CTRL and ASCEMS during control and chondrogenic conditions. In control conditions, ASCEMS were characterized by increased mitochondrial fission in comparison to ASCCTRL. We found that extensive remodeling of mitochondrial network including fusion and fission occurs during early step of differentiation. Moreover, we observed mitochondria morphology deterioration in ASCEMS. These conditions seem to cause autophagic shift in ASCEMS, as we observed increased accumulation of LAMP2 and formation of multiple autophagosomes in those cells, some of which contained dysfunctional mitochondria. “Autophagic” switch may be a rescue mechanism allowing ASCEMS to clear impaired by ROS proteins and mitochondria. Moreover it provides a precursors-to-macromolecules synthesis, especially during chondrogenesis. Our data indicates that autophagy in ASCEMS would be crucial for the quality control mechanisms and maintenance of cellular homeostasis ASCEMS allowing them to be in “stemness” status.Equine metabolic syndrome (EMS) is mainly characterized by insulin resistance, obesity, and local or systemic inflammation. That unfriendly environment of adipose tissue has huge impact on stem cells population (ASC) residing within. In the present study, using molecular biology techniques and multiple imaging techniques , we evaluated the impact of EMS on ASC viability and chondrogenic differentiation. Moreover, we visualized the mitochondrial network and dynamics in ASC Equine metabolic syndrome (EMS), the most common metabolic disorder in horses, is characterized by insulin resistance, obesity and abnormal fat deposition, chronic or past laminitis, and finally local and/or systemic inflammation. According to the current statistics, obesity in horses affects over 45% of the population and is steadily growing . Increas inter alia by their paracrine/autocrine activity. In particular, synthesis and secretion of extracellular microvesicles (ExMVs) that are rich in growth factors including bone morphogenetic protein (BMP-2), vascular endothelial growth factor (VEGF), and finally fibroblast growth factor (FGF) [β1), and interleukins 4 and 13  [Currently, the adipose tissue has become the most popular source of mesenchymal stem cells used for cellular therapies in the wide field of regenerative veterinary medicine of both small and large animals. These cells are characterized by the presence of specific surface markers including CD90, CD105, and CD44 and lack of CD45 expression. Other features of MSCs include their elevated proliferative potential, increased viability, and high clonogenic potential (CFU-fs) in conjunction with high ability to self-renew , 8. Theior (FGF) . All the, IL-13) –16.The MSC may influence on adjacent cells not only by the production of the ExMVs carrying proteins and lipids, but also with ExMVs enriched in nucleic acids, most importantly mRNA and miRNA . CirculaThe progressive oxidative stress and inflammation that are characteristic of insulin resistance conditions induce in progenitor cells of EMS horses the natural mechanism that protects them against cellular damage and apoptosis. Recently, the autophagy has been reported as such mechanism that prevents the metabolically affected progenitor cells from extracellular and intracellular stresses and allows them to maintain their natural multipotency. Autophagy, as a dynamic process, might be divided into few stages including (i) induction of the process, (ii) autophagosome formation, (iii) autophagolysosome formation, and (iv) delivery and degradation of the autophagic body. However, it seems that the autophagy process is not always associated with autolysosome formation and does not necessarily lead to apoptosis. The hypothesis proposed is that EMS conditions might temporarily induce autophagy in stem progenitor cells, allowing them to keep physiological functions and survive . In the EMS cells to use them in clinical practice.Bearing in mind postulates mentioned above, we were interested whether adipose-derived progenitor cells of EMS horses that are affected by elevated oxidative stress—of both mitochondrial and endoplasmatic reticulum—exhibit impaired multipotency and are capable of developing automechanism that protects them from molecular quiescence and allow them to maintain their functionality. Our hypothesis is based on an assumption that macroautophagy and selective mitophagy are the mechanisms maintaining cellular homeostasis of ASC derived from EMS horses during their differentiation into chondrogenic cells, thus keeping their functionality and maintaining regenerative potential. Another proposed explanation is the horizontal transfer of ExMVs containing miRNA that regulate gene expression in dysfunctional cells and promoting their multipotent recovery. Here, we demonstrated that EMS conditions impair chondrogenic differentiation potential of ASCs and finally cause extracellular matrix formation dysfunctions. Moreover, we have fund that EMS horses suffer for reduced mitochondrial biogenesis, limited fusion, and abundance of autophagosomes and autolysosomes formation that affects their “stemness.” Still, more investigations and effort are required to stop or what is more required reverse the senescence stage of ASCAll reagents used in this experiment were purchased from Sigma Aldrich (Poland), unless indicated otherwise.All experimental procedures were approved by the II Local Ethics Committee of Environmental and Life Sciences University .n = 6) and control, healthy horses (n = 6). Detailed characterization of animals used in this study is shown in All horses were age-matched  and divided into two groups: EMS group . The supernatant was discarded and the pellet was resuspended in the culture medium. The cell suspension was then transferred to a culture flask.White, subcutaneous adipose tissue (2 grams) was collected from the horses' tail base, according to the standard surgical procedure and ethical standards, as previously described. After harvesting, specimens were placed in a sterile Hank's balanced salt solution (HBSS). Cells were isolated under aseptic conditions following the previously described protocol by Grzesiak et al. . Briefly2, and 95% humidity). DMEM containing 4500 mg/L glucose supplemented with 10% FBS and 1% of PSA was used as a culture medium. The media were changed every two days and the cells were passaged using trypsin solution  after reaching 80% confluence. Prior to the experiment, EqASCs were passaged three times. All experiments were performed after 7 days of ASC propagation.During the experiment, the cells were cultured under aseptic and constant conditions in an incubator  and fluorescein isothiocyanate (FITC) . At least ten thousand stained cells were acquired and analysed by Becton Dickinson FACSCalibur flow cytometer. The samples were analysed using CellQuest Pro software.Equine ASC were recognized by immunophenotyping using fluorochrome conjugated monoclonal antibodies specific for CD44, CD45, CD90, and CD105. Due to immunophenotyping ASC were detached using TrypLE Express solution, washed with HBSS, and resuspended at total of 5 4 and the media (500 μL/per well) were changed every two days. Osteogenesis and chondrogenesis lasted 10 days, while adipogenesis lasted 9 days. Extracellular mineralized matrix was visualized with Alizarin Red while formation of proteoglycan-rich matrix was confirmed with Safranin O staining. Lipid droplets formed during adipogenesis were stained with LipidTox (Life Technologies).To confirm multilineage differentiation potential of ASC, cells were cultured in in StemXVivo kits (R&D Systems) in accordance to manufacturer's instructions. In order to perform the test, the cells were seeded in a 24-well plate at the initial density of 1 × 104 per well in 24-well culture plates. The proliferation rate was evaluated after the 1st, 5th, 7th, and 10th days of the experiment. First, culture medium was replaced with a medium containing 10% of resazurin (Alamar Blue). Next, cells were incubated with dye for 2 hours at 37°C. Then, supernatants were collected and transferred to 96-well plates. Dye reduction was determined using a spectrometer (BMG Labtech) at the specific wavelengths, that is, 600 nm for resazurin and 690 nm as a background absorbance. Cell number was obtained from test data. To prepare the curve, cells were seeded at the density of 2 × 104, 4 × 104, and 8 × 104 per well and dye absorbance was measured in relation to certain cells number. Linear trendline equation allowed estimating cells number throughout the experiment. Proliferation factor (PF) of cells was determined in relation to the proliferation activity of ASC isolated from healthy individuals. The value equal to 1 is normative unit, showing proliferative activity of ASC from control group.Cell proliferation rate was established with resazurin-resorufin test. In order to perform the assay, cells were seeded at concentration 2 × 10Detailed cells morphology was evaluated using SEM. Cells were fixed with 4% paraformaldehyde (PFA), rinsed with distilled water, and dehydrated in a graded ethanol series . Samples were then sprinkled with gold , transferred to the microscope chamber, and observed using SE1 detector, at 10 kV of filament tension.For transmission electron microscopy (TEM), cells were fixed in 2.5% glutaraldehyde at 4°C. After fixation cells were (1) centrifuged at 2000 ×g for 10 min and rinsed with PBS for 30 min, (2) centrifuged again, parameters as above, (3) incubated with 1% osmium tetroxide in HBSS for 2 hours, (4) washed with HBSS, (5) centrifuged as described above, (6) dehydrated in graded acetone series (30% to 100%), and (7) embedded using Agar Low Viscosity Resin Kit . Ultrathin sections (80 nm) were collected on copper grids. Uranyl acetate and lead citrate were used for contrasting for 30 and 15 min, respectively. The observations were carried out using Auriga60 Zeiss STEM, at 20 kV filament tension. z-axis were captured for each experimental group, giving the coarse picture of about 2400 nm × 1800 nm × 600 nm (x-axis  ×  y-axis  ×  z-axis) of volume.To analyse the volume of cell interiors at the mitochondria sites, FIB-SEM analysis used modified method previously developed by Knott et al. . For ultIn order to visualize the mitochondria, cells were incubated with MitoRed dye (1 : 1000) in 37°C for 30 minutes. Following fixation with PFA, cells were rinsed three times with HBSS and cells' nuclei were counterstained with diamidino-2-phenylindole  for 5 minutes.Prior to the analysis of LAMP2 localization, cells were fixed in 4% PFA for 30 min and washed three times with HBSS. Then, cells' membranes were permeabilized with 0.5% Triton X-100 for 20 min at room temperature. After washing with HBSS three times, unspecific binding sites were blocked with blocking buffer  for 45 min. Cells were then incubated overnight at 4°C with primary antibodies against LAMP2 (Abcam) diluted 1 : 500 in HBSS containing 1% Goat Serum and 0.2% Tween-20. Cells were then washed