean annual precipitation (MAP), mean annual temperature (MAT), July and January mean temperature, vegetation type and dominant plant taxon . If no dominant taxon is listed, the surrounding vegetation is too diverse to determine dominant taxon. ‘NA’ – data not available.File: oo_456694.txthttps://binary.pensoft.net/file/456694Kathleen R. Stoof-Leichsenring, Sisi Liu, Weihan Jia, Kai Li, Luidmila A. Pestryakova, Steffen Mischke, Xianyong Cao, Xingqi Liu, Jian Ni, Ulrike Herzschuh87B698CD-C244-5185-928D-C4EBB37719A210.3897/BDJ.8.e57089.suppl2Supplementary material 2Taxa list of identified plant sequencesData typeTaxonomic list of plant species derived from metabarcodingBrief descriptionPoaceae). 'root' indicates that the DNA sequence could not be assigned to a reference in the appropriate database. ‘occurrence_in_PCR’– total number of occurrences in the PCR samples.A taxa list of identified plant sequences with either a 100% match to the embl138 or arctborbryo taxonomic database. The table contains the ‘ID’ – unique identifier for a cluster on the sequencing flow cell, ‘best_identity_arctborbryo’ – best identity with the arctborbryo database, ‘best_identity_embl138’ – best identity with the embl138 database, ‘scientific_name_by_arctborbryo’ – best taxa name within the arctborbryo database, ‘scientific_name_by_embl138’ – best taxa name within the embl138 database, ‘DNA sequence’ – Nucleotide sequence of the DNA sequence type detected, ‘best_match_in_arctborbryo’ – accession number of the best reference entry in the arctborbryo database, ‘best_match_in_embl138’ – accession number of the best reference entry in the embl138 database, ‘count’ – total count of sequence type in the total sequencing project. The scientific name 'PACMAD clade' indicates the taxonomic assignment to a true group of grasses . 'root' indicates that the DNA sequence could not be assigned to a reference in the appropriate database. Samples are sorted according to the four sequencing runs, which is indicated by the short name of the sequencing run at the beginning of the sample name. Sample batches  belonging together are numbered. Sample names that include A and B were sediment samples taken from the same bulk surface samples and share the same environmental data as given in Suppl. material 1.A taxa list of identified plant sequences with either a 100% match to the embl138 or arctborbryo taxonomic database. The table contains the ‘ID’ – unique identifier for a cluster on the sequencing flow cell, ‘best_identity_arctborbryo’ – best identity with the arctborbryo database, ‘best_identity_embl138’ – best identity with the embl138 database, ‘scientific_name_by_arctborbryo’ – best taxa name within the arctborbryo database, ‘scientific_name_by_embl138’ – best taxa name within the embl138 database, ‘DNA sequence’ – Nucleotide sequence of the DNA sequence type detected, ‘best_match_in_arctborbryo’ – accession number of the best reference entry in the arctborbryo database, ‘best_match_in_embl138’ – accession number of the best reference entry in the embl138 database, ‘count’ – total count of sequence type in the total sequencing project. The scientific name 'PACMAD clade' indicates the taxonomic assignment to a true group of grasses (File: oo_456687.txthttps://binary.pensoft.net/file/456687Kathleen R. Stoof-Leichsenring,Sisi Liu, Weihan Jia, Kai Li, Ulrike Herzschuh


Post-Transplant erythrocytosis (PTE) has not been studied in large recent cohorts. In this study, we evaluated the incidence, risk factors, and outcome of PTE with current transplant practices using the present World Health Organization criteria to define erythrocytosis. We also tested the hypothesis that the risk of PTE is greater with higher-quality kidneys.We utilized the Deceased Donor Study which is an ongoing, multicenter, observational study of deceased donors and their kidney recipients that were transplanted between 2010 and 2013 across 13 centers. Eryrthocytosis is defined by hemoglobin> 16.5 g/dL in men and> 16 g/dL in women. Kidney quality is measured by Kidney Donor Profile Index (KDPI).p < 0.001], more likely to have polycystic kidney disease , have received kidneys from younger donors [36 ±13 vs 41±15 years], and be on RAAS inhibitors . Recipients with PTE were less likely to have received kidneys from donors with hypertension , diabetes , and cerebrovascular event . Higher KDPI was associated with decreased PTE risk [HR 0.98 (95% CI: 0.97–0.99)]. Over 60 months of follow-up, only 17 (36%) recipients had sustained PTE. There was no association between PTE and graft failure or mortality,Of the 1123 recipients qualified to be in this study, PTE was observed at a median of 18 months in 75 (6.6%) recipients. Compared to recipients without PTE, those with PTE were younger [mean 48±11 vs 54±13 years, The incidence of PTE was low in our study and PTE resolved in majority of patients. Lower KDPI increases risk of PTE. The underutilization of RAAS inhibitors in PTE patients raises the possibility of under-recognition of this phenomenon and should be explored in future studies.The online version contains supplementary material available at 10.1186/s12882-021-02231-2. Post-transplant erythrocytosis (PTE) has a reported incidence between 8 and 20% and is a potentially serious complication following kidney transplantation (KT) with increased morbidity and risk for thromboembolic events –7. SeverMost of these studies lacked standardized definition of PTE. Some studies used cut off values of hematocrit (50 to 53.5%), or hemoglobin (16.5–18 g/dL), whereas others used a cut off value of actual red cell mass –9, 11–13In 2016, the World Health Organization (WHO) revised the cutoff values for diagnosing erythrocytosis to include hemoglobin > 16.5 g/dL in men and > 16 g/dL in women, hematocrit > 49% in men and > 48% in women, or red cell mass > 25% above the mean normal predicted value , 16. TheThe study cohort is derived from the Deceased Donor Study (DDS), which is an ongoing multicenter, observational, cohort of deceased donors and their kidney recipients –20. BriePTE was defined as having at least one measurement of hemoglobin > 16.5 g/dL in men and > 16 g/dL in women during the 5 years follow-up after transplant. Persistent PTE was defined as having at least two qualifying measurements of hemoglobin. Every recipient had hemoglobin abstracted from clinical charts at each time point  regardless whether the patient qualifies for PTE or not, eliminating any lead-time bias. Diagnosis of PTE was based on laboratory criteria and not based on documentation of PTE diagnosis in clinical chart. Possible causes of secondary erythrocytosis were not available in the abstracted data. Baseline characteristics were collected at the time of transplant per the DDS protocol. The primary outcome of interest is PTE up to 5 years after transplant and the time to first diagnosis of PTE. The secondary outcomes include death-censored graft failure, death only, or all-cause graft failure with median post-transplant follow-up of 4  years. Death censored graft failure was defined as primary non-function of kidney after transplant, re-transplantation, or return to dialysis during follow-up time before death. Death only was defined as all-cause mortality before graft failure. All-cause graft failure was defined as all-cause mortality, primary non-function, return to dialysis, or re-transplantation.We used KDPI as the primary exposure. KDPI is derived from the kidney donor risk index (KDRI), which is calculated using the 10-variable equation generated by Rao, et al. . The var2), and preemptive transplant. We conducted a complete case analysis because < 2% of data for all covariates was missing. SAS 9.4 software for Windows  was used for all analyses. All statistical tests and confidence intervals were 2-sided with a significance level of 0.05.We reported descriptive statistics as means (standard deviation) or medians (inter-quartile range) for continuous