Brassica campestris L.) straw (RS), apple  tree branches (ATB), and pine (Pinus sp.) sawdust (PS), were carried out by pyrolysis under dynamic conditions (30 to 900 °C) at different heating rates of 5, 10, and 15 °C·min−1. Correlation analysis showed that intrinsic physicochemical properties play distinct roles in different stages of pyrolysis. The ash content was negatively correlated with the temperature range (R2) of the second stage (190–380 °C) of pyrolysis. The lignin content and the amount of pyrolysis residues (RSS) were positively correlated. Kinetic triplets, including the activation energy (Ea), pre-exponential factor (A), and reaction model [f(α)], were obtained using different methods, including the Flynn–Wall–Ozawa (FWO), Freidman, Kissinger–Akahira–Sunose (KAS), and Starink methods. The mean activation energy (Ea[mean]) for RS, ATB, and PS calculated by the different methods ranged from 167.15 to 195.58 kJ·mol−1, 195.37 to 234.95 kJ·mol−1, and 191.27–236.45 kJ·mol−1, respectively. Correlation analysis of the intrinsic physicochemical characteristics and kinetic factors of agroforestry waste showed that the minimum Ea (Ea[min]) was significantly positively correlated with heat capacity (C0) and negatively correlated with thermal diffusivity (D). The Ea[mean] and the maximum value of Ea (Ea[max]) significantly positively correlated with the sum content of cellulose and lignin, indicating that the contents of cellulose and lignin determines the energy required for the pyrolysis process of agroforestry waste. The mechanism of degradation involves the diffusion model , the growth model (A4), and the geometrical contraction model (R3). These results indicate that the pyrolysis of agroforestry waste is a complex process due to the heterogeneity of its intrinsic physicochemical properties.To obtain a comprehensive understanding of the qualitative and quantitative effects of the intrinsic properties of biomass on its pyrolysis characteristics and assess the behavior of agroforestry waste, thermogravimetric analyses of three representative agroforestry wastes, namely rape ( X, NOX, CH4, and SO2, but also result in economic losses ), mean (Ea[mean]), and maximum (Ea[max]) values of Ea are shown in Ea[mean] values determined by the FWO, Friedman, KAS, and Starink methods during the conversion process of RS were 167.15, 195.58, 177.90, and 178.60 kJ·mol−1, respectively; those of the ATB were 195.37, 234.95 207.19, and 207.57 kJ·mol−1, respectively; and those of the PS were 200.58, 236.45, 191.27, and 201.53 kJ·mol−1, respectively. It is worth nothing that Ea[mean] values determined for the three types of biomasses were different, which could indicate that the thermal decomposition mechanism of the whole pyrolysis process was different. The Ea[min] and Ea[max] values were obtained from Ea[min] essentially occurred at the initial stage of pyrolysis, while the occurrence of Ea[max] was dependent on the feedstocks and the calculation model.The minimum  and negatively correlated with D (p < 0.05). High fixed C and C0 do not facilitate the start of biomass pyrolysis, but high thermal conductivity values are favorable to the start of biomass pyrolysis. Aup-Ngoen et al. noted that the rhizome biomass of cassava (Manihot esculenta) with high fixed C withstood more decomposition compared to the other types of biomass residues  and Ea[max] obtained by the FWO, Freidman, and Starink methods significantly positively correlated with the sum content of cellulose and lignin, indicating that the contents of cellulose and lignin determines the energy required for the pyrolysis process of agroforestry waste. It also confirmed that the Ea[max] during the pyrolysis process was caused by the simultaneous decomposition of cellulose and lignin, which requires a large amount of energy. The maximum activation energy values significantly negatively correlated with the contents of moisture, ash, N, and hemicellulose. The reason is that hemicellulose and the N-containing substances in biomass have a weak bond strength and are easily decomposed  values were significantly positively correlated with C0 and negatively correlated with D. The Ea[mean] and Ea[max] were significantly positively correlated with the sum content of cellulose and lignin, indicating that the contents of cellulose and lignin determine the energy required for the pyrolysis process of agroforestry waste.The (4)A value and several reaction models  involved in the pyrolysis process indicate that the pyrolysis of agroforestry waste is a complex process due to the heterogeneity of its intrinsic physicochemical properties.Vast variations in In this study, the effect of intrinsic physicochemical properties of agroforestry waste on its pyrolysis characteristics was comprehensively investigated. The results of the study showed that:In summary, based on these findings, it can be concluded that agroforestry wastes have good pyrolysis potential, and the pyrolysis of agroforestry waste is a complex process due to the heterogeneity of its intrinsic physicochemical properties. The qualitative and quantitative effects of these intrinsic properties on the pyrolysis characteristics and behavior obtained from this study will help researchers and agroforestry industries directly estimate pyrolysis rates and control the efficiency of the pyrolysis process.