again and incubated for 1 hour with goat anti-mouse secondary antibodies conjugated with atto-488 , avoiding direct light. Subsequently, nuclei were counterstained by incubation with DAPI for 5 min. Cells were observed and photographed using confocal microscope  and analysed using ImageJ software.To investigate the expression of LAMP2 and Ki67, cells were detached from culture plate and centrifuged (350 ×g for 5 minutes) followed by the 10-minute fixation in 4% ice cold PFA. After washing, cells were then incubated in 0.1% Tween-20 in HBSS for 20 min. Then cells were washed again and incubated with anti LAMP2 (Abcam) and Ki67 (Abcam) antibody, diluted 1 : 200 in HBSS containing 10% Goat Serum for 30 min at 22°C. Following another wash, cells were incubated with Alexa 488 goat anti-mouse secondary antibodies  for 30 minutes at 22°C. At least five thousand stained cells were acquired and analysed by FACSCalibur flow cytometer. The samples were analysed using CellQuest Pro software.To estimate the mitochondrial membrane potential, cells were detached from culture dishes and incubated with 1 mM JC1 (Life Technologies) for 30 min in 37°C. After washing, cells were analysed in FACSCalibur flow cytometer. At least five thousand stained cells were acquired and analysed using CellQuest Pro software.To evaluate the serum levels of FGF-21, enzyme-linked immunosorbent assay (ELISA) was performed. In order to evaluate the amount of FGF-21, blood was harvested from investigated individuals and centrifuged prior to serum isolation. The ELISA test was purchased from MyBioSource and performed in accordance to the manufacturer's instructions.Nitric oxide concentration was assessed using commercially available Griess Reagent Kit (Life Technologies). Superoxide dismutase (SOD) activity was measured using a SOD Assay kit (Sigma Aldrich). Reactive oxygen species (ROS) were estimated by incubating cells with an H2DCF-DA (Life Technologies). All procedures were performed in accordance to the manufacturer's instructions.TM Real-Time PCR Detection System (Bio-Rad). Reaction mixture contained 2 μL of cDNA in a total volume of 20 μL using SensiFAST SYBR & Fluorescein Kit (Bioline). The concentration of primers in each reaction equaled 500 nM; primer sequences used in individual reactions are listed in After the 7th day of culture (viability test) and the 11th day (chondrogenic differentiation), the cells were rinsed with HBSS and homogenized by TriReagent®. Total RNA was isolated using phenol-chloroform method as previously described by Chomczynski and Sacchi . The obtTotal RNA was isolated by TRI Reagent (Sigma Aldrich) extraction method. The precipitation was performed overnight at −20°C in order to increase the yield of small RNAs. Concentration and purity (A260/A280 ratio) of total RNA were measured using nanospectrometer (WPA Biowave II). Traces of genomic DNA (gDNA) were digested with DNA-free™ Kit (Ambion). To determine miRNA expression, 375 ng of RNA was reverse-transcribed using Mir-X miRNA First-Strand Synthesis Kit . The matrices were used for quantitative PCR with SYBR Advantage qPCR Premix . Specific miRNA primers used for detection of miR-140, miR-146a, miR-223, and miR-489 are listed in MVs were isolated in accordance to protocol presented by Shin et al. , which dt-test. Differences with a probability of p < 0.05 were considered significant.All experiments were performed in triplicate or more. Statistical analysis was performed using GraphPad Prism 5 software . Differences between groups were determined using unpaired Student's Isolated cells presented typical for ASC surface antigens characteristics including expression of CD44, CD90, and CD105 and the lack of expression of CD45 surface antigen . ObtaineEMS, displayed lowest proliferation rate in comparison to control group, and on the 1st and 7th days of the experiment, difference between groups was statistically significant (p < 0.001). Moreover, using flow cytometry, the expression of KI67 was estimated and confirmed the lower proliferation rate of ASCEMS  in accordance to the manufacturer's protocol. Throughout the experiment, ASCf ASCEMS . Cell mof ASCEMS . Observaed cells .p < 0.01, EMS . Additionally using SEM we evaluated the number and size of cartilaginous nodules within investigated cells. Hence we observed that ASCEMS were characterized by the formation of lowest number of nodules (p < 0.001). Moreover, those nodules presented decreased size in comparison to ASCCTRL , Decorin (p < 0.001), and Sox-9 (p < 0.05) was significantly reduced in ASCEMS.ASC were cultured under chondrogenic conditions in order to evaluate their chondrogenic differentiation potential on a functional level. A significantly lower proliferative activity was observed in chondrocytes precursors (Chp) derived from ASC ASCCTRL . Stainin ASCCTRL . To provEMS (p < 0.05).On the last day of chondrogenic differentiation, we estimated the miRNA expression in investigated cultures. Interestingly, no differences were observed in the expression of mir-140  and mir-EMS after the 7th day (p < 0.05), while SOD activity was significantly decreased after the 10th day (p < 0.01).Oxidative stress factors, for example, ROS and NO, and the activity of SOD were evaluated after the 1st, 7th, and 10th days of culture. Obtained results shows that, during osteogenesis, the amount of ROS is similar between groups . Interesα (α in control and chondrogenic conditions between groups. The expression of FOXO1 in control condition was comparable between groups. During chondrogenesis it was significantly decreased in ASCEMS (p < 0.001) after the 2nd and 10th days, respectively.Using RT-PCR the mean mRNA levels of HIF-1-α  and FOXOα  were est in vivo and become a cell source for regenerative medicine, that underlines their importance in the field of veterinary regenerative medicine [EMS) are marked by excessive molecular aging, decreased viability, displayed senescence associated features, and abundantly accumulated oxidative stress factors [EMS are characterized by impaired chondrogenic differentiation potential, since those cells are commonly used for the regenerative medicine purposes. Here we demonstrated that ASCEMS displayed slightly impaired chondrogenic differentiation potential, but abundance of auto- and mitophagy are the processes that allowed ASCEMS to maintain multipotency abilities. Selective autophagy in the course of equine metabolic syndrome might become one of the fundamental mechanisms that protects progenitor cells of adipose tissue from cellular senescence and cytophysiological impairment.Mesenchymal stem cells harvested from adipose tissue (ASCs) contribute to peripheral tissue repairmedicine , 22. Howmedicine . As it w factors . Hence, EMS in native culture are characterized by decreased viability and clonogenic potential as well as exhibit lower percentage of Ki67 positive cells when compared to the healthy ASC. Moreover, among typical for mesenchymal stem cells surface antigens  we have found the significant higher fraction of CD44+ cells among investigated ASCEMS population. It was recently demonstrated that CD44+ is overexpressed in inflammatory cells in obese patients adipose tissue and serum CD44+ cells level is positively correlated with insulin resistance and glycemic control [EMS characterization. Simultaneously, with decreased ASCEMS proliferative potential, we have noticed their impaired growth pattern and limited extracellular microvesicles (MVs) secretion. Moreover, the MVs derived from ASCEMS were characterized by the decreased amount of transferred mir-223 and mir-146 with simultaneous upregulation of mir-456 mRNA level. This stands in good agreement with other authors' previous finding, since mir-223 and mir-146 were found to be characteristic for obese, hypercholesterolemic individuals [EMS were characterized by upregulation of p21, p53, and cas-9 as well as BAX expression in comparison to healthy ASC [Disease and aging are two major factors that have profound influence on adipose tissue progenitor cells characteristics. Here we have found that ASC control . Additio control . Thus, oividuals , 26. WhaEMS might be directly linked to oxidative stress factors (OS) accumulation. However, one of the mechanisms that protects progenitor cells against excessive accumulation of OS factors and its harmful effects is called mitophagy. In the current research, we observed the reduced number of mitochondria with parallel higher expression of lysosomal associated membrane protein 2 (LAMP2) in ASCEMS. Observed mechanism impinges on “autophagic flux” in ASCEMS when compared to the control cells. Moreover, we observed significantly decreased mitochondrial membrane potential (JC-1 test), which may be partially responsible for entering of ASCEMS in autophagic pathway due to mitochondria impairment. Interestingly, ASCEMS displayed lower expression of MnSOD, which can lead to decreased antioxidative protection of mitochondria and in consequence switch autophagy in those cells on. In native, nonchondrogenic culture in both Beclin1 and LC3 was downregulated in ASCEMS that partially excludes autophagy and indicates regular lysosomic fusion. Moreover, observed upregulation of mir-140, which exerts effect on mitochondrial fission and apoptosis through targeting mitofusin 1 (Mfn1), may be directly correlated with frequently observed in ASCEMS mitochondrial fission. What is interesting, we simultaneously observed increased amount of serum fibroblast growth factor 21 (FGF21) in EMS horses. It was shown that FGF21 is a hormone that mediates an adaptive response to starvation and becomes also a long-standing marker of mitochondrial disease in humans [The impaired proliferative activity, cell growth pattern, and apoptosis observed in ASCn humans , 28. Then humans . Becausen humans  showed Fn humans , 31–33.EMS culture. The situation is changed, when chondrogenic differentiation potential is considered.However, lysosomal degradation of impaired mitochondria, as a process of damaged mitochondria elimination, seems to be characteristic for native ASCEMS. Activation of autophagy in those cells may help to clear damage due to ER stress proteins and impaired by ROS mitochondria. Therefore, autophagy is crucial for ASCEMS to maintain their “stemness,” although the role of that process in regulation of stem cells metabolism is still poorly understood. Available data suggest that unique properties of stem cells including pluripotency, self-renewal, and differentiation depend on autophagy activation [EMS to fulfil their function in the unfavorable environment of adipose tissue inflammation. Autophagy was also shown to participate in differentiation, as a cell remodeling mechanism that promotes morphological and structural changes. Here we have found that during chondrogenesis expression of Beclin1 and LC3 increases which stands to the contrary of the data presented by Oliver et al. [EMS characterized by mitochondria impairment and ROS accumulation undergo