variables, and frequencies (percentages) for categorical variables. Continuous variables were compared with Wilcoxon rank sum or Kruskal-Wallis tests, and categorical variables were compared with chi-square tests or Fisher exact tests. Cox proportional hazards models were used to evaluate the association between KDPI and the outcome of PTE. We accounted for the cluster effect of paired kidneys from the same donor using robust sandwich estimates We used Kolmogorov-type supremum tests to evaluate proportional hazards assumptions . We alsop < 0.001).Fourteen recipients were excluded from the 1137 due to missing hemoglobin or KDPI data, leaving the study cohort of 1123 patients. The median follow-up time was 60  months. PTE was observed in 75 (6.6%) patients and the median time to development of PTE was 18 months . Mean peak hemoglobin in patients with PTE was 17.12 ± 0.77 compared to 13.32 ±1.79 in patients without PTE  as the cause of their end stage renal disease .Compared to recipients without PTE, those with PTE were younger  and basiliximab were used for induction immunosuppression in 928 (83%) and 163 (15%) of recipients, respectively. Triple maintenance immunosuppression regimen consisting of a calcineurin inhibitor, mycopthenolate and prednisone was utilized in 82% of recipients. There was no difference in the choice of induction or maintenance immunosuppression at discharge, 12 months, and 24 months after transplant between those with and without PTE  versus 8.1 (4.9) mg, p = 0.01] and at 24 months . Renin-angiotensin-aldosterone system (RAAS) inhibitors were prescribed in 182 (16%) and 257 (23%) of the recipients in this cohort within 12 and 24 months following KT respectively. Within the first 12 months, 24% of those with PTE were on RAAS inhibitors during at least one follow-up visit compared to 16% of those without PTE (p = 0.05). Within the first 24 months, 35% of recipients with PTE were on RAAS inhibitors at some point compared to 22% of those without PTE (p = 0.01). Regarding the timing of starting RAAS inhibitors, 31 (41%) of patients with PTE were started on RAAS inhibitors after meeting criteria for PTE and 10 (13%) were already on RAAS inhibition at the time they met criteria for PTE. This leaves 34 (45%) patients with PTE who did not receive any RAAS inhibitors despite meeting criteria for PTE.The average daily mycophenolate dose in the first 12 and 24 months following KT between PTE and non-PTE groups was similar . We also observed a continuum of risk across the KDPI score such that, higher KDPI was associated with lower risk of PTE [HR 0.98 (0.97–0.99)]  deaths and 8 (11%) graft losses in recipients with PTE compared to 103 (10%) and 228 (22%) respectively in the non-PTE group. PTE was not associated with increased risk of death [unadjusted HR 0.88 ], all cause allograft failure [unadjusted HR 0.81 ], or death censored allograft failure [unadjusted HR 0.72 ] over the 5 year follow up period Table .Table 3There was no difference in the rate of hospitalization for any cause, hospitalization due to cardiac event, or acute rejection between the two groups Table .Table 4Recipients with PTE achieved higher estimated glomerular filtration rate (eGFR) compared to those without PTE at all points of follow-up within the first 24 months.Only 52% (31 out of 59) of men and 18% (3 out of 16) of women with PTE were observed to have persistent PTE. There was no difference in the use of RAAS inhibitors between those with and without persistent PTE [21% versus 18% at 12 months (p 0.64) and 35% versus 34% at 24 months (p 0.91)].This large multicenter study provides significant updates to the existing knowledge in PTE. We utilized longitudinally collected data over a long follow-up period to re-examine risk factors and outcomes of PTE in the era of modern immunosuppression, and using standardized definitions with consideration for gender differences.Earlier studies established PTE as an erythropoietin-dependent phenomenon , 25, 26.RAAS inhibitors have been shown to be effective in the treatment of PTE and therefore are used as first-line therapy , 27–31. Concerning PTE impact on transplant outcomes, our study showed that those with PTE did not achieve superior patient or allograft survival over a follow-up period of 5 years despite having better kidney quality, therefore, arguing the possibility that PTE may mitigate the expected favorable outcomes of receiving a higher-quality kidney. It is worth recalling here that a previous study by Kiberd et al. showed that recipients with PTE achieved superior overall survival but had inferior renal allograft survival , 32, 33.Our study is the first to assess the natural course of PTE using the updated WHO criteria to define erythrocytosis with lower hemoglobin thresholds in a multi-center seting . DespiteConsistent with previous studies, we found an association between PTE and recipient sex, renal allograft function, and end-stage renal disease due to polycystic kidney disease , 12, 34.Some limitations exist in the current study. All patients in this cohort were recipients of deceased-donor kidney transplants which limit generalizability to living donor kidney transplant recipients. We could not assess rates of certain PTE-related complications such as thromboembolic events because the occurrence of these events was not systematically collected during follow-up. For the same reason, the presence of secondary causes of PTE such as tobacco use, malignancy, lung disease, sleep apnea, and renal artery stenosis were not assessed. Other therapies that have been traditionally used as second line treatments for PTE such as therapeutic phlebotomy , and theIn conclusion, our study demonstrated a low incidence of PTE in the current era of immunosuppression and an association between PTE and quality of donated kidneys. This association is driven by traditional risk factors for chronic kidney disease in the donor. Despite the low incidence, the underutilization of RAAS inhibitors in patients with PTE raises the possibility of under-recognition of this phenomenon. We could not demonstrate any associations between PTE and recipient outcomes, but lack of improved graft survival in recipient receiving higher quality kidneys is concerning. Our findings need to be confirmed in larger cohorts to allow for improved recognition and management of PTE.Additional file 1: Table 1S. Association of Post Transplant Erythrocytosis and Patient & Allograft survival After Exclusion of Events in First 18 Months


Streptococcus pneumoniae (pneumococcus) in vitro biofilm formation, colonization of human middle ear epithelium cells (HMEECs) as well as mouse nasal cavity and its transition to the middle ear and lungs. The in vitro biofilms and planktonic growth of S. pneumoniae were evaluated in metal ion free medium in the presence of UP. Biofilms were quantified by crystal violet (CV) microplate assay, colony forming unit (cfu) counts and resazurin staining. Biofilm structures were analyzed using a scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs were evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected using the Ezcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using the Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization was evaluated using microarray. In vivo colonization of pneumococci in the presence of UP and transition to middle ear and lungs were evaluated using an intranasal mice colonization model. The UP exposure significantly increased (*p < 0.05) pneumococcal in vitro biofilms and planktonic growth. In the presence of UP, pneumococci formed organized biofilms with a matrix, while in absence of UP bacteria were unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilm formation and quorum sensing were up-regulated in pneumococci biofilms grown in the presence of UP. The HMEECs viability was significantly decreased (p < 0.05) and bacteria colonization was significantly elevated (p < 0.05) in co-treatment (UP + S. pneumoniae) when compared to single treatment. Similarly, increased apoptosis and ROS production were detected in HMEECs treated with UP + pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, and immune response, were up-regulated in co-treatment and were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. The in vivo study showed an increased pneumococcal colonization of the nasopharynx in the presence of UP and a higher transition of bacteria to the middle ear and lungs in the presence of UP. The UP exposure elevated S. pneumoniae in vitro biofilm and colonization of HMEECs, and in vivo mouse nasopharyngeal colonization, and increased dissemination to mouse middle ear and lungs.Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism by which this occurs is not well understood. This study evaluated the effect of urban particles (UP) on  Urban particles (UP) are a major component of air-pollution and are a mixture of liquid and suspended atmospheric particles that consists of various size particles with an average size of 10 µm , along with fine (PM2.5) and ultrafine particles (PM0.1)5. These UP also contain various metal ions such as iron, calcium, manganese, sodium, magnesium, zinc, copper etc.6. The UP are inhaled and deposited on various parts of body including the nose, pharynx, larynx, trachea, bronchi, and distal lungs. Epidemiological studies have revealed that vehicle emissions and cigarette smoke containing PM exposure increased the vulnerability to respiratory infections including otitis media (OM) and pneumonia10. Previously, PM including UP have been implicated in enhancing respiratory tract and nosocomial microbial infection such as OM, bronchitis, bronchiolitis, tuberculosis, atypical mycobacterial infections, pneumonia, and meningitis12.World-wide air-pollution is a major problem due to toxic particulate matter (PM) contents that exert a negative effect on human health. In 2012 alone, approximately 7 million deaths have been reported, particularly in developing countries where pneumonia was one of the most common diseaseStreptococcus pneumoniae is a Gram positive commensal opportunistic pathogen that colonizes the nasopharynx and is responsible for two million deaths annually14. Pneumococci initially colonizes the nasopharyngeal cavity in the form of biofilms, and from these, transits to other sterile sites causing chronic otitis media (COM), meningitis, pneumonia, bacteremia and sepsis in immune-compressed patients, children and the elderly16. The initial interaction of PM such as UP and nosocomial bacteria such as S. pneumoniae occurs in the nasopharyngeal cavity, and PM exposure increases the risk of infection and alters host’s defense18. Indeed, severe pneumonia and OM were reported in cigarette smoker’s exposure to PM and bacteria such as Streptococcus and Haemophilus12. The adverse effects of PM, including UP alone on human health, including pneumonia, OM and various other diseases is known21. Similarly various studies have reported that S. pneumoniae is implicated in nosocomial infections such as pneumonia, OM, meningitis and bacteria16. The UP and S. pneumoniae are the two risk factors for pneumonia and OM that interacts in the nasopharyngeal cavity. However, the interaction of S. pneumoniae in the presence of UP or vise-versa is not well understood. Whether the pre-exposure of UP to the nasopharynx increases pneumococcal colonization or if the presence of UP reverts commensal pneumococci to the pathogenic kind needs to be understood. We hypothesize that the presence of UP favors S. pneumoniae biofilm formation, colonization of epithelial cells, and the nasopharynx and risk factor AOM and pneumonia. Therefore, in this study, we evaluate the effects of UP exposure on; (i) S. pneumoniae in vitro biofilm and planktonic growth, (ii) pneumococcal colonization and toxicity to human middle ear epithelium cells (HMEECs) and alteration of global gene expression (iii) pneumococcal colonization of mouse nasopharynx and dissemination to the middle ear and lungs.The animal experiments were carried out as per the Institutional Animal Care and Use Committee (IACUC) Korea University Seoul, South Korea. Animal protocols were approved by the Institutional Review Board of Korea University, college of medicine, Seoul, South Korea with IRB number KOREA -2017-0190. The HMEECs used in this study was kindly provided by Dr. David J. Lim .Streptococcus pneumoniae D39 is Avery’s virulent serotype 2 (NCTC 7466) strain obtained from the Health Protection Agency Culture Collection 22. It is a fully sequenced and highly virulent strain in animal models, even after many years of its isolation from a patient24. Other S. pneumoniae such as serotype 3 (ATCC 6303) 19A and 19F (ATCC 49619) were obtained from ATCC, USA. The antibiotic resistant strain of S. pneumoniae (CCARM 4001) was purchased from . The bacterial strains were grown on blood agar plates (BAP) supplied with 5% sheep blood purchased from a local supplier . The UP was purchased from NIST (NIST 1604a), and stock solutions were prepared in phosphate buffered saline (PBS) and sonicated for 5 minutes and sterilized using an autoclave.S. pneumoniae D39 planktonic and biofilm growth was evaluated in a metal ion free medium in the presence of different concentrations of UP (50–200 µg/mL). The metal ion free medium was prepared by treating brain heart infusion (BHI) broth medium with chelex-100 (Sigma) that chelate metal ions25. The bacteria’s planktonic growth was detected by measuring optical density at 600 nm, resazurin staining and cfu counts at various time points. The effects of UP on in vitro biofilm growth of S. pneumoniae were evaluated using the static microtiter plate assay, resazurin staining and cfu counts26. Briefly, the bacterial colony was grown overnight on BAP that were scraped and subsequently grown in BHI medium till log-phase and pelleted by centrifugation. A 1 × 105 cfu/mL cell suspension was prepared in chelex-100 treated medium, and 200 µL was inoculated in 96-well plates or 1 mL in 24-well plates. The UP solutions were vortexed and final concentrations of 50–200 µg/mL were added. The plates were incubated at 37 °C in 5% CO2 for 48 hr. After incubation, the medium was removed and the plates were washed twice with sterile water and stained with 0.1% CV for 15 min. The plates were washed twice and CV was dissolved in ethanol and absorbance was measured at 570 nm using a spectrophotometer. The metabolically active cells within the biofilms grown with UP (200 µg/mL) were detected by resazurin staining. Resazurin, is a non-fluorescent blue colored dye that is reduced to a pink colored fluorescence (Resorufin) by metabolically active cells allowing quantification of viable cells. Resazurin staining was performed by a procedure previously reported27. For resazurin staining the biofilm were washed and incubated with (0.02%) resazurin dye in 100 μL BHI-broth for 30 min at 37 °C. The fluorescence was measured  using a multimode microplate reader . The viable bacteria within the biofilms were detected by cfu counting. The biofilms were washed and dissolved in sterile water and serially diluted and plated on BAP. The bacteria colonies were counted after over-night incubation at 37 °C.The effect of UP on S. pneumoniae D39 biofilms were grown in 24-well microplates by the procedure mentioned above for 48 hr. Biofilms were washed and fixed with 2% glutaraldehyde and 2.5% paraformaldehyde. Post-fixation, the biofilms were treated with 1% osmic acid for 2 hr, followed by dehydration by treatment with different concentration of ethanol from 60–95%. The biofilms were immediately washed three times and preserved in t-butyl alcohol at −20 °C. The biofilms were freeze dried and coated with platinum. The images were captured using a field emission scanning electron microscope .The morphology of biofilms grown in the presence of 200 µg/mL UP were evaluated using SEM. The In-vitro