The characteristics and differences of microbiota between the well-nourished (W) and malnourished (M) groups were compared. We studied the genera and the operational taxonomic units (OTUs) within the genus of our patients, initially comparing the malnourished and the well- nourished groups and later on reanalyzing the whole group using these OTUs. At the OTU level, 6 bacteria were significantly correlated with the serum albumin level. The abundances of 2 OTUs (OTU208 Lachnospiraceae_incertae_sedi and OTU4 Bacteroides) were more in W group. Meanwhile, 4 OTUs  displayed higher abundance among individuals in M group. Notably, the OTU168 Clostridium_sensu_stricto was the only bacteria that significantly correlated with serum albumin , pre-albumin , and SGA . The higher the OTU168 Clostridium_sensu_strictu, the lower serum albumin and pre-albumin and a higher score of SGA signifying a worse nutritional status. Our preliminary findings suggested a relationship between the nutrition status and microbiota in PD patients. Our results provide a basis for further exploration of the interactions between malnutrition and intestinal flora in PD patients with potential interventions using probiotics and prebiotics.Malnutrition is a common complication in the dialysis population, both hemodialysis and peritoneal dialysis (PD). We report our exploratory study on the characteristics of intestinal microbiota and nutritional status in PD patients. The nutritional status of our PD patients were evaluated, and their feces were collected for  Approximately 11% of patients with ESKD are treated with PD in China2. Malnutrition is a common complication in patients undergoing PD and the incidence ranges from 18 to 75%4. The malnutrition is a consequence of a complex mechanism related to multiple factors including not only patients factors such as loss of residual kidney function, intestinal dysfunction5 and inadequate nutritional intake6, but also factors associated with PD per se such as inadequacy of dialysis, protein loss with PD fluid and continuous micro-inflammation status7. Malnutrition is also a key factor that affects patient quality of life and prognosis9. Patients receiving PD who become malnourished have higher hospitalization rates and longer hospital stays, as well as significantly elevated risks of peritonitis and death11. Therefore, malnutrition in these patients deserves more attention in clinical practice.Peritoneal dialysis (PD) is a standard modality of kidney replacement therapy for patients with end-stage kidney disease (ESKD) with increasing use worldwide13. Disturbance of the gut microbiota may upset gut homeostasis and result in abnormal nutrient absorption and even malnutrition in patients undergoing PD14.The disturbance of gut microbiota in patients with chronic kidney disease (CKD), especially ESKD, has gained increasing attention in recent years. Dysbiosis in CKD patients generally manifests as a loss of beneficial microorganisms and an increase in pathogenic microorganisms and conditional pathogensThe objective of this study is to investigate the composition and distribution characteristics of gut microbiota in patients undergoing PD. We particularly looked into the operational taxonomic units (OTUs) of the microbiome and their relationships with nutritional status. We hope this can provide a scientific basis for the exploration of potential intervention to alleviate malnutrition in this patient cohort in future.15; (3) able to independently collect fecal samples; and (4) voluntary participation in the study with provision of written informed consent. Patients were excluded for the following reasons: (1) cognitive or hearing impairment; (2) other concomitant severe diseases, such as active tuberculosis, malignant neoplasms, severe hepatitis, liver cirrhosis, and active autoimmune disease or other chronic diseases; (3) administration of oral or intravenous antibiotics, microecological preparations, or lactulose within two weeks before sample collection; (4) transfer to other centers on a temporary or long-term basis during the course of PD treatment; (5) poor compliance and the inability to adhere to dialysis prescriptions and execute sample collection instructions; (6) major gastrointestinal surgery within the last 3 months; (7) gastrointestinal endoscopy within the last month; (8) gastrointestinal bleeding within the last month; (9) severe cardiac dysfunction (New York Heart Association class IV); and (10) confirmed diagnosis of