mitochondria clearance via mitophagy as observed with TEM, during both control and chondrogenic conditions. Thus, considering that some amino acids generated through the autophagic pathway may be used for protein, nucleotides, sterols, and energy synthesis, it is tempting to speculate that autophagy may help ASCEMS undergo differentiation. The “autophagic switch” may lead to synthesis of proteins and other cellular constituents, in addition to degrading proteins related to pluripotency maintenance that may impair differentiation. It has been shown that adipose-specific deletion of Atg7 leads to decreased adipose mass and enhanced insulin sensitivity, and autophagy is important in normal adipogenesis [EMS autophagy may result from ER stress at it was shown that autophagy is activated for cell survival during such condition [α pathway [EMS. Despite being merely speculation, autophagy could help ASCEMS maintain their self-renewal and pluripotency capacity or also differentiate by providing ATP and precursors for protein synthesis.Besides its recycling functions, in response to energy or nutrients deficiency, autophagy is also recognized as a quality control mechanism for proteins and organelles including mitochondria –36. It mtivation ; thus wetivation . Thus, ir et al. , who obsr et al. . Here weogenesis . On the ogenesis . Increasondition . Recentl pathway . It stanEMS which may be correlated with apoptosis and decreased proliferation of those cells. Our data stands with a good agreement with Forni et al. [ in vivo [Mitochondrial dynamics was recently recognized as an important constituent of cellular quality control ; thus hei et al.  who obse in vivo . HoweverEMS. As stem cells destined for regenerative medicine applications may be generated from individuals of advanced age or suffering from metabolic disorders (autophagic/mitophagic), quality control processes would need to be carefully examined and controlled to ensure long-term safety of any cellular grafts. It is tempting to speculate that modulation of autophagy may have important outcomes, such as an increase in the efficiency of cell proliferation and differentiation.The past decade has witnessed significant growth in interest regarding stem cells and autophagy. However, research of those two exciting areas is very much in its infancy. Here, for the first time, we evaluated autophagy, ER stress, and mitochondria dynamics in ASC during both control and chondrogenic conditions. During chondrogenesis we observed “autophagic switch” in both healthy and EMS groups, while basal autophagy was increased in ASC


P < 0.05). Immunoglobulin A, IgM and IgG levels in colostrum did not differ between the two groups (P >0.05). Colostrum yield was lower in the control than that in YD group . Although the YD supplementation did not change fecal bacteria diversity in sow, more beneficial and fermentative bacteria  were found in the YD fed group (P <0.01) while, some opportunistic pathogens, including Proteobacteria, especially the genera Desulfovibrio, Escherichia/Shigella and Helicobacter, were suppressed. Piglets at one week of age from sows fed YD had more beneficial microbial populations with significant diversity and fewer opportunistic pathogens. Additionally, we established a Pearson’s correlations between CY, colostrum components, piglet birth weight and fecal microbiota. Therefore, YD added to the sow diet during pregnancy increases colostrum availability and its energy content for neonate piglets, also promoting beneficial maternal microbial sources for neonate.Dietary supplementation with yeast derivatives (YD) contributes to the health and physiology of sows and piglets, but few studies have focused on how it influences gut health and performance of sows and piglets. The goal was therefore to examine whether YD, based on brewer’s yeast hydrolysate added to pregnancy diet, would affect colostrum composition, yield (CY) and gut microbiota of sows and piglets. Sows were allocated to either a control diet (n = 19) or a control diet supplemented with 2g YD/kg (n = 18) during the pregnancy. Piglets suckling belonging to the control sows (n = 114) and supplemented sows (n = 108) were also included in the study. Gut microbiota populations of sows at farrowing and piglets at one and four weeks of age were assessed using 16S rRNA gene sequencing. Colostrum samples were examined for nutritional composition and immunoglobulin (Ig) content. All piglets were individually weighed at birth and 24 hours later in order to calculate CY, and later at four weeks to calculate average daily gain (ADG). Protein, lactose and dry matter content of colostrum did not significantly differ between the two groups, while sows fed YD had higher levels of fat in their colostrum ( Saccharomyces cerevisiae, brewer’s yeast. The YD used in the study was derived from brewer’s yeast (Saccharomyces cerevisiae) by acid hydrolysis, containing both cell wall and extract fractions and a reasonably high level of soluble compounds such as oligosaccharides and peptides  are widely used in animal nutrition as natural additives ,2. YD ty piglets –11. YD h piglets –15. The The experimental protocol was approved by the National Animal Experiment Board in Finland . The experiment was carried out on a commercial pig farm in southern Finland from October 2015 to January 2016, the owner of the farm gave written permission to conduct the study in his farm. The experiment was repeated four times using four different batches of sows that farrowed during that time.®, Table A in ®, Table A in ®, Hankkija Oy/Suomen Rehu, Hyvinkää, Finland, product details in ®, Vilomix, Finland) after they had been weighted for colostrum intake analysis. Six piglets from each litter were selected and ear-tagged based on body weight at birth (BWB) in a block of three categories, 2 piglets weight <1 kg, 2 piglets 1.4–1.8 kg and 2 piglets >1.8 kg, representing small, normal and large sized piglets, respectively. Cross-fostering was allowed only after 24 h weighing, it was only carried out within a treatment, except the six selected ear-tagged piglets. Litters were balanced according to the number of functional teats. Altogether 114 piglets belonged to sows fed with CON diet and 108 piglets belonged to sows fed with YD diet. Piglets were supplied with a dry creep feed after one week, in addition to maternal milk  were balanced between the treatment groups. They were selected on the basis of start of farrowing by first come first sample–principle. Sow farrowed spontaneously and researcher (one of the authors) sampled all sows upon availability at farrowing. During gestation, sows were housed in groups of 15 to 20. The group housing rooms were equipped with individual feeding stalls and sows were fed a pregnancy diet , weight at 17 to 24 h of age , duration of CI (t in min and 17 h ≤ t ≤ 25 h), and time between birth and first suckling . The regression equation was: CI = -217.4 + 0.217 × t + 1861019 × BW24/t + BWB × (54.80-1861019/t) × (0.9985–3.7 × 10−4 × tFS + 6.1 × 10−7 × t2FS). The tFS was estimated to be 35 min, which was based on our observations from previous studies  were prellulose ,53. Howeammation –56. Thuset al. [Desulfovibrio was shared between mother and piglets. Lactobacillus, Eubacterium and Clostridium are among other bacteria identified that could be transferred from mother to piglet gut via feces or suckling. In humans, Lactobacillus fermentum has been confirmed to represent vertical transfer between mother and neonate via breast milk [Lactobacillus at one week of age. Interestingly, different species of Lactobacillus have been used as a growth-promoting feed supplement preventing and treating diarrhea in piglets and maximizing the average daily gain, crude protein apparent digestibility and serum specific IgG level [Lactobacillus from the mother’s gastrointestinal tract or milk may colonize the piglet’s gastrointestinal tract and promote piglet growth. However, Flavonifractor, Barnesiella and Roseburia have a positive influence on the daily growth of the piglets [Oscillibacter, Clostridium IV, Blautia and Gemmiger in their gastrointestinal tracts. It has been suggested that species from these genera are abundant in the neonate gastrointestinal microbiota because they are adapted to utilize a wide range of milk oligosaccharides as a unique carbon source [PCA analysis revealed that piglets at one week of age have unique microbiota. Many studies, however, report similar results, that the suckling piglet has unique microbiota acquired from the mother. The maternal dietary treatment impacted the composition of the microbiota in piglets, which was distinct from the sow’s fecal microbial alterations. This was also observed when feeding sows with inulin, prebiotics or probiotics ,58. Moreet al.  reportedast milk . IrrespegG level . Thus, i piglets . Pigletsn source –63.Overall, this study demonstrated that addition of YD in the gestation diet was able to enhance colostrum production, and especially colostrum fat, ensuring enhanced colostrum intake per kg live born piglets, more energy and sustained piglet immunity. Supplementing the gestation diet with YD may change the gut microbiota, alleviate farrowing stress, improve sow physiology and, therefore, produce more viable piglets with a stable gut.S1 Fig(A) Shannon Index for sows fed YD and control group. (B) Shannon Index for piglets at one week of age with two different sow treatment groups. (C) Shannon Index for piglets at four weeks of age with two different sow treatment groups. (D) Simpson’s Index for sows fed YD and control group. (E) Simpson’s Index for piglets at one week of age with two different sow treatment groups. (F) Simpson’s Index for piglets at four weeks of age with two different sow treatment groups.(TIF)Click here for additional data file.S2 FigSample dissimilarity within each group is indicated in the graph and overall dissimilarity is indicated as “Between”. (A) Sow, piglets at one week and piglets at four weeks of age. (B) Sow treatment groups. (C) Piglets at one week of age with two different sow treatment groups. (D) Piglets at four weeks of age with two different sow treatment groups.(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S1 TableP values are based on the results from the Mann-Whitney test.(DOCX)Click here for additional data file.S2 TableP values are based on the results from the Mann-Whitney test.(DOCX)Click here for additional data file.S3 TableP values are based on the results from the Mann-Whitney test.Values are presented in normalized square root transformed abundance. (DOCX)Click here for additional data file.S4 TableP values are based on the results from the Mann-Whitney test.Values are presented in normalized square root transformed abundance. (DOCX)Click here for additional data file.S5 TableValues are presented in normalized square root transformed abundance. P values are based on the results from the Mann-Whitney test.(DOCX)Click here for additional data file.


Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs ( Brucella, established in 1920 by Meyer and Shaw, currently comprises twelve specieshttp://www.bacterio.cict.fr/b/brucella.html) that are historically differentiated by host tropism, pathogenicity and phenotypic traits. A decade ago the genus consisted of six “classical” Brucella species , which are also referred to as the “core” Brucella. Three of these species  are important zoonotic pathogens that infect humans and a variety of other mammals, causing a disease called brucellosis. This is considered to be one of the most important zoonoses worldwide, with 500,000 human cases reported each yearBrucella was one of the first microorganisms weaponized by the U.S. military in 1950sThe genus Brucella species was extended in 2007 to include B. ceti and B. pinnipedialis, isolated from marine mammalsB. microti, was initially isolated from common voles6B. microti is different from the classical species in that it is fast-growing and metabolically very active, resembling Ochrobactrum, the closest phylogenetic neighbour of BrucellaB. microti (strain CCM 4915T) is nearly identical to B. suis 1330B. microti is closely related to the other core Brucella, it represents a distinct basal lineageB. papionis, isolated from baboons, phylogenetically clustering with the core Brucella spp.12B. vulpis from red foxes, forming a long-branched sister clade to the classical species1The group of classical Brucella isolates have been described. The first, B. inopinata (strain BO1), was isolated from a breast implant wound infection15BrucellaB. inopinata (BO1) is as active as B. microti7B. inopinata (BO1)B. inopinata (BO1), are united in a clade that is separate from the core Brucella spp.Additional atypical Brucella isolates have been reported from cold-blooded hosts, i.e. African bullfrogs Leptopelis vermiculatus)Ceratophrys ornata)B. inopinata (BO1) and strain BO2 described as B. inopinata-like have been reported15In the past few years, more and more Brucella species for mammalian hosts both in in vitro and in vivo models of infection.The present work is a comprehensive molecular and phenotypic study of a large collection of isolates from African bullfrogs building on a previous preliminary description of two of these strains2 requirement, H2S production, urea hydrolysis, agglutination with monospecific sera, dye sensitivity, and phage lysis are determined for the identification and sub-differentiation of Brucella spp. To gain deeper insight into the composition of this novel group of amphibian Brucella sp., all currently available bullfrog isolates have been characterized using a comprehensive combination of molecular and classical microbiological approachesHistorically, COO. anthropi by API®-20NE and Vitek2® Compact Brucella agar at 37 °C without supplementary CO2. After 24 h, typical raised, convex and circular colonies, 1–2 mm in diameter, were visible. Seventeen of these strains produced translucent to whitish colonies, but four strains  developed colonies with a markedly brownish pigmentation.Primary isolation from various tissues of dead or moribund African bullfrogs revealed Gram-negative coccoid rods that were identified as Brucella. They produced H2S, and urea was hydrolyzed within 10 to 45 min by all but two of the strains. Strains 10RB9215 and 10RB9213 had a prolonged hydrolysis time of 80 and 90 min, respectively, revealing reduced urease activity  serum. All the bullfrog isolates presented a smooth lipopolysaccharide phenotype, but none agglutinated with either anti-M or anti-A monospecific sera. In three of the strains  a slight agglutination was seen using high concentrations (1:10 or 1:20) of anti-A sera.4x RTD  nor at 104x RTD .® BfR Brucella assay (Merlin Diagnostika) was used for biochemical profilingB. inopinata (BO1) and B. microti, but in contrast to the relatively low activity of classical Brucella species (B. papionis), i.e. H-hydroxyproline-βNA (HP: +), Glu(pNA)-OH (ENAOH: −), Pyr-pNA (PYRNA: −), suggest an allocation of the bullfrog strains to the genus Brucella. Hierarchical cluster analysis performed by the Ward’s linkage algorithm positioned the amphibian Brucella sp. strains between B. inopinata (BO1) and B. microti, close to B. suis biovars 1–4 and B. canis .The presence of the B. inopinata whereas type B contained a 44 nt insertion apparently derived from Ochrobactrum species. A recA-gene based phylogenetic reconstruction revealed three main clusters and two strains with a unique sequence type each  and B. inopinata-like isolates . A recently described amphibian isolate from the UKMultilocus sequence analysis (MLSA) showed the amphibian isolates to be different from all previously described hrough E . Clusterproaches . PhylogeB. inopinata (BO1), forming a separate cluster within the genus Brucella  that have not yet been previously identified in more than 3,500 other Brucella strains. On the basis of the MLVA-11 data set, seven individual genotypes could be identified, numbered 196 to 202.In the comparative multilocus variable-number tandem repeat (VNTR) analysis (MLVA), the African bullfrog strains grouped together with Brucella . Brucell related . By use Brucella  that is substantially increased by inclusion of these lineages in phylogenies suggest substantial uncertainty for the topology of the amphibian and related Brucella strains.Whole genome sequencing of nine isolates, at least two each from the three major clades and the two strains found in separate lineages, confirmed their affiliation with the genus Brucella . PhylogeBrucella genomes. These regions are either shared across all of the African bullfrog strains, shared across some of the strains, or found in a specific genome  found in the BO2Brucella genomes share the same 5′ flanking region  genomes or to the novel configuration that strain BO2 has.As each bullfrog strain had a unique S-LPS profile, we specifically searched for the nineteen genes in the g region , and eac5 genome . None ofrmlACBD region is shared across two of the bullfrog strains (10RB9213 and 10R9215) as well as the previously described BO2 and B13-0095. Strains 10RB9215 and B13-0095 also share four genes upstream of this region. Two of these (rfbD and tagH) have been previously noted in the B13-0095 and BO2The wbk region is strikingly similar between B. microti and B. inopinata (BO1), as well as highly similar in strains 10RB9215 and B13-0095. In contrast, the bullfrog isolates and BO2 have unique genes in this region  and all of the classical species.Two other genes, B. microti CCM 4915 revealed an interesting pattern of pseudogenization, loss, or in-frame deletions of many of the flagellar genes. The examination was expanded to look across the major species and strains of Brucella as previously described2930An initial examination of the genes from the African bullfrog strains, the BO1 and BO2 strains, and Brucella examined had pseudogenized at least one, if not many of the flagellar genes. Brucella microti was the only exception, having no pseudogenes, but it did have two genes with internal deletions (fliG and flgI) and turned out to be immotile. There is no pattern of pseudogenization in the other species of the classical clade, but instead loss of function of different flagella genes appears in each species. Within the species, some strains revealed pseudogenes that appear to be functional in their close relatives . The observed reduction in viable intracellular counts for at least one out of four bullfrog strains (09RB8471) at 30 h post-infection may be explained by very rapid growth, resulting in macrophage lysis and killing of released bacteria in the gentamicin-containing medium. In contrast, strain 09RB8910 reached higher bacterial loads, maybe because the infected macrophages were more efficiently protected from apoptosisB. inopinata (BO1), and slightly higher than that of B. microti CCM 4915 . Remarkably, at the end of the experiment (84 d post infection) strain 09RB8913 showed persistence in the spleen comparable to B. suis 1330.The pathogenic potential of the nization  as well nization  from daynfection , similarB. suis 1330 and those infected with the bullfrog strains, except for 10RB9213 in the spleen three and five days post infection, and for 10RB9213 and 09RB8913 in the liver 12 weeks post infection.In the course of infection, the colonization rate of the target organs was significantly different between BALB/c mice infected with Brucella sp. strains indicating a very mild inflammatory reaction.Neither spleen nor liver weight significantly increased due to the infection with the amphibian Brucella species , BO2, and the Pacman frog strain B13-0095, as well as four of our bullfrog strains 09RB8918, 09RB8913, 10RB9210, 09RB8471. As part of these two subclades, our bullfrog strains thus span much of the known diversity within the atypical Brucella species. Interestingly, B. vulpis is related but is basal to all of these other atypical species. Combined, these results suggest substantial undiscovered diversity in the genus.Our analysis expands the previously described ancestral clade of Brucella species but found in soil bacteria such as Rhizobium and Agrobacterium (data not shown). In contrast to their soil- and plant-associated relatives, which harbour virulence factors such as plasmids and temperate phages necessary to survive in fast changing harsh environmental conditions, having these type of genes seems to be superfluous for an organism adapted to intracellular survivalB. inopinata that was very similar to a temperate phage of O. anthropi and to several prophages identified in rhizobialesBrucella sp. isolated from African bullfrogs may hint that horizontal gene transfer does occur between Brucella and other bacteria in the same niche, such as soil associated bacteria.Comparative whole genome analysis revealed a set of genes present on the chromosomes of the African bullfrog strains that are not found in other wbk region .This notion that the basal species more readily exchange DNA with each other and other microbes is strengthened by the great diversity of new genomic regions found in the five strains more deeply analyzed, and is also apparent in the heterogeneity of the k region . Across k region , althougBrucella in mammalian hosts. Brucella melitensis has been shown to produce a sheathed flagellum during the early stages of exponential growth29B. melitensis with mutated flagellar genes were not attenuated in cellular models of infection, BALB/c mice were able to clear them within 12 weeks of infectionBrucella and the sequences of the genes that produce the flagellum, an extensive pattern of pseudogenization is apparent . In contrast to the hepatosplenomegaly observed after B. suis infection, intraperitoneal infection of BALB/c mice with the African bullfrog strains proceeded in the form of asymptomatic persistence. This might be explained by differences in the expression of flagellin, which is known to play a key role in the immunological stand-off between Brucella and its hostBrucella sp. strains may contribute to the stealthy strategy of this pathogen remains unknown. Interestingly, motility of the flagellum necessary to cope with environmental stress and the flagellum as a virulence factor for persistent infection in a mammalian host do exist in parallel in the bullfrog isolates.Although primarily isolated from a cold-blooded host, the African bullfrog strains revealed long-term survival in mammals comparable to the classic Brucella virulence is the capacity of the bacteria to replicate inside macrophages and their ability to escape the host immune system. We tested the pathogenic potential of the novel amphibian isolates in both in vitro and in vivo infection models. The African bullfrog strains showed an enhanced capacity to replicate in macrophages at a level comparable to B. microti, B. inopinata, and the Australian rodent isolates33B. suis. The high replication rate in phagocytic cells correlates well with the fast growth of the bullfrog strains in culture medium. Specific metabolic pathways might be responsible for the faster growth and metabolism of the bullfrog isolates differed essentially from other brucellae  and Brucella sp. strain BO2.In the last decade, the emergence of several new Brucella members have emerged from soil bacteria that are characterized