biofilms grown in the presence of UP (200 µg/mL) were analyzed using confocal microscopy and fluorescence in-situ hybridization (FISH) by the procedure reported earlier28. The S. pneumoniae D39 biofilms were grown in μ-slides  for 48 hr and washed and hybridized with a peptide nucleic acid (PNA) universal bacterial probe EUB338 (5′-TGCCTCCCGTAGGA-3′)29. The PNA probe was labeled at the N terminus with AlexaFluor488 via a double 8-amino-3,6-dioxaoctanoic acid (AEEA) linker and was synthesized from Panagene . The biofilm samples were prefixed in 4% paraformaldehyde, dehydrated with ethanol and hybridized with a probe at 46 °C for 3 hr in hybridization buffer . The biofilms were washed in washing buffer at 48 °C. The probe labeled biofilm was examined using a Nikon A1 confocal microscope  with FITC channel.luxS), competence related genes  and toxin related genes  were evaluated using real-time RT PCR. S. pneumoniae D39 biofilms were grown in metal ion-free medium by procedure mentioned above, and total RNA was extracted using RNeasy Total RNA Isolation System Kit . The RNA was quantified using Nano-drop, and cDNA was synthesized using Bioneer cDNA synthesis kit . The cDNA amplification was carried out in a 20 µL reaction mixture consist of 10 µL 2x SYBR Green PCR mastermix, 4 µL cDNA, 2.5 pmol forward and reverse primers. The PCR reaction was performed for 45 cycles, with an initial denaturation step at 95 °C for 2 min, followed by 45 cycles of denaturation at 95 °C for 30 s and annealing and extension at 60 °C for 1 min. The primers used in real-time PCR are given in Supplementary Table −ΔΔCT method.The gene expressions of biofilm related gene  containing medium (antibiotic free medium) and further incubated for 8 hr. The HMEECs pre-exposed to UP were infected with S. pneumoniae (MOI 10) for 15 hr. The HMEECs viability was detected using the EZ-cytox cell viability kit  as per manufacturer’s instruction.The HMEECs were maintained and routinely cultured in airway epithelial cell growth medium  supplemented with bovine pituitary extract (0.004 mL/mL), epidermal growth factor (10 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/mL), epinephrine (0.5 µg/mL), triiodo-L-thyronine (6.7 ng/mL), transferrin (10 µg/mL), retinoic Acid (0.1 ng/mL) and 1% fetal bovine serum and 5% COS. pneumoniae treatment were detected by the Annexin-V/PI double staining apoptosis detection kit according to the manufacturer’s protocol . Briefly, 1 × 106 HMEECs were seeded overnight in a six-well plate and the medium was replaced with fresh medium containing UP (200 µg/mL) and incubated for 8 hr. Thereafter, the HMEECs were treated with S. pneumoniae (MOI 10) for 15 hr. After treatment, cells were collected and washed twice with PBS and re-suspended in 1× binding buffer . The cells were stained with Annexin-V for 15 min followed by PI staining and evaluated using a flow cytometer . The apoptosis rate was calculated using Beckman Coulter software.The apoptosis of HMEECs pre-exposed to UP followed by S. pneumoniae were detected by the OxiSelect ROS assay kit  according to the manufacturer’s procedure. Briefly, 1 × 104 HMEECs were grown in a 96-well dark-well clear bottom plate for 24 hr. The cells were washed twice with PBS and exposed to 100 µL of 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) in culture medium at 37 °C for 40 min. The cells were treated with UP (200 µg/mL), followed by S. pneumoniae (MOI 10) treatment. The ROS was detected measuring fluorescence using a microplate reader at 480/530 nm. Hydrogen peroxide was used as a positive control.The ROS generated in HMEECs exposed to UP and in situ hybridization (FISH). The PNA-probe used for bacterial detection is mentioned above in the confocal microscopy section. Briefly, 5 × 105 HMEECs were seeded in a 4-chamber glass-slide with a confocal compatibility surface. The cells were grown for 24 hr and treated with UP (200 µg/mL) for 8 hr followed by S. pneumoniae (1 × 107) treatment for 12 hr. The slides were washed and pre-fixed in 4% paraformaldehyde and hybridized with the PNA bacterial probe at 46 °C for 3 hr followed by washing. The background nuclei of HMEECs were counter stained with Hoechst dye. The cells were analyzed under a confocal microscope. The bacteria were visualized with the FITC (green) channel and the background HMEECs nuclei with the DAPI (blue) channel.Pneumococcal biofilm or aggregations on HMEECs in the presence of UP were evaluated using PNA-probe fluorescent S. pneumoniae, gene expression analysis was performed using microarray. Global gene expression of HMEECs treated with UP (200 µg/mL), S. pneumoniae (MOI 10) or co-treatment (UP + S. pneumoniae) were analyzed using the Affymetrix microarray chip. The total RNA of HMEECs was extracted using the Trizol reagent , RNA quality was assessed using the Agilent 2100 Bioanalyzer , and quantity was determined using the ND-1000 spectrophotometer . The gene expression of HMEECs was detected by the Affymetrix GeneChip Human Gene 2.0 ST oligonucleotide array using the manufacturer’s procedure (http://www.affymetrix.com)30. The labeling and hybridization protocols used were standardized by e-biogen  as per GeneChip expression analysis technical manual 30. Briefly, 300 RNA was converted to cDNA using T7-oligo (dt) promoter primer, and through in vitro transcription the cRNA was synthesized from cDNA using Invitrogen kit . The fragmented cDNA was end-labeled through terminal transfer reaction incorporating a biotinylated dideoxynucleotide and hybridized to the GeneChip Human Gene 2.0 ST array 30. The hybridization reaction was performed by incubating samples at 45 °C for 16 hr by procedures described by Affymetrix 30. The hybridized and Streptavidin Phycoerythrin stain chips were scanned using Affymetrix Model 3000 G7 scanner and the raw.cel files and expression data were generated by Affymetrix Command Console software1.1 30. The expression data were normalized by Robust Multi-Average. The 2-fold change of gene expression in treated samples with respect to the control sample (untreated) was considered significant. The gene ontology (GO) and KEEG pathway analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/), string and Medline databases (http://www.ncbi.nlm.nih.gov/). Microarray data were submitted to NCBI GEO with access number GSE138091. The microarray gene expressions of ten genes were differentially expressed in microarray and were confirmed by real time (RT) PCR using the procedure mentioned above (biofilm gene expression). The primers are shown in Supplementary Table To educate the molecular mechanism underlining the elevated toxicity of UP + moniae MO 10 or coIn vivo colonization of S. pneumoniae in the presence of UP and subsequently transit to the middle ears and lungs was evaluated using intranasal mice models33. Twenty-seven, pathogen-free C57BL/6 mice, 10–14 weeks of age were obtained from Koatech . Upon arrival, mice were examined for any abnormality, and housed isolated for 2 weeks for acclimatization in an infection free zone. Animals were divide into five groups according to the treatment; vehicle control , UP only , S. pneumoniae only , UP + S. pneumoniae (n = 6), and untreated . The UP was suspended in PBS, and the suspension was sonicated for 5 min before being used. Fifteen microliters of UP or bacterial suspension were intranasally administrated by dropping in mice nares without anesthesia. The mice were kept in an invert position for 5 min to pass the solution through the nasal cavity of the mice. Two hundred microgram per mice UP and 5 × 106 bacteria were inoculated twice so that the final UP quantity were 400 µg/mice and bacteria were 1 × 107/mice, respectively. The mice were incubated for 5 days, and were sacrificed under deep anesthesia and cervical dislodging. The nasal lavages were collected by washing the nasal cavity with PBS by inserting a cannulated needle into the trachea. Bacterial load in nasal lavages were detected