PD-related peritonitis, lung infection, or acute infection of other organs or tissues within the last month.Patients who underwent continuous ambulatory peritoneal dialysis (CAPD) at the Peritoneal Dialysis Center, Department of Nephrology, General Hospital of Ningxia Medical University between May 2017 and Dec 2018 were included. The inclusion criteria were as follows: (1) age ≥ 18 years; (2) stable CAPD treatment for ≥ 3 months16 was used to divide the CAPD group to the well-nourished group (W group) and the malnourished group (M group).The cut off serum albumin level of 35 g/L as suggested by Chinese National Renal Data System (CNRDS)A cross-sectional observational study design was adopted. The study was approved by the medical research ethics committee of Ningxia Medical University . We confirmed that all methods were performed in accordance with the relevant guidelines and regulations.17. BMI was calculated using the following formula: BMI = Body weight (kg)/height2 (m2).The following patient information was collected: age, sex, PD vintage, body mass index (BMI), primary disease , biochemical parameters and 24-h residual urine volume. Subjective Global Assessment (SGA) was used to classify for every CAPD patients to level A, B, and C successively18. The lean tissue index (LTI) and fat tissue index (FTI) normalized to 1.73m2 body surface area (kg/m2) were used to present muscle and fat content of patient respectively. The item of overhydration (OH) were used to determine fluid status, with a normal reference between plus and minus 1.Multi-frequency body composition measurement  were performed to measure the nutrition status and hydration status for each participantPatients who underwent PD at our center received PD treatment using the PD-2 system  with a daily peritoneal dialysate dose of 6L to 10L. Dialysis was performed with a lactate-buffered dialysate with 2.5 or 1.5% glucose concentration, 132 mmol/L sodium, 1.75 or 1.25 mmol/L calcium, 0.25 mmol/L magnesium, 96 mmol/L chlorine, and 40 mmol/L lactate.Each fecal sample collected was placed in an ice box immediately and transported to a − 80 °C freezer within 30 min to store until use. Genomic DNA of each fecal sample were extracted with the E.Z.N.A. Stool DNA Kit . The V3–V4 regions of 16S rRNA genes were amplified using primer 341F and 805R . After purification, the polymerase chain reaction products from each sample were indexed and mixed at equal ratios for sequencing by Shanghai Mobio Biomedical Technology Co. Ltd. through the MiSeq platform . The total sequencing readouts was 941,946 and mean readouts was 31,398 for each sample.P was < 0.05.Numerical data are expressed as mean ± standard deviation, and categorical data are expressed as number and percentage. Intergroup comparisons of numerical and categorical data were performed using the independent samples t-test and Fisher’s exact test. Statistical analysis was performed using SPSS 20.0 , with differences considered statistically significant when To our knowledge, there is no report on relationship between nutrition status and microbiota in PD patients. Since the present study was exploratory, no reference available for the primary study end-point in calculation of sample size.http://drive5.com/usearch) based on the following criteria: (1) the sequences of each sample were extracted by allowing zero mismatches in each index; (2) sequences with overlap < 16 bp were discarded; (3) sequences with an overlap error rate > 0.1 were discarded; and (4) sequences with a length < 400 bp after merging were discarded. The optimized sequences obtained after processing were clustered into operational taxonomic units (OTUs) using UPARSE , as follows: (1) non-repetitive sequences were extracted from the optimized sequences; (2) single-copy non-repetitive sequences were deleted; (3) OTU clustering was performed using a similarity threshold of 97%; and (4) the representative OTU sequences were generated. Using a confidence threshold of 70%, the representative OTU sequences were compared with sequences in the SILVA database  to determine the taxonomic position of each 16S rRNA sequence. After the OTUs were binned with the UPARSE pipeline, we counted the gross OTUs at each taxonomic level . The compositions of gut microbiota at each taxonomic level in two groups were compared by using the Mann–Whitney U test.For analysis of sequence data, clean data were extracted from the raw data using USEARCH 8.0  and overhydration.The study was reviewed and