by motility and a broad metabolic activity. Brucella evolved to become a highly virulent but host specific clonal pathogen by genome reduction and adaptation. However, the apparent competence of the amphibian strains to exchange DNA may allow Brucella to adapt to changing environments quickly and give it a broad range of hosts.Our data support the hypothesis that the classical core A total of 21 strains  isolatedBrucella agar and Columbia agar for 48 h at 37 °C both with and without 10% CO2. Molecular analyses  as well as phenotypic characterization  were performed as described previously for B. microti isolatesThe bacteria were grown on 2 requirement, H2S production, urea hydrolysis, agglutination with monospecific sera , dye sensitivity (basic fuchsin and thionine), and phage lysis  for the identification and sub-differentiation of the African bullfrog strainsClassical microbiological methods were employed, that determine COBrucella species display lipopolysaccharide (LPS) heterogeneity25Since members of the atypical group of novel emerging ® BfR Brucella assay tests for 29 aminopeptidases, two phosphatases, four glucosidases, one esterase, and the metabolism of eleven monosaccharides, three disaccharides, seven sugar derivates, 15 amino acids, eleven organic acids, one salt, one amino acid derivate, one peptide, one base, and six classical reactions . Biotyping experiments were carried out in triplicates. Hierarchical cluster analysis was conducted by Ward’s linkage algorithm using binary coded data based on empirically set cutoffs . All characters were considered equal within the data set.Metabolic activity was evaluated using a commercial biotyping system  as described previouslyBrucella sp. strain 10RB9206 was harvested after 12, 24, 36, 48, 60, 72, and 84 h of culture to analyze the ultrastructure of a prospective flagellum using transmission electron microscopy (JEM-1010 electron microscope). Bacterial samples were prepared for microscopic examination by negative staining with 1% solution of uranyl acetate dissolved in distilled water (pH 4.2 to 4.5).Motility of the bacteria was tested using a 0.3% semisolid agar including TTC  to visualize dynamics of bacterial swarming. Exemplarily, A single colony of each strain was transferred from the agar plate to 200 μl 5x lysis buffer D  diluted 1:5 in distilled water, supplemented with 0.5% Tween 20  and 2 mg/ml proteinase K . The bacterial samples were incubated at 56 °C for 1 h and inactivated at 95 °C for 10 min, before crude DNA samples were purified using the QIAamp DNA Mini kit  according to manufacturer’s instructions.High quality genomic DNA (gDNA) was prepared for whole genome sequencing by using a Qiagen genomic extraction kit and Qiagen Genomic-tip 100/G  according to manufacturer’s recommendations. Bacterial lysis was optimized by three freeze/thaw cycles in a lysozyme/lysostaphin buffer.Brucella-specific bscp31 gene and insertion element IS711 was determined711 and its copy number were also assessed by Southern blot analysis as described previouslyThe presence of both the rrs) and recA (recombinase A) genes were amplified and sequenced as described previously48rrs sequence was amplified using universal primers, i.e. 27f and 1492r recA-BrucOchro-f and recA-BrucOchro-r generated a fragment including the whole recA gene . 15 pmol of each primer were added to 50 μl Ready-To-Go master mix  and PCRs were carried out in a Perkin-Elmer GeneAmp 2400 thermal cycler . A total of 30 cycles were performed: 30 s of denaturation at 94 °C, 30 s of annealing at 58 °C (rrs) or 65 °C (recA), and elongation at 72 °C for 90 s (rrs) or 60 s (recA). The run was completed with a final elongation step of 7 min at 72 °C. PCR products were analyzed by agarose gel electrophoresis (1% [wt/vol] in Tris-Acetate-EDTA [TAE] buffer). The purified recA and rrs fragments were sequenced with an ABI Prism 3100 genetic analyzer  using recA-BrucOchro-f/recA-BrucOchro-r primers and an internal primer set consisting of 341fw, 518r, and 926 f, respectively. ClustalW, version 1.8, was used  to generate multiple sequences alignments.16S rRNA Ochrobactrum outgroup) were concatenated as described previously19Multilocus sequence analysis was performed as described previouslypanel 1: bruce06, bruce08, bruce11, bruce12, bruce42, bruce43, bruce45, bruce55) and eight microsatellite markers . Alleles were called according to version 3.6 of the Brucella table for allele assignment  into number of repeats (u)).Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA-16) was carried out as described by Le Flèche and colleagueshttps://github.com/TGenNorth/NASP) using default settings for the programs implemented in the pipeline. Briefly, assemblies of the amphibian Brucella sp. in FASTA format were aligned to the B. abortus 2308 reference genome  and analyzed for SNPs with MUMmer 3.23data not shown). Reads were aligned to the reference with BWA v. 0.3.7http://tree.bio.ed.ac.uk/software/figtree/). Eight of the classical Brucella species were included in the tree for phylogenetic context and to contrast the amounts of genetic diversity among the various clades. Ochrobactrum anthropi ATCC 49188T (GenBank accession numbers CP000758-CP000763) was used as an outgroup.Comparative genomic analyses were conducted following previously described methodology53B. inopinata BO1B. inopinata BO1. Brucella sp. BO2 and B. microti CCM 4915T were also chosen for the initial comparison. All genomes were annotated with RASTtk to provide consistency across the results. A list of all genomes used, as well as their annotation statistics, is provided  was conducted to analyze the wbk region. Contigs from each of these genomes were re-annotated using the current version of RASTtk53wbk region was selected from genome, with special attention paid to the flanking genes and the contigs they were isolated on, as well as the strength of the BLASTP hit, which included the % Sequence Identity, the % Sequence Coverage, and the directionality (uni or bi) of the hits. Only bidirectional hits were used. The PLFams were noted for all of the genes that matched these criteria. If there was a discrepancy between the PLFam assigned to each gene, a multiple sequence alignment was generated in PATRIC and examined. Following this examination, it was determined that genes with a Sequence Identity greater than 70% would be considered homologs.A detailed comparison of specific genomes  and B. inopinata (strain BO1). For macrophage and murine infections, all strains have been cultivated on Tryptic Soy Broth  for 48–72 h.The replication of brucellae within macrophages is a prerequisite for virulence of the pathogen in a given host species. Using macrophage-like murine J774-cells, we therefore compared the behaviour of in vitro infection experiments; namely strains 09RB8471, 09RB8910, 09RB8913, and 10RB9213. Infection experiments were performed as described previously5 cells/ml in RPMI 1640 cell culture medium supplemented with 10% foetal calf serum (FCS), and incubated for 24 h at 37 °C with 5% CO2 prior to infection. The cells were infected at a multiplicity of infection (MOI) of 20 with early-stationary phase bacteria cultured in TS medium. After 30 min, cells were washed twice with phosphate-buffered saline (PBS) and incubated in RPMI 1640/10% FCS with gentamicin (30 μg/ml) for at least 1 h to kill non-phagocytosed bacteria. Ninety minutes, 4, 24, and 30 h post-infection, cells were washed twice with PBS and lysed in 0.2% Triton X-100. CFUs were determined by plating serial dilutions on TS agar, followed by incubation at 37 °C for 2–3 days. All experiments were performed twice in triplicate.Based on molecular and biotyping data, 4 of the most diverse of the 21 bullfrog isolates were selected for B. suis 1330 as a control were intraperitoneally injected into the mice using 104 CFU in a single dose. Five animals per group were euthanized by CO2 asphyxiation after 3, 5, 7, 14, 28, 56, and 84 days. Spleens and livers were aseptically removed, weighed, homogenized, serially diluted in PBS, and plated onto Blood Agar Base (BAB) plates to count bacteria. Student’s t-test was applied to test for significant differences between the groups in the course of time, with p values ≤ 0.05 considered significant.The mouse experiments were approved by the ethical review committee of the Centro de Investigación y Tecnología Agroalimentaria, Unidad de Sanidad Animal, Zaragoza, Spain . Well-established experimentation guidelines were followed33How to cite this article: Al Dahouk, S. et al. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts. Sci. Rep.7, 44420; doi: 10.1038/srep44420 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Persons with chronic hepatitis B (CHB) infection were reported to suffer severe disease after hepatitis E virus (HEV) superinfection, but the studies regarding HEV seroprevalence in this population were limited. A recent study in Vietnam found higher HEV seroprevalence among CHB patients compared with healthy controls.A community-based case-control study was conducted in two counties of Shandong province, China, where hepatitis E incidence was at the highest (Rushan) and lowest (Zhangqiu) in the province based on data from routine public health surveillance. Four townships were selected randomly from each county and all residents in these townships were tested for hepatitis B surface antigen (HBsAg). Those tested positive for HBsAg (CHB group) and the 1:1 age and sex-matched HBsAg-negative residents (control group) were included. Anti-HEV IgM and IgG were tested and positive rates of IgG and IgM were compared between the CHB group and the control group.CI: 7.47–11.09) in Zhangqiue and 38.06% (95% CI: 35.07–41.19) in Rushan (P < 0.001); the corresponding rates of IgM were 0.1% (95% CI: 0.002–0.54) and 1.57% (95% CI: 0.90–2.53), respectively (P < 0.001). HEV IgG seroprevalence was similar between CHB group and the control group in both counties  and the same results were found for the positive rate of IgM . Multivariable analysis showed the status of HBsAg was not independently associated with the status of anti-HEV IgG in either Zhangqiu or Rushan .In total, 2048 CHB participants and 2054 controls were included in the study. In the CHB group, HEV IgG seroprevalence was 9.16% (95% The seroprevalence of HEV varies greatly in different geographic areas, but the seroprevalence is similar between populations with and without CHB. CHB patients residing in high HEV endemic areas might be at higher risk for HBV-HEV superinfection. Hepatitis E virus (HEV) was discovered in 1980’s and has been documented to be prevalent in many countries , 5.3% inAlthough HEV is primarily transmitted by the faecal-oral route, its transmission by transfusion has already been documented . HEV RNABoth hepatitis B and hepatitis E are endemic in China. The seroprevalence of hepatitis B surface antigen (HBsAg) and anti-HEV IgG was 7.18% and 23.1%, respectively according to a national survey in 2006 , 18. TheWe conducted this study to evaluate HEV seroprevalence among persons with and without CHB in Shandong province, China.Shandong province is located in eastern China. The province has 140 counties and a population of 97 million. The average seroprevalence of anti-HEV IgG was 11% among the general population . This stA face-to-face interview was conducted by the staff from the county level Center for Disease Control and Prevention (CDC). The information was collected on age, gender, education attainment, special occupations , health habits including washing hands before dining, drinking boiled water and not-eating out of home and the histories of chronic diseases such as hypertension, heart disease, stroke, etc.Blood samples of 5 ml were collected from each participant. HBsAg was detected using Xinchuang ELISA kits . Anti-HEV IgG and IgM were detected using the Wantai ELISA kits . HEV RNA was tested by real-time PCR for the serum positive for anti-HEV IgM . All tests were performed following the manufacturers’ instructions and were conducted by staff at Shandong Provincial CDC.Further medical examination was carried out for HBsAg-positive participants in local hospitals, including physical examination, Ultrasound examination of liver, testing of HBV serological markers including antibody against HBsAg (anti-HBs), antibody against hepatitis