by plating serially diluted nasal lavages on BAP. The lungs and both bullae were aseptically harvested. The lungs and bullae were homogenized and the lysate was serially diluted and plated on BAP. Pneumococci cfu were counted after overnight incubation at 37 °C.Biofilm experiments were carried out in triplicate, repeated at least three times and results from independent experiments were averaged and standard deviations were calculated. Other experiments were performed in replicates and repeated to calculate statistically significance. The mean of the replicates and the statistical significance were calculated using student’s t test and one way ANOVA and p-values less than 0.05 was considered significant.S. pneumoniae in the presence of different concentrations of UP, showed an increased cell growth in metal ion free medium supplemented with UP. Initially, the pneumococcal growth was slow, however, at 36 and 48 hr the pneumococci growth was significantly elevated (p < 0.05) in the presence of UP in comparison to the control  at 100 and 200 µg/mL UP  were also significantly elevated (p < 0.05) in the presence of 100 µg/mL UP . These results indicate that the presence of UP increased pneumococcal plankton and in vitro biofilm growth. UP Fig. . The res UP Fig. . The via UP Fig. . The in in vitro biofilms grown in metal ion free medium supplemented with 200 µg/mL UP revealed a differentiated morphology in comparison to the control. In control biofilms (without any supplement), pneumococci were unable to form an organized biofilm, bacteria formed long chain like-structures, and the cell size appeared smaller than normal (pointed) with no visible matrix  and competence related genes such as luxS, comA, comB, and ciaR were up-regulated more than 2-fold in biofilms grown with 200 µg/ml UP  when compared to single treatment of UP or S. pneumoniae  were attached to the bottom of the surface and on the HMEECs. However, large cell aggregates or clumps were absent   Fig. . In Fig.S. pneumoniae showed 1927 genes (753 upregulated and 1160 down-regulated) that were significantly differentially regulated. However, HMEECs treated with UP + S. pneumoniae revealed 2945 genes (1122 upregulated and 1806 down-regulated) that were significantly differentially regulated  that were significantly differentially regulated in HMEECs treated with UP Fig. . The HMEted Fig. . Interested Fig. . The gents Table .Figure 7S. pneumoniae treatment. Whereas genes involved in cytokine-cytokine receptor interactions, arachidonic acid metabolism, and viral carcinogenesis were exclusively expressed in UP treatment. The JAK-STAT signaling pathway genes were expressed in UP and co-treatment. The MAPK signaling pathway, microRNAs in cancer and FoxO signaling genes were expressed in S. pneumoniae and co-treatments. A large number of genes differentially expressed in HMEECs treated with UP + S. pneumoniae indicates that co-treatment influences number of cellular processes and pathways.The KEGG pathway analyses of differentially regulated genes are shown in Supplementary Fig. 2. The KEGG pathway analysis revealed that genes involved in prostate cancer, adherens junction, Notch signaling pathway, pathways in cancer, glycerolipid and glycerophospholipid metabolism were exclusively differentially regulated in co-treatment. Whereas genes involved in legionellosis, salmonella infection, estrogen signaling pathway, spliceosome, transcriptional mis-regulation in cancer, antigen processing and presentation, and toxoplasmosis were exclusively differentially expressed in S. pneumoniae. For example, the genes involve in angiogenesis and DNA repair were exclusively expressed in co-treatment and S. pneumoniae treatment. Similarly, the RNA splicing related genes were differentially expressed in co-treatment and not expressed in UP and only up regulated in S. pneumoniae treatment. Genes involved in apoptosis, cell cycle, immune response and inflammatory response, cell repair, cell growth, cell differentiation, cell proliferation, etc. were differentially expressed in all three treatments . The number of genes involved in apoptosis and cell death that were upregulated in co-treatment, UP and S. pneumoniae were 32, 10 and 22 respectively, and the number of genes that were down-regulated were 61, 9 and 14 respectively . The number of genes involved in immune responses that were up-regulated in co-treatment, UP and S. pneumoniae were 26, 8 and 28 respectively, while number of genes down-regulated were 57, 10, and 15 respectively . The number of genes related to inflammatory response that were up-regulated in co-treatment, UP and S. pneumoniae were 4, 1 and 8 respectively, while the number of genes down-regulated in co-treatment, UP and S. pneumoniae were 14, 1 and 3 respectively . Similarly, the number of genes related to cell cycle that were up-regulated in co-treatment, UP and S. pneumoniae were 14, 3, and 9 respectively, while the number of genes up-regulated were 66, 2, and 24 respectively . Interestingly, genes involved in DNA repair were differentially regulated in co-treatment, UP and S. pneumoniae were 23, 1 and 12 respectively. The important genes involved in apoptosis and cell death, immune response and inflammatory responses that were significantly expressed in co-treatment and respectively expressed in single treatment of UP or S. pneumoniae are shown in Supplementary Table S. pneumoniae indicates that inflammatory and toxicity of co-treated was significantly higher when compared to single treatment of UP or S. pneumoniae.The gene ontology analysis of differentially expressed genes in all three treatments are shown in Supplementary Fig. 3. The GO analysis showed that some gene categories exclusively expressed in co-treatment were not expressed in UP or In vivo mice experiment results showed an elevated colonization of pneumococci in the presence of UP in the nasopharynx of mice and a significant number of bacteria disseminated to the lungs and middle ear. The cfu counts of nasal lavages were significantly increased (p < 0.05) in mice exposed to UP and bacteria  revealed that a marked number of bacteria transited to the mice middle ear. The cfu counts of mice bullae inoculated with bacteria only were 1.02 × 1030% Fig. .Similarly, the pneumococcal counts in mice lungs revealed a significantly high (p < 0.05) number of bacteria that were able transit to the lungs in the presence of UP. In the presence of UP 22% more pneumococci were detected in mice lungs inoculated with UP and bacteria Fig. . These rS. pneumoniae asymptotically colonizes the nasal cavity and causes infections such as OM and pneumonia in immune-compromised individuals. Similarly, UP are inhaled during respiration and deposited in the nasal cavity. It was previously detected that cigarette smokers exposed to PM are more susceptible to microbial infection, including OM, which has been associated with microbial biofilms20. However, the biofilm formation and colonization of S. pneumoniae in the presence of UP is not well understood. In this study, we evaluated the effects of UP on S. pneumoniae in vitro biofilm formation, colonization of HMEECs and the nasopharynx and transit to the middle ear and lungs.34. Furthermore, the in vitro biofilm growth results revealed elevated pneumococcal biofilm growth in the presence of UP, and bacteria formed thick biofilms that were organized in a 3–dimensional honey-comb structure as previously reported35. Interestingly, in the absence of UP (control) the bacteria were unable to form organized biofilms and the bacterial cell appeared small in size and formed a chain like structure. These results indicate that probably in the absence of UP, normal bacterial growth was disrupted, and in the presence of UP, pneumococci were able to maintain normal grown34. The primary reason for elevated biofilm growth is due to an increase in planktonic cell growth, and the second reason could be that UP provides favorable