approved by the General Hospital of Ningxia Medical University institutional review board.All the participants provided a written informed consent.In total, 30 CAPD patients were enrolled with 19 in W group and 11 in M group. The mean patient age in the entire CAPD patient group was 46.3 ± 13.6 years, and there were 12 men (40%). The mean vintage was 26.6 ± 19.8 months. Sixteen patients (53.3%) had primary glomerulonephritis, 6 (20%) had diabetes mellitus, and 8 (26.7%) had hypertensive nephropathy.P > 0.05).Table P = 0.03). (PERMANOVA and PCoA plot in the supplemented files)   Fig. . The abuoup Fig. .Figure 2OTU208 Lachnospiraceae_incertae_sedis: mean log2 fold change 2.66, padj = 0.013 and OTU4 Bacteroides: mean log2 fold change 3.91, padj = 0.025, respectively). Meanwhile, 4 OTUs displayed higher abundance among individuals in M group .  while 2 presented positive correlation (OTU208 Lachnospiraceae_incertae_sedis and OTU4 Bacteroides) , pre-albumin , and SGA . The higher the OTU168 Clostridium_sensu_stricto, the lower serum albumin and pre-albumin and also a higher score of SGA which also signified a worse nutritional status.Notably, the OTU168 Lachnospiraceae_incertae_sedis was related to residual urine volume and overhydration besides albumin level. The OTU4 Bacteroides was also correlated with multi-items including age, vintage, and residual urine volume. The OTU225 Akkermansia was positively correlated with overhydration and negatively with serum albumin. OTU87 Megasphaera was also positively correlated with dialysis vintage and overhydration while having a negative correlation with albumin and pre-albumin. OTU31 Peptostreptococcaceae_incertae_sedis had a negative correlation with serum albumin and did not show any significant correlation with other confounding factors. Finally, the OTU168 Clostridium_sensu_stricto was significantly associated with nutrition markers presented as albumin, pre-albumin and SGA. Meanwhile, it did not show any significant correlation with other confounding factors. Table In Table 20. In the present study, sequencing analysis was performed on the gut microbiota of patients undergoing PD, and the gut microbiota characteristics of patients with different nutritional statuses were compared. Our results indicated that there were differences in the species abundance and gut microbiota compositions of patients undergoing CAPD based on nutritional status, suggesting that changes in bacterial taxa and microbiota structure might be related to malnutrition.Recent research on the relationship between intestinal microecology and chronic diseases suggests the presence of severe dysbiosis within the gut microbiota of ESKD patients, which is associated with disease progression and the onset and development of complications21. The increased intraperitoneal pressure, long-term retention of dialysate in the peritoneal cavity and decreased gastrointestinal motility caused by PD together with the use of iron supplements and antibiotics can damage the intestinal barrier, leading to changes in the abundance and diversity of gut microbiota24.Physical, chemical, biological, and immune barriers exist in normal intestinal mucosa. In patients undergoing PD, several factors may lead to ischemia, edema and injury in the mucosa which can damage the intestinal barrier and result in gastrointestinal dysfunction and gut microbiota dysbiosis26. Our study is probably the first focusing on the relationship between gut microbiota and nutritional status in patients undergoing PD. Studies indicated a gut microbiota imbalance in patients undergoing PD and this imbalance is characterized by a decrease in beneficial bacterial populations and an increase in pathogenic or conditional pathogenic bacteria29. In the present study, OTUs across the M and W CAPD subgroups showed that the fecal microbiota of the two subgroups had similar numbers of bacteria but different bacterial populations.Previous studies reported changes in the gut microbiota composition among patients with CKD and ESKDAkkermansia, Defluviitaleaceae_uncultured, Megasphaera, Peptostreptococcaceae_incertae_sedis, Ruminococcaceae_incertae_sedis, and Coprobacter were significantly different between the two groups. In a recent study trying to define dysbiosis for a cluster of chronic diseases including urinary stone disease, obesity, diabetes, cardiovascular disease, and kidney disease, each disease state was associated with a loss of microbial diversity in the gut30. They reported that among