B core antigen (anti-HBc), hepatitis B e antigen (HBeAg) and antibody against hepatitis B e antigen (anti-HBe), and HBV DNA level and Alanine aminotransferase (ALT) level. The participants were classified into HBV carrier, CHB patients, cirrhosis and hepatocellular carcinoma (HCC) according to the above medical examination. HBV carrier was defined as: (1) Tested positive for HBsAg; (2) had no signs and symptoms suggestive of hepatitis; (3) ALT level was within normal limit (< 40 IU/ml) ; (4) B uP value < 0.05 was considered to be statistically significant.The seroprevalence of anti-HEV IgG and IgM across different groups was assessed with Pearson Chi-square test, trend Chi-square test and fisher’s exact test as appropriate. Multivariable logistic regression model was built to estimate the independent association between status of anti-HEV IgG and HBsAg. The analyses were conducted with STATA 10.0 and the This study was approved by Ethics Committee of Shandong Provincial CDC and a written informed consent form was signed by each participant.P = 0.232, 0.742; Rushan: P = 0.462, 0.471). In Zhangqiu, the HBsAg-positive group was consisted of 658 HBV carriers, 102 chronic hepatitis, 22 cirrhotic cases, 1 HCC case and 243 participants with unknown clinical type. In Rushan, the corresponding numbers were 838, 74, 8, 3 and 99.As shown in Tables CI 7.47–11.09) in CHB group in Zhangqiu and was lower in comparison with that in Rushan . IgG seroprevalence increased with age in both counties (P < 0.01), although the seroprevalence was significantly higher in Rushan than in Zhangqiu in all age groups except those above 70 years (P < 0.05). IgG seroprevalnce was similar in males and females in Zhangqiu (P = 0.938), but was significantly different in Rushan (P < 0.001). The rate was much higher among HBV carriers in Rushan than in Zhangqiu (P < 0.001) and the same result was observed among hepatitis cases (P < 0.001). No significant difference in HEV IgG seroprevalence was found among different CHB groups in Zhangqiu (P = 0.714) and the same result was found in Rushan (P = 0.267). The details are shown in Tables HEV IgG seroprevalence was 9.16% (95% an P = 0.67. The dan P = 0.67. The dP = 0.001), and the same trend was found in the proportion of participants with HBV DNA > 103 copies/ml . No significant difference was found between the participants positive for anti-HEV IgG and negative for anti-HEV IgG in the proportions of participants with elevated ALT level and the liver damage identified by ultrasound (P > 0.05). The details are shown in Table In the CHB group, the positive rate of HBeAg among anti-HEV IgG (+) participants was significantly lower than the rate among anti-HEV IgG (−) participants . The corresponding rate was 1.57%  in Rushan, which was much higher than the rate in Zhangqiu (P < 0.001). The positive rate of IgM in Rushan was not statistically significant by age, gender and clinical types (P > 0.05). HEV RNA has been tested for all serum positive for anti-HEV IgM, but none was positive. The details are shown in Table Only one participant was positive for anti-HEV IgM in CHB group in Zhangqiu and the positive rate was 0.1% . Age-specific and sex-specific seroprevalence of HEV IgG was similar between the CHB group and the control group except in age-groups of 0–29 years and 50–59 years in Zhangqiu and 0–29 years in Rushan. The positive rate of anti-HEV IgM was 0.1% and 0.48% respectively among CHB group and the control group in Zhangqiu (P = 0.103). The corresponding rates in Rushan were 1.57% and 2.24% and the difference was not significant too (P = 0.262).HEV IgG seroprevalence was 9.16% and 10.88% in the CHB group and the control group, respectively, in Zhangqiu and the corresponding rates were 38.06% and 39.71% in Rushan. No significant difference between the two groups was found in either county (P < 0.05), and gender, age and education attainment were independently associated with anti-HEV IgG in Rushan (P < 0.05). The status of HBsAg was not independently associated with the status of anti-HEV IgG in either county (P > 0.05). The details are shown in Table Multivariable analysis showed that only age was independently associated with anti-HEV IgG in Zhangqiu , it suggests a high risk to attack HEV acute infection among HBsAg-positive patients in the county because this result came from a cross-section study instead of a cohort study.Some studies showed that HEV infection could result in severe disease in patients with underlying CHB even liver failure , 29–31, Hepatitis E vaccine was licensed in China in 2011 and is the unique commercially available hepatitis E vaccine in the world till now . The immTwo counties with different HEV prevalent level were included in the study, giving the study more depth and improved the generalisability of the findings. However, due to the community-based design of the study, most HBsAg-positive participants were carriers in our study and the conclusions should be further documented in clinical cases.The HEV seroprevalence is similar between HBsAg-positive and HBsAg-negative participants in the same area. The risk of HBV-HEV superinfection could vary greatly in different areas and the HBsAg-positive persons living in HEV hyperendemic areas might be at higher risk for HBV-HEV superinfection. Further study should be conducted to determine the epidemiological characteristics and clinical outcome of HEV-HBV superinfection in HEV hyperendemic areas.


The change of serum platelet indices such as platelet distribution width (PDW) has been reported in a series of inflammatory reaction and clinical diseases. However, the relationship between PDW and the incidence of persistent organ failure (POF) in acute pancreatitis (AP) has not been elucidated so far.  A total of 135 patients with AP admitted within 72 hours from symptom onset of AP at our center between December 2014 and January 2016 were included in this retrospective study. Demographic parameters on admission, organ failure assessment, laboratory data, and in-hospital mortality were compared between patients with and without POF. Multivariable logistic regression analyses were utilized to evaluate the predictive value of serum PDW for POF. P < 0.001). After multivariable analysis, high PDW level remained a risk factor for POF . A PDW value of 16.45% predicted POF with an area under the curve (AUC) of 0.870, a sensitivity with 0.867, and a specificity with 0.771, respectively.  30 patients were diagnosed with POF. Compared to patients without POF, patients with POF showed a significantly higher value of serum PDW on admission (14.88 ± 2.24 versus 17.60 ± 1.96%,  Our results indicate that serum PDW on admission could be a predictive factor in AP with POF and may serve as a potential prognostic factor. Acute pancreatitis (AP) is a common urgent abdominal condition with symptoms of upper abdominal pain, abdominal distention, nausea, and vomiting at onset. According to the revised 2012 Atlanta criteria, AP can be classified as mild (MAP), moderately severe (MSAP), and severity (SAP) through the occurrence of local/systemic complications and/or transient/persistent organ failure . It is aIn addition to the important role in hemostasis and thrombosis, platelet also contributes to the inflammatory process . SeveralWe know that AP is a systemic inflammatory response involving multiple organs. Platelet undergoes a series of changes during inflammation and reflects this process through various platelet indices. Here, we analyzed whether PDW could be a predictive factor in AP patients who have POF. In addition, we compared it with the Rason and SIRS scoring systems and other clinical parameters.We retrospectively analyzed the data from 135 patients admitted in the Pancreatic Surgery Department of Wuhan Union Hospital with confirmed diagnosis of AP from December 2014 to January 2016. The diagnosis criteria of AP are based on two or more of the following conditions: (1) upper abdominal pain of acute onset, (2) serum amylase or lipase activity three times greater than normal, and (3) finding on cross-sectional abdominal imaging consistent with AP [2/FiO2 < 300 mmHg; (2) serum creatinine > 170 μmol/L; and (3) systolic blood pressure < 90 mmHg not responsive to fluid resuscitation [The severity of disease was determined by the revised Atlanta definition criteria 2012. Organ failure (OF) was defined by (1) PaOThe etiologies of AP were generalized to be the following four classifications: (1) biliary, (2) alcohol, (3) hyperlipidemia, and (4) idiopathic. Biliary factor was considered if biliary tract stone was found through the detection of CT, ultrasound, magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde cholangiopancreatography (ERCP), or operation. If one drinks regularly at least 100 g/day or 250 g/week for more than 5 years, alcohol is the etiological factor of AP. For AP due to hyperlipidemia, plasma triglycerides or cholesterol level higher than 2.3 mmol/L and 5.17 mmol/L was necessary. If lack of any causes above or unexplained reasons, an idiopathic AP was considered.t-test. Categorical variable comparisons were performed by using the chi-square test. Univariable analysis used a log-rank test. All variables with statistically significant predictive value in univariable analysis were selected for further multivariable analysis. A Cox regression model was used in multivariable analysis. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were presented. Receiver-operator curves (ROC) were presented to evaluate the sensitivity and specificity of the parameters in predicting POF in AP. P value < 0.05 showed a statistical difference.Statistical Production and Services Solution 19.0  was used in statistical analysis. Continuous data and categorical variables were presented as the means ± standard derivation (SD) and frequency, respectively. Baseline characteristics were compared between study and control groups by using Student's n = 50) and low-PDW group  was presented in P < 0.001), PNec , and in-hospital mortality . Overall, 26 of these patients had solitary POF  and 4 patients were observed with multiple POF  according to A total of 135 patients with confirmed AP admitted to the Union Hospital (Wuhan) from December 2014 to January 2016 were included in this study. The basic characteristic of high-PDW group . There was also no statistically significant difference between the gender, alcohol, smoke, and etiology of the groups.The clinical and the demographic characteristics of patients with and without POF were demonstrated in P < 0.05) between patients with and without POF  above were performed in multivariable analysis. The PDW level (>16.45%) could be a prognostic factor of POF   . As show< 0.001) . These rIn our research, we found the correlation between the PDW and the incidence of POF in AP. Our result showed that PDW detected on admission was significantly higher in patients with POF than patients without POF. After multivariable analysis, PDW was classified as an independent predictive factor for POF. Therefore, PDW could be a relevant risk factor of the occurrence of POF which was superior to some traditional inflammation biomarkers. According to Seker et al.'s report , ESR mayAs a common biomarker in platelet indices, PDW can act as an indicator of platelet volume variability and increases in the presence of platelet anisocytosis . In addiThe value of PDW is usually calculated by a function of standard deviation of log platelet volume. PDW can directly measure variability in platelet size and reflect the heterogeneity in platelet morphology –21. RepoPOF, the main cause of death in AP, develops in 10%–20% of AP patients within the onset two weeks of disease, with a mortality rate between 20% and 50% , 20. So In summary, this is the first time to evaluate PDW as a marker of incidence of POF in AP. Our finding suggests that PDW could be a potential predictive factor of POF in AP. However, there are still several limitations of the present research. First, as an observational study, the causality role of PDW and POF in AP requires to be investigated further in a prospective validation study. Second, the sample size of our study is not large enough to detect the differences between all other markers effectively. Multicenter prospective studies with large sample size are necessary in the future.