scaffold for biofilm growth31. In addition, the elevated expression of biofilm related genes such as luxS, comA, comB, ciaR in the presence of UP was attributed to increased biofilms. Pneumococcal biofilm formation is regulated by LuxS mediated quorum sensing (QS) and two component QS36. Here, our results demonstrated increased expression of LuxS, ciaR, comA, comB in biofilms grown in the presence of UP, which contain a number of metals including iron (3.92%). These results indicate that the metal contents of UP induced expressions of biofilm and competence related gene expression and elevated biofilms growth. Previously, various studies reported that the presence of metal ions, such as iron and zinc, elevated pneumococcal biofilm growth, and iron up-regulated luxS gene expressions and controlled biofilm formation and fratricide37. Overall, these results indicate that S. pneumoniae forms elevated biofilm growth in the presence of UP and regulated gene expression of genes involved in biofilm, competence and toxin production.Here, we detected an elevated pneumococcal planktonic growth in the presence of UP, indicating that the UP composition induced a biological effect that favored pneumococcal growth. Planktonic growth was examined in metal ion free medium, which are critical components of bacterial growth medium. These UP contain various metals such as iron (3.92%), sodium (4240 mg/kg), silicon (12.8%), zinc (4800 mg/kg), magnesium (0.81%), manganese (790 mg/kg), calcium (5.84%), etc. . The increased growth in samples supplied with UP potential indicate that pneumococcal growth was attributed to the metal components of UP. Iron is an essential nutrient for bacterial growth and many studies reported it crucial for pneumococcal energy production, nucleotide synthesis, and regulation of gene expressionS. pneumoniae produces various toxins including pneumolysin (encoded by the ply gene) that induces DNA damage and cell cycle arrest, and hydrogen peroxide mediated by pneumococcal autolysin LytA39. We detected up-regulation of ply and lytA genes in pneumococcal biofilms in the presence of UP. Similarly, the PM contents of UP also exerts cytotoxicity through oxidative stress, DNA oxidative damage, mutagenicity, and stimulation of pro-inflammatory factors41. These results indicate that UP and S. pneumoniae treatment individually are toxic to epithelium cells, which is mediated by elevated ROS production, and in co-treatment, ROS production was amplified43. Possibly, various factors including elevated ROS, hydrogen peroxide and toxins produced by pneumococci, increased the apoptosis rate in HMEECs43. Overall, these results indicate that exposure of HMEECs to UP increased cell susceptibility to S. pneumoniae infection, and amplified inflammation, ROS production and apoptosis that results in decreased HMEECs viability.The results of this study showed decreased HMEECs viability, increased apoptosis, ROS production and increased bacteria colonization/aggregation to HMEECs in the presence of UP. S. pneumonia treatment was more toxic to HMEECs. In our previous study, we detected a concentration dependent decreased HMEECs viability and increased inflammatory related gene expression in the presence of UP41. Here, we detected the up-regulation of pro-inflammatory cytokines such as Il 24, HMOX1 and PTGS2 in co-treatment indicated an increased inflammation46. The down-regulation of CD44 in co-treatment indicated that immune responses and defense was suppressed. It has been reported that a defect in CD44 gene expression obstruct bacteria clear during otitis media47. The up-regulation of apoptosis related genes  and down-regulation of anti-apoptosis genes  in HMEECs indicates that co-treatment induced apoptosis in HMEECs. The AIFM2 gene encodes an apoptosis inducing factor mitochondrion associated protein, which plays a vital role in the caspase-independent cell death pathway48. In addition, the CASP4 protein plays an important role in the clearance of bacterial toxins, and the down-regulation may hamper bacterial clearance49. The BCL2L11 gene encodes for anti-apoptosis proteins that play a vital role in apoptosis and is associated with OM50. Similarly, the NOTCH1 gene down-regulation has been reported to induce apoptosis and inhibit cell proliferation and metastasis in laryngeal squamous cells and is a risk factor for AOM51. The IFI6 gene encodes protein that negatively regulates apoptosis and has been down-regulated on virus infection52. Another important gene down-regulated in co-treatment was CXCL10, which encodes an important anti-microbial peptide (AMP) capable of directly killing bacteria and can act as a chemoattractant for natural killer cells54. The down-regulation of CXCL10 indicates decreased defense of HMEECs. Here, our results showed up-regulation of five genes that encode metallothionein proteins in co-treatment and less than 2 fold expressed in UP or S. pneumoniae treatment. The metallothioneins are stress response proteins induced during inflammation, bacterial infection, restraint stress and agents that generate reactive oxygen species55. In addition, the cell growth related genes were also reduced as suggested by down-regulation of CIT and MKI67. The significantly low viability of HMEECs in co-treatment could be due to the amplified inflammation, which may result in HMEECs injury and resulting in cells becoming more susceptible to pneumococci infection and cell death .Global gene expression using microarray revealed up-regulation of a number of genes involved in apoptosis, inflammation and cell death. Down-regulation of genes involved in DNA repair, anti-apoptosis and cell growth in co-treatment indicates that UP + 56. Our in vivo results showed increased pneumococcal colonization in the mouse nasopharynx in the presence of UP. Probably the presence of UP decreased the host defense and obstructed bacterial clearance that favored pneumococcal colonization57. S. pneumoniae is known to form biofilms in vivo during nasopharyngeal and middle ear colonization59. Moreover, elevated recovery of pneumococci in the presence of UP from the nasal lavage indicated that perhaps the UP provided a favorable surface for pneumococci colonization/biofilms31. Biofilms in the nasopharynx act as a reservoir of bacteria, and planktonic bacteria from those biofilms can transit to other typically sterile anatomical sites such as middle ear causing OM, in blood causing bacteremia, or meningitis, and in lungs causing pneumonia63. Here, our results demonstrate that UP impacts in vivo colonization of pneumococci and promotes the spread from nasopharynx to the middle ear and the lungs in mouse intranasal model. Recovery of increased pneumococci in middle ear and lungs suggest that UP induces S. pneumoniae dissemination and subsequently colonization to the middle ear or lungs, and that could be a key factor in how PM induced microbial infections such as pneumonia and OM64.A significant percentage of the population carries pneumococci asymptomatically in the nasopharynx, and after inhalation the UP find their path in the nasopharynx where they interact with epithelium cells and bacteria. Previous studies reported enhance respiratory tract and nosocomial microbial infection including OM and pneumonia in the presence of PM containing air-pollutionin vitro biofilms and planktonic growth. Pre-exposure of HMEECs to UP decreased cell viability, increased apoptosis, ROS production and bacteria aggregation on HMEECs. The co-treatment impacts a number of genes involved in inflammatory response, apoptosis, immune responses and cell death. The presence of UP increased pneumococcal in vivo colonization of the nasopharynx and dissemination to the middle ear and lungs.The results of this study showed that UP increased pneumococci Supplementary figure 1 .Supplementary figure 2.Supplementary figure 3.Supplementary figure 4.Supplementary table I.Supplementary table II.Supplementary table III.