the taxa that were most consistently found to be depleted in disease individuals were the Bacteroides, Coprococcus, Prevotella, Ruminococcus, and Sutterella. However, their subsequent analysis suggests that the association of some of these taxa with dysbiosis is closely tied to their number of constituent OTUs in the gut30. Some taxa, primarily Bacteroides, Corynebacterium, and Anaerococcus were identified as dysbiotic more than expected given their diversity, indicative of a more specific physiological interaction between these taxa and disease. It was previously reported that Bacteroides either has a health-protective31 or health-antagonistic response32. Results from their analysis show that this genus is strongly associated with both health and disease in terms of the number of independent populations it was found to be associated with. Thus it is likely that some OTUs within the genus provide more of a protective effect and others more of a detrimental health impact30. Thus our analysis focused more on the OTU level of our patients, initially comparing the malnourished and the well- nourished groups and later on reanalyzing the whole group of the 30 CAPD patients. Notebly, the OTU87 Megasphaera and OTU168 Clostridium_sensu_stricto were both negatively correlated with the serum albumin and pre-albumin. Especially, the OTU168 Clostridium_sensu_stricto was the only bacteria that significantly correlated with SGA, pre-albumin and serum albumin at the same time. The higher the OTU168 Clostridium_sensu_stricto, the lower serum albumin and pre-albumin and also a higher score of SGA which also signified a worse nutritional status. These two OTUs may have significantly adverse association with the nutritional status of PD patients.We found that 6 genera, including Clostridium sensu stricto are grouped around the type species Clostridium butyricum and belong to the Clostridium cluster I within the Clostridiaceae family. It has been reported that up to 72 Clostridium spp. have been detected in the human gastrointestinal samples, of which 30 belong to the Clostridium sensu stricto33. A recent study suggested that the change in the abundance of Clostridium sensu stricto was positively associated with IL-10 and LDL-cholesterol, while a negative correlation was observed with changes in insulin levels34. Studies also showed that CD4 + T regulatory cells (Tregs) are abundant in intestinal lamina propria, and their accumulation in the small intestine and colon is differentially regulated. The induction of colonic Tregs is dependent on commensal microorganisms with specialized properties. Among the indigenous commensal bacteria, Clostridium spp belonging to clusters IV and XIVa are outstanding inducers of Tregs in the colon35. Notably, Clostridium clusters IV and XIVa constitute a smaller proportion of the fecal community in patients with human inflammatory bowel diseases than in healthy controls36. Another recent study of microbiota analysis on the liver steatosis and fibrosis in obese patients found the relative abundance of fecal Clostridium sensu stricto was significantly decreased with the presence of liver fibrosis and was negatively associated with liver stiffness measurement and myosteatosis37.The Clostridium_sensu_stricto association with a worse nutrition status in PD patients with a lower serum albumin and pre-albumin and also a higher score of SGA would warrant further investigation. It is of note that this OTU did not show any correlation with residual kidney function or hydration status, pointing that this OTU168 may have a particular significant value in the nutritional status in PD patients.Our finding of OTU168 Megasphaera was also negatively correlated with the serum albumin and pre-albumin but not with SGA. It also did not show significant correlation with residual kidney function but had a positive correlation with overhydration. The negative association with the albumin and prealbumin may be partly related to the overhydration status in patients with a higher abundance of this OTU87 Megasphaera. Despite this, it is of note that human gut isolates of Megasphaera have been found to harbor a wide range of carbohydrate active enzymes alongside with oligopeptide transport systems allowing them to use carbohydrates as well as a wide range of amino acids as carbon source38. Besides the production of propionate, human gut isolates of Megasphaera, have been shown to produce butyrate from glucose38.OTU87 Clostridiales and Akkermansia muciniphila, which