Finally, reactive oxygen species (ROS) production was significantly accentuated in S+MI group concomitant with a significant increase in NOX-4 protein levels. In conclusion, smoking aggravates murine acute renal damage caused by MI at the structural and molecular levels by exacerbating renal dysfunction.Cigarette smoking (S) is a risk factor for progressive chronic kidney disease, renal dysfunction, and renal failure. In this study, the effect of smoking on kidney function was investigated in a mouse model of myocardial infarction (MI) using 4 groups: control (C), smoking (S), MI, and S+MI. Histological analysis of S+MI group showed alterations in kidney structure including swelling of the proximal convoluted tubules (PCTs), thinning of the epithelial lining, focal loss of the brush border of PCTs, and patchy glomerular retraction. Molecular analysis revealed that nephrin expression was significantly reduced in the S+MI group, whereas sodium-hydrogen exchanger-1 (NHE-1) was significantly increased, suggesting altered glomerular filtration and kidney functions. Moreover, S+MI group, but not S alone, showed a significant increase in the expression of connective tissue growth factor (CTGF) and fibrotic proteins fibronectin (FN) and  Cardiovascular diseases (CVD) are the leading cause of mortality worldwide, in both men and women , 2. In tAccording to the World Health Organization (WHO), the number of smokers has increased to almost a billion in 2012  and smokS is an important risk factor in the development of atherosclerosis, starting from endothelial cell (EC) dysfunction to acute clinical events. Early studies have shown that death from coronary heart disease (CHD) is more common in smokers than nonsmokers and that CVD is the major cause of smoking-related premature death . In factFree radicals are generated continuously; however, protective mechanisms in the body regulate their concentration leading to a well-balanced equilibrium . Once thSeveral studies showed that S induces renal tubular injury and glomerular damage, leading to kidney diseases and dysfunction . In addiThis study aims to mimic the clinical concept of smoking cessation post-MI following hospitalization by investigating the impact of acute smoke exposure on early kidney injury following acute cardiac injury. Indeed, we sought to determine the effect of S and MI on kidney function by measuring key functional proteins and proinflammatory and oxidative stress markers, in addition to alterations in kidney structure, in a mouse model of acute exposure to S. We hypothesize that S followed by MI aggravates the renal damage caused by S or MI alone, leading to renal dysfunction. Results of this study should raise awareness about S as a risk factor, especially when followed by MI.This study was approved by the Institutional Animal Care and Utilization Committee (IACUCC number: 17-04-410) of the American University of Beirut (AUB) for the project entitled “Assessment of Renal Changes after Smoking and Myocardial Infarction: A Comparative Study.” Male C57BL6 mice weighing 20–25 g were purchased from Charles River Laboratories  and housed under pathogen-free conditions with constant temperature and humidity control at the AUB animal care facility. Mice had sterile bedding and ad libitum access to water and rodent feed. A total of 20 mice, at 16 weeks of age, were divided into 4 groups and used in this study. All surgical procedures were performed under deep anesthesia.A total of 20 mice, at 16 weeks of age, were used in this study. Four groups of 5 mice each were divided during the 2-week study period as follows: controls (C), smoking , myocardial infarction , or both smoking and myocardial infarction (S+MI). For the last group, one week subsequent to S, MI was performed by permanently ligating the left anterior descending (LAD) coronary artery. Mice were then sacrificed one week post-MI.Briefly, age-matched male C57BL/6J mice were exposed to cigarette smoke (S) using a cigarette smoking exposure apparatus . This apparatus includes a smoke generator with a mixing/conditioning chamber and a “nose-only” rodent exposure carousel. It allows for exposure to mainstream smoke from a cigarette in conscious, restrained rodents. This system has been extensively used to study smoking-related diseases. Mice in the smoking groups  were exposed to the smoke of 3R4F research-grade cigarettes , which are scientifically prepared cigarettes concentrated with toxins and chemicals rendering the study timeline suitable to observe the effects of smoking on the mice. Cigarettes were placed into the cigarette puffer, and a peristaltic pump was used to generate puffs at a frequency of 1 puff/min, a duration of 2.5 seconds/puff, and a volume of 5 ml/puff. Animals received 10.9 mg total particle matter (TPM), 9.4 mg tar, and 0.726 mg nicotine per cigarette. The amount of TPM were measured on the smoke filters before and after the experiment. Mice received S twice daily for 1 week preceding MI. Previously, plasma cotinine measurements were conducted in the S group by competitive chemiluminescent immunoassay  in order to verify systemic exposure to aerosol constituents using the nose-only exposure system. Animals were found not to be previously exposed to any substance other than clean air, and therefore the measured cotinine levels were strictly due to exposure during the protocol. Plasma cotinine measurements conducted for 5 animals in the S group after 1 day of exposure were found to be 76 ± 7.6 ng/ml .Induction of MI by LAD ligation leads to the development of heart failure. Briefly, the mouse was placed on a heating pad to prevent hypothermia, and orotracheal intubation was performed by placing a needle into the trachea, connected to a mini ventilator (Harvard Apparatus). Following skin incision, pectoralis muscles were retracted and intercostal muscles carefully dissected. Small incisions were made in order to expose the left atrium (LA), left ventricle (LV), and the LAD coronary artery. MI was performed by ligation of the LAD coronary artery 1–3 mm from the tip of the LA, followed by closure of the thorax. Occlusion of LAD was confirmed by the appearance of a pale color in the anterior wall of the LV within a few seconds after ligation. Infarction was defined by blanching of the LV and ST elevation on the electrocardiogram using Indus MouseMonitor system. Before surgery, mice were given tramadol as analgesic then anesthetized with isoflurane (2-3% in oxygen). During recovery, mice were placed in a warm incubator and monitored closely until freely grooming. Mice were then monitored on a daily basis.After induction of MI, animals were checked every other day by ultrasounds, for 7 days, to assess cardiac remodeling. Briefly, transthoracic echocardiography was performed using SonixTouch ultrasound system  equipped with a high-frequency linear array transducer (L40-8). Mice were anesthetized with 1-2% isoflurane in an oxygen mix and placed on an electrical body temperature heated table. 2D (B-mode) images of the left ventricle were acquired from the parasternal long axis view in supine position. Indeed, parasternal long- and short-axis views were taken by directing the ultrasound beam at the midpapillary muscle level. Continuous interface of the anterior and posterior walls was visualized and well defined before measurements. The transducer was placed on the left thorax, and M-mode and 2D echocardiography images and mean calculations were obtained from three or more consecutive cardiac cycles. Body temperature, heart rate, and respiratory rate were continuously monitored throughout the procedure to adjust the depth of anesthesia. Linear internal measurements of the LV were acquired from the 2D parasternal long-axis images at the midpapillary level. Left ventricular volumes and ejection fraction were calculated according to a cube function formula .At the end of the experimental period, mice were anesthetized with isoflurane then euthanized by cervical dislocation. Kidneys were isolated and divided into 2 parts: either fixed in formalin and embedded in paraffin for histopathology examination or snap frozen in liquid nitrogen then stored at −80°C to be used later for RNA studies and protein analysis.μg/ml Leupeptin, 2 μg/ml aprotinin, and 1 μl/ml DTT), followed by a centrifugation at 12000 ×g for 10 min at 4°C. Total protein concentrations were then determined using “DC™ Protein Assay II” kit Bradford method .Snap frozen kidneys were ground in liquid nitrogen by a mortar and pestle and then homogenized in lysis buffer  containing protease and phosphatase inhibitors  were loaded into the wells of 10% SDS-PAGE gel and run until the dye front reached the bottom of the gel. Gels were then transferred to PVDF membranes at 4°C at 30–40 volts overnight. The membrane was then blocked in 5% fat-free milk, prepared in Tween PBS, for 1 h at RT. Proteins were detected by monoclonal primary antibodies against sodium hydrogen exchanger (NHE-1), nephrin, connective tissue growth factor (CTGF), fibronectin (FN), α-smooth muscle actin (SMA), NOX-4, and GAPDH ; the latter was used to ensure equal loading of samples. Immunoblots were then probed with the appropriate secondary antibodies  for 1 h at RT. Bands were detected with ECL chemiluminescence kit (Thermo Fisher scientific). Intensity of bands was then determined by densitometry, using the ImageJ software (https://imagej.nih.gov/ij/).Protein samples . The quantity of RNA was measured using the NanoDrop® ND-1000 UV-Vis spectrophotometer. RNA purity was assessed using the absorbance ratio of 260 to 280 nm, where a value of 1.8–2.0 indicated good-quality RNA. Finally, RNAs were treated by Deoxyribonuclease I  to remove contaminating genomic DNA.