Lactobacillus reuteri, Lactobacillus fermentum, and Bacillus subtilis sp. natto)-loaded chitosan nanogels were prepared from the culture of corresponding cultures. The chitosan nanogels were previously characterized by Zetasizer, FTIR, and TEM. The prepared formulations' effectiveness and dressing activity were assessed by evaluating wound closure and histological trials in Sprague-Dawley rats. The results indicated that all probiotic lysate formulations have advantages over the wound healing process. However, Bacillus subtilis natto has a better wound healing quality, which is well known in pathology examination. The favorable effects of probiotic lysate nanogels, including the reasonable wound closing rate, good wound appearance, and satisfactory histological observation via in vivo examination, suggest it as a promising nominee for wound healing purposes.Wound healing is a physiological reaction to tissue injuries which plays a crucial role in replacing the destroyed tissues. Probiotics produce valuable compounds that possess antibacterial and anti-inflammatory activities, immunomodulatory effects, and angiogenesis traits leading to the promotion of wound healing. Chitosan nanostructures have versatile properties making them quickly produced into gels, scaffolds, nanoparticles, beads, and sponge structures that can be incorporated into wound healing processes. In the current study, three formulations from nanogel consisting of probiotic supernatant ( Wound healing, a network of complex biological processes involving many extracellular and intracellular macromolecules, plays a significant role in skin remodeling and reestablishing the skin barrier following damages , 2. Manaw/w distilled water. CS-NG also visualized a sound polymeric bioagent delivery system having a high drug loading capacity, stability, and switching property against physical changes, including ionic strength, pH, and temperature. Therefore, they have especially been recommended for various pharmaceutical applications, including drug delivery, antimicrobial activity, and wound healing [Chitosan is a natural linear polysaccharide composed of poly 1, 4), B-D-glucopyranose amine units that can be obtained from the deacetylation of the crushing of crustacean chitin shells. This natural polymer has attractive features such as biologic activity, low toxicity, bioadhesive, biodegradability, biocompatibility, low cost, and antibacterial, antiviral, and antifungal properties , B-D-glu. Chitosa healing , 7.As live microorganisms that confer a health benefit on the host, probiotics can modulate the immune system, reduce inflammation, and promote wound healing processes . Wound hin vivo wound healing evaluations in the rat model were then performed, and the histological consequences have been assessed.In the present study, a novel chitosan-based nanogel containing a probiotic supernatant complex was developed for skin delivery to promote the wound healing process. The prepared nanogels were characterized by their particle size, zeta potential, and morphology properties. The The chitosan was purchased from Katokichi Co., Japan. MRS  broth, tryptic soy broth (TSB), and yeast extract were prepared from Difco . Maltose was purchased from Merck .Bacillus subtilis sp. natto , Lactobacillus fermentum (ATCC 9338), and L. reuteri (ATCC 23272) strains were used in the experiment.The probiotic bacteria of B.S. natto was cultured using 10 g soy-peptone, 2 g potassium hydrogen phosphate, 1 g magnesium sulfate, 20 g maltose, 10 g yeast extract, and 2 g glucose at 37°C for 48 h. The pH was adjusted to 7.2 using acetic acid 0.2 M or sodium hydroxide 0.2 M. Inoculum of bacteria containing approximately 1 × 108 to 109 CFU/ml was prepared [μm membrane filter to remove the remaining bacteria and remains and then lyophilized before storage at −20°C. Bacterial counting on MRS agar plates showed no evidence of lactobacilli growth. The presence of lipopolysaccharide (LPS) in L.S. had been excluded with a diagnostic kit from Cambrex Corporation .The Lactobacilli bacteria were cultured using MRS  broth medium  for 48 h at 37°C under microaerophilic conditions until the stationary phase was achieved. prepared . The numw/v) was added to 7 ml of the chitosan suspension  in acetate buffer (pH = 4) in a drop-wise manner, over a 2 min period under the continuous magnetic stirring (1500 rpm) at 28°C. Nanoparticles were obtained after centrifugation at 12,000 × g for 15 min. The supernatant was dropped, and the mass was resuspended in the deionized water [CS-NG were fabricated via ionic gelation of chitosan employing a polyanion, sodium triphosphate (TPP). In pleasing status, 1 ml of the polyanionic TPP solution  powder and compressed into a tablet to perform Fourier transform infrared spectroscopy (FT-IR). The sample was scanned by the FT-IR spectrometer  using a resolution of 4 cmμl of the sample was placed on a carbon film coated on 300 mesh copper grids (EMS) for 2 min, and the excess liquid was absorbed with a filter paper. The sample was then negatively stained with 20 μl of 2% uranyl acetate solution for less than a minute, and the excess stain was absorbed again with a filter paper. The sample was allowed to air dry and examined using a Transmission Electron Microscope  at an accelerating voltage of 100 kV.The morphology and size of the prepared nanogels were determined using TEM. A total of 20 The particle size distribution, mean hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the CS-NG were obtained by a Malvern Zetasizer  at a fixed scattering angle (90°) and using a He-Ne laser (633 nm). The refractive index and viscosity of pure water were utilized for data analysis.w/w) was obtained.One milligram of various lyophilized probiotic supernatants was individually added into 10 grams of the CS-NG (2.5%) for 5 minutes at room temperature to prepare different probiotic supernatant-enriched nanogels as tabulated in In the present study, 25 mature Sprague-Dawley rats weighing 200-300 g were used. The rats were purchased from the Center of Comparative and Experimental Medicine at the Shiraz University of Medical Science and treated according to the discipline of animal management and welfare of the University of Helsinki, Finland. Animals were placed in a standard cage with free access to tap water and a rodent's pellet chow diet . To eliminate any effects of stress conditions on the experimental rats, they were allowed to be adapted to the new laboratory conditions for 15 days. Then, the rats were divided into five experimental groups, as presented in 2 and 2 mm depth were made via cutting out of a skin layer from the shaved area. All medications in the form of topical formulations (each containing 1 g of probiotic lysate in 100 g of CS-NG (1% w/w)) were