both displayed lower abundance among 134 Danish adults with prediabetes when compared with 134 age- and sex-matched individuals with normal glucose regulation39. Like what we have performed for the whole group of 30 CAPD patients, they went on further to study the association between differentially abundant OTUs and clinical biomarkers relevant for diabetes in the entire group of 268 individuals39. They found that two OTUs (OTU 3856408 and OTU 193129) classified as Lachnospiraceae, two OTUs (OTU 4364405 and OTU 819353) classified as Ruminococcaceae and OTU 4465124 classified as Clostridium correlated particularly strongly and negatively with fasting plasma levels of glucose, insulin, C-peptide, triacylglycerol and hsCRP, as well as HOMA-IR, BMI and waist circumference39.The importance of OTUs has been studied in a group of pre-diabetes patients. They found that the two OTUs that differed the most were a member of the order Lachnospiraceae_incertae_sedi and OTU4 Bacteroides) presented positive correlation with serum albumin. OTU4 Bacteroides also has a positive correlation with residual kidney function making it difficult to discern the effects whether it is more related to the residual kidney function or the nutritional status per se. It is obvious that PD patients with good residual kidney function can have a good nutritional status40. Despite such complex interactions, recent review suggested that metabolites secreted by different Bacteroides spp. assist in maintaining stability of the immune system41. These species are primary producers of short-chain fatty acids in the human gut, mostly in the form of acetate and propionate which are important for the maintenance of intestinal homeostasis41. Both acetate and propionate are potent anti-inflammatory mediators as they inhibit the release of pro-inflammatory cytokines from neutrophils and macrophages. Also, butyrate increases expression of tight-junction proteins in the gut to reduce potential gut hyperpermeability. This, in turn, decreases inflammation and endotoxemia that are associated with leaky gut42. It would be interesting to study further the OTU4 Bacteroides effect on the immune regulation and endotoxemia in future.We also found two OTUs . OTU225 Akkermansia was shown to have a positive correlation with overhydration. OTU31 Peptostreptococcaceae_incertae_sedis showed no relationship with residual kidney function or overhydration. The significance of these 2 OTUs on nutrition required further study.In our study, another 2 OTUs showed negative correlation with serum albumin but not with serum pre-albumin or SGA (OTU225 It should be noted that our findings only provide a snapshot of the complex fecal microbiomes in PD patients and nutritional subgroups, and only the bacterial composition was analyzed. There are several limitations of this study. First, the population in this study comprised patients who were undergoing PD. Dialysis in the peritoneum around the gut may have effects on intestinal functions and environment, thereby affecting microbiota. Additionally, the results suggest that the composition and structure of intestinal microflora are related to nutritional status, but it is not clear whether the relationship is causal. Finally, the small sample size of malnourished patients may have magnified the effects of individual differences.Clostridium_sensu_stricto was associated with a worse nutritional status in PD patients with a lower serum albumin and pre-albumin and also a higher score of SGA. The results of this study indicate the presence of a possible gut microbiota imbalance in patients undergoing PD with malnutrition. Further research is required to elucidate the association of the gut microbiota with malnutrition in patients undergoing PD and look into the pathogenetic mechanisms by which gut microbiota causing malnutrition in PD patients. This may help in future to study the potential use and effectiveness of probiotic formulations in improving nutritional status in PD patients.In conclusion, this is an exploratory study looking into the association of gut microbiome with nutritional status of patients on peritoneal dialysis. We have focused our study on the OTU level of the microbiota which should have advantage over studies on genus level alone. Significant differences were also found in the compositions of gut microbiota between malnourished and well-nourished patients undergoing PD. In particular, OTU168 Supplementary Figure 1.Supplementary Information 1.