® Green PCR Master Mix  to quantify the gene expression of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) genes. GAPDH expression was used to normalize gene expression between the different samples. During RT step, cDNA was synthesized from 1 μg RNA, using Revert Aid 1st Strand cDNA synthesis kit , followed by real-time PCR analysis in a CFX96 real-time PCR system . For the qPCR reaction, the synthesized cDNA was loaded in duplicates with 5 μM of each of the forward and reverse primers of the gene of interest and mixed with SYBR Green. The PCR conditions were 95°C for 5 min, then 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 15 sec, and extension at 72°C for 10 sec. The plate reading was set after the 72°C step. The amplification cycles were followed by a melt curve set from 70°C to 100°C, with 0.5°C increment every 5 seconds. Primers used were the following: IL-6, F: GAACAACGATGATGCACTTGC and R: TCCAGGTAGCTATGGTACTCC; TNF-α, F: AATGGGCTCCCTCTCATCAGTTC and R: TCTGCTTGGTGGTTTGCTACGAC; and GAPDH, F: GGGGCTCTCTGCTCCTCCCTG and R: CGGCCAAATCCGTTCACACCG. Negative control (water instead of DNA) was used to check for nonspecific amplification. Normalized fold expression relative to the control was calculated and plotted using Bio-Rad CFX Manager to compare differential gene expression.To quantify differences in mRNA expression, gene expression was monitored using SYBRKidney tissues were fixed in 10% neutral formalin in a biopsy cassette for 2 days at RT, then dehydrated and embedded in paraffin. Tissue sections from paraffin blocks were mounted on glass slides, deparaffinised, rehydrated, and then stained with haematoxylin and eosin. The slides were then examined under a light microscope.μM of DHE were applied to dewaxed and hydrated paraffin-embedded kidney sections and incubated in a humidified chamber in the dark for 15 min at 37°C, followed by washing with PBS. Cell Nuclei were stained by Bisbenzimide (Hoechst stain). Tissue images were acquired using a laser scanning confocal microscope . Quantification was done on kidney sections obtained from 5 different animals per condition. A total of 3 different fields of view were quantified per section. The “particle analysis” plugin in the ImageJ software (https://imagej.nih.gov/ij/) was used to obtain DHE fluorescence integrated density (FID), which represents ROS generating cells, and the Hoechst FID, which represents total cells in the tissue section. A ratio was calculated for DHE FID/Hoechst FID for each image. Finally, ratios from all images were averaged and results were analyzed by 2-way ANOVA followed by multiple pairwise analysis using Tukey's post hoc test.The levels of reactive oxygen species (ROS) were assessed using the DHE staining method  for superoxide anions. Briefly, 10 This dye is commonly used to stain carbohydrates and polysaccharides, such as glycogen, producing a pink color. Briefly, after dewaxing and hydration steps of the paraffin-embedded kidneys, the tissue was incubated with 0.5% periodic acid for 10 minutes followed by the Schiff reagent for 10–20 min and then washed for 5 minutes, according to manufacturer's protocol . Various steps of dehydration with increased percentage of ethanol  were done, followed by clearing using xylol, and finally slides were mounted using a mounting medium. Slides were then observed using light microscopy.Trichrome stain is usually used to highlight collagen fibers in a tissue. Briefly, after dewaxing and hydration steps, the tissue was soaked in Bouin solution for 1 hr at 56°C, washed in running tap water, and then rinsed in distilled water, according to manufacturer's protocol . Tissues were then incubated in hematoxylin for 10 min, washed, and then stained in Biebrich scarlet acid fuchsin for 10 min. After washing, sections were differentiated in phosphomolybdic-phosphotungstic acid solution for 10 min, then transferred to aniline blue solution, stained for 5 minutes, and then mounted and observed using light microscopy.p value was determined and values for p < 0.05, p < 0.001, and p < 0.0001  were considered significant. Microsoft Excel and GraphPad Prism software were used to perform statistical analysis.Results are expressed as the mean ± SEM. Statistical comparisons were performed using 2-way ANOVA followed by multiple pairwise analysis using Tukey's post hoc test in order to determine statistical significance. The This study aimed to mimic the clinical concept of smoking cessation post-MI following hospitalization by investigating the impact of acute smoke exposure on early kidney injury following acute cardiac injury. Indeed, mice were exposed to cigarette smoking daily, for 7 days, prior to myocardial infarction (MI) induction via LAD ligation, which also lasted for 7 days. Animals of similar age  were used according to an experimental design that mimics acute smoke exposure with or without myocardial infarction . FollowiThe H&E stain showed normal cellular glomeruli with preserved tubular structures and absence of any arterial abnormality in the control group . HoweverThe PAS stain also showed preserved tubular structures and absence of any increased mesangial matrix in the control (C) group . HoweverThe trichrome stain showed normal glomerular structure, normal tubules with no interstitial fibrosis or inflammation, and normal vessels in the control group . In addi∗∗p < 0.001) in S, MI, and S+MI groups, in comparison to controls , indicating that smoking significantly exacerbates MI-induced changes in nephrin expression (Nephrin is the first protein identified in slit diaphragms (SD), cytoplasmic extensions of podocytes, and a key molecule to maintain SD structure . Results Figures . This depression .+-H+) exchanger-1 (NHE-1), encoded by the SLC9A gene, is a 12-transmembrane protein expressed in renal basolateral proximal tubules and thick ascending epithelia and plays a key role in maintaining homeostatic intracellular volume and pH [∗∗p < 0.001) ~2-fold increase in NHE-1 protein levels in the kidney, in comparison to controls , which plays a crucial role in extracellular matrix (ECM) formation leading to kidney fibrosis , 42. In ∗p < 0.05) in FN renal protein expression  increase in its expression, in comparison to controls , in comparison to controls, whereas MI and S+MI groups showed a significant increase  inflammatory markers were assessed in kidneys, using qPCR. Results showed that only the S+MI group had a significant increase in the renal transcriptional expression levels of IL-6 and TNF-α , in comparison to C and S groups  of ROS generation, in comparison to controls  was then assessed in the different mice groups. Data showed that kidneys in the groups of mice  revealed statistically significant increase  increase in NOX-4 protein expression levels in S, MI, and S+MI groups, in comparison to controls  ROS production was significantly more accentuated and prominent in the cigarette smoking followed by MI (S+MI) group, in comparison to S or MI groups alone. In fact, a significant increase in NOX-4 protein levels was demonstrated in S, MI, and S+MI groups, in comparison to controls.In this study, we report that myocardial infarction (MI) aggravates the ischemic acute renal damage caused by cigarette smoking (S) leading to renal disintegration and function impairment in kidneys of a mouse model. The above statement is supported by the following: (1) Histological analysis of the cigarette smoking followed by MI (S+MI) group showed alterations in kidney structure such as proximal convoluted tubules (PCTs), swelling of the PCT cells with thinning of the epithelial lining, focal loss of the brush border of PCTs, and patchy glomerular retraction. (2) Cigarette smoking (S), as well as MI, altered nephrin and NHE-1 protein expression, in comparison to controls. (3) Nephrin expression was significantly reduced in the S+MI group, in comparison to the S group, whereas NHE-1 was significantly increased. Thus, cigarette smoking followed by MI (S+MI) induces altered glomerular filtration and kidney functions which leads to kidney fibrosis. (4) Cigarette smoking followed by MI (S+MI) group, but not S alone, showed a significant increase in protein expression levels of CTGF, FN, and SMA, in comparison to controls, suggesting accumulation of ECM. (5) MI and cigarette smoking followed by MI (S+MI) groups showed a significant increase in the mRNA expression levels of inflammatory markers IL-6 and TNF-In this study, nephrin expression was significantly reduced in the cigarette smoking followed by MI group, implying that MI worsens cigarette smoking induced renal damage. Nephrin is required to maintain slit diaphragm (SD) integrity and PI3K/AKT signaling, which provides a strong survival signal protecting renal cells from stress-induced death . Indeed,+ binding to an extracellular NHE-1 site triggers a conformational change that translocates Na+ across the membrane. This structural alteration simultaneously creates a cytoplasmic binding site for intracellular H+, hence permitting H+ binding and export [+. The apoptotic stress, generated by S in the kidney, activates NHE-1 [In renal proximal tubules, Nad export . NHE-1 pes NHE-1 . We havees NHE-1 –55. ThisHigh extracellular glucose induces CTGF expression in mesangial cells and increases the production of FN and COL4 leading to ECM production , 42. In α inflammatory genes in the kidneys of MI and S+MI groups. Also, the expression of the fibrotic marker FN was altered, which has an important role in the initiation and progression of atherosclerosis through the promotion of hypertrophy of vascular smooth muscle cells. Many studies have reported a strong association between smoking and CVD occurrence [2O2 component of ROS participates in signaling pathways related to cellular proliferation, migration, and apoptosis [Smoking induced the expression of IL-6 and TNF-currence –59 wherecurrence . Smokingpoptosis , 22. On poptosis , 61. A mpoptosis . In thisIn summary, our results suggest that MI aggravates the acute renal damage caused by cigarette smoking leading to renal damage and function impairment in kidneys of a mouse model.