administered to the wounds every day. The contralateral injury in the same animal receiving CS-NG without any probiotic lysate and rats receiving no treatment served as a control group.The rats were anesthetized using a mixture of ketamine : xylazine 80 mg/kg : 10 mg/kg. Excision wounds of approximately 226 mmThe percentage of wound healing was measured according to the following equation:After 14 days, the animals were sacrificed by spinal cord injury under anesthesia. Wound skin tissues (each in 3.5 cm × 1.2 cm) with full thickness were removed, and paraffin-embedded sections were prepared. The sections (each with 2 mm thickness) were cut with a microtome, cutting perpendicular to the width of the skin surface. The sections were all stained with hematoxylin-eosin. The degrees of epithelialization, fibrosis, inflammation, and granulation were assessed for all of the specimens. In order to calculate the wound healing process quantitatively, histopathological changes of skin tissue  were blindly scored by an expert clinical pathologist as follows:0: there is no apparent change in histopathological features of skin tissue+1: mild changes could be diagnosed in histopathological features of skin tissue by the pathologist+2: moderate changes could be diagnosed in histopathological features of skin tissue by the pathologist+3: severe change could be diagnosed in histopathological features of skin tissue by the pathologistP value of less than 0.05  was considered statistically significant. Results were stated as mean ± standard deviation (S.D.). Scores for tissue histopathological changes are represented as median and quartiles for five random pictures per group. The analysis of tissue histopathological alterations was performed by the Kruskal-Wallis followed by the Mann-Whitney U test.The data gathered from each group after three selected days were statistically analyzed by the Statistical Package for the Social Sciences (SPSS) software version 18.0  using analysis of variance (ANOVA) and Duncan's mean comparison test. Each group contained five animals, and a −1, which corresponds to the overlapping of primary amine and hydroxyl groups stretching vibration. Also, deforming –NH2 vibration of hydroxyl peaks appeared at 1654 cm−1. The peak at 2925 cm−1 was attributed to the aliphatic carbon stretching vibrations. A characteristic peak at 1382 cm−1 was due to the C-N stretching vibration of amine groups, and the peak found at 1165 cm−1 belonged to the asymmetric stretch of the C-O-C bonds. Therefore, the FT-IR spectra showed the characteristics of CS-NG bonds, and it was concluded that the inter- and intramolecular bonds of nanogel were well formed. The synthesized chitosan was a smooth, uniform nanogel with semisolid consistency and white colour . The zeta potential of nanogels was obtained as 13.07 ± 0.17 mV.P value < 0.05). The wound healing rates were higher in the groups receiving the L. Fermentum supernatant-loaded CS-NG and L. reuteri supernatant-loaded CS-NG than the other groups. The wound healing process in these two groups was completed by day 10. Even after 14 days (the end of the experiment), the healing process in the control group was not completed  [L. reuteri enhanced wound healing via the PI3K/AKT/β-catenin/TGFβ1 pathway [L. fermentum, Brandi et al. stated that treatment of wounds with L. fermentum increases wound healing processes via exerting anti-inflammatory and antipathogenic influences [The trend of wound healing during 14 days for topically treated rats by probiotic supernatants, chitosan, and untreated rats is shown in ompleted . Khodaii pathway . As for fluences . Chitosafluences . HoweverB.S. natto groups did not show granulation criteria, while L. fermentum and L. reuteri demonstrated a degree of inflammation. The highest inflammation was related to the control group. Although the degree of fibrosis was observed in all experimented groups, this factor is significantly higher in the control group than the others. The boosted effects of the probiotic supernatant-loaded CS-NG in wound healing may be attributed to their metabolites , which provoke proteoglycan deposition and angiogenesis as well as reduction of inflammation along with invigorating various growth factors like EGF, PDGF, FGF, and TGFβ [The histological assessment in rats offers essential facts for the situation of a wound. Skin tissue histopathological alterations are presented in and TGFβ , 17–23.Shewanella putrefaciens on the wounded marine animal model was reported after dietary administration [In the past, researchers believed that probiotics were only useful in gastrointestinal problems and diseases . Gradualstration . Due to stration –29. Thisstration . In a ststration .It should be noted that because of the effect of CS-NG together with probiotic lysates, our formulation may entail antimicrobial effects, and the pH regulation of wound local space avoids the adverse effects of broad-spectrum antibiotics. It seems that this formulation provided a three-dimensional network with desirable swellability for maintaining wound moisture and rheological properties to exhibit excellent wound healing capability. Further in-depth studies about various aspects of this formulation, the mechanisms involved in its effects, and more molecular information are needed before using this product for further applications. Accordingly, this novel formulation may open a new window on wound healing treatment in the future after deep consideration and clinical trials.B.S. natto has a better wound healing quality than other groups, which is well known in pathology examination. The reason for the slow wound closure rate in the group receiving supernatants of B.S. natto may be the presence of the enzyme nattokinase, which has anticoagulant properties [The chitosan was chosen as an intrinsically biocompatible and antimicrobial substance for further derivation to offer qualities to improve the antimicrobial activity and attain superb biocompatibility and good water maintenance nature. The FT-IR spectrum of nanogels pointed out that the inter- and intramolecular bonds of nanogel were well-formed. The morphological observation designates that the nanogels had an acceptable uniformity in size distribution with a size range of 10–50 nm and good physical stability of nanogels. Wound healing trials employing an animal model illustrated that incorporating the prepared novel probiotic lysate nanogel on to an open wound provokes a special wound closing rate compared to usual chitosan and gauze. In general, each of the probiotic lysate groups has advantages over the other groups. For example, the rate of wound closure in the group receiving lactobacilli is higher than that in the other groups. However, the group receiving operties . All the