DMPK gene. The human skeletal actin long repeat (HSALR) mouse model reproduces several aspects of the disease, but the muscle-wasting phenotype of this model has never been characterized in vivo. Herein, we used quantitative MRI to measure the fat and muscle volumes in the leg compartment (LC) of mice. These acquired data were processed to extract relevant parameters such as fat fraction and fat infiltration (fat LC/LC) in HSALR and control (FBV) muscles. These results showed increased fat volume (fat LC) and fat infiltration within the muscle tissue of the leg compartment (muscle LC), in agreement with necropsies, in which fatty clumps were observed, and consistent with previous findings in DM1 patients. Model mice did not reproduce the characteristic impaired fat fraction, widespread fat replacement through the muscles, or reduced muscle volume reported in patients. Taken together, the observed abnormal replacement of skeletal muscle by fat in the HSALR mice indicates that these mice partially reproduced the muscle phenotype observed in humans.Myotonic dystrophy type 1 (DM1) is a severe autosomal dominant neuromuscular disease in which the musculoskeletal system contributes substantially to overall mortality and morbidity. DM1 stems from a noncoding CTG trinucleotide repeat expansion in the  DM1 protein kinase (DMPK) gene. DM1 is autosomal dominant and can affect all ages, from newborns to elderly adults. Although DM1 is the most common form of muscular dystrophy in adults and the prevalence of the mutation is 1/2100 births1, there are currently no treatment options for this disease. Adult-onset patients face risks of physical disability and a shortened lifespan. Characteristic neuromuscular symptoms include muscle stiffness and weakness, impaired muscle relaxation capacity (myotonia), and progressive loss of muscle mass (atrophy). However, DM1 is not merely a skeletal muscle disease but a multisystemic disease, as other tissues and organs, such as the smooth muscle, brain, and heart, are also affected2. Premature deaths among DM1 patients are caused by respiratory failure due to muscle weakness and myotonia that affect the peripheral respiratory system. Additionally, failure of the central respiratory drive and the upper airway muscles contributes to an increased risk of pulmonary infections in patients4.Myotonic dystrophy type 1 (DM1) is a debilitating chronic disease caused by an expansion of the unstable CTG triplet repeat in the 3' untranslated region of the 5. The principal factor consists of DMPK transcripts carrying the DM1 mutation. Mutant RNA is retained in the cell nucleus in several tissues, including the heart, brain, and skeletal muscle, where it aggregates into insoluble, stable, membraneless structures known as foci. Ribonuclear foci of expanded CUG repeat hairpins can bind and sequester RNA-binding proteins, thus preventing them from performing their normal functions6.Muscle atrophy and wasting are critical signs of DM1, and various factors have been hypothesized to lead to these phenotypes8. Consequently, this technique helps diagnose neuromuscular pathologies, as it can reveal disease-specific patterns of muscle involvement9. Furthermore, as MRI is a noninvasive technique that does not expose patients to ionizing radiation, it can be used to track the evolution of muscular diseases. In musculoskeletal disorders, MRI is being used increasingly often for longitudinal evaluation of candidate drugs10 and validation of clinical trial outcomes11.Magnetic resonance imaging (MRI) is the gold-standard technique in muscle pathology. The benefits include its capacity to discriminate muscular from adipose tissue accurately and to identify fat infiltration into skeletal muscle12. Patients also show significantly reduced skeletal muscle volume, especially in the gastrocnemius medialis and soleus, accompanied by increased T2 water relaxation time, indicating muscle atrophy and edema, respectively13. Other MRI studies have shown that over 70% of DM1 patients display fatty infiltration in the lower extremities, with especially severe defects in the gastrocnemius and tibialis anterior during the initial stages of the disease14. Interestingly, the authors reported the possibility of detecting affected muscles even before weakness was clinically noted. Recent studies have shown that an increased fat fraction in the muscles of DM1 patients is correlated with muscle weakness, reduced specific strength, and high disease severity16. Taken together, these data support the idea that muscle impairment as determined by quantitative MRI is strongly correlated with strength, supporting its feasibility as an approach to predict muscle dysfunction in DM1.Clinical research on DM1 has categorically demonstrated that patients display muscle defects that can be quantitatively analyzed using MRI. Specifically, DM1 patients have almost 4 times more fatty infiltration in the skeletal musculature than unaffected controls. Additionally, fat infiltration correlates with scores on the 6-min walk test and the Muscular Impairment Rating Scale17, but there are insufficient noninvasive methods for preclinical evaluation of these candidate drugs in DM1 model mice; such methods would allow dynamic evaluation of phenotype progression. Therefore, we decided to optimize quantitative MRI to these circumstances and present an in vivo method to quantitatively measure muscle defects in a murine model expressing 250 CUG repeats in the skeletal muscles, driven by a human skeletal actin transgene18 (HSALR). The HSALR murine model accurately reproduces key DM1 phenotypes such as aberrant alternative splicing, centrally located nuclei in muscular fibers, variable muscle fiber size, and myotonia20. Some further studies performed in young HSALR mice have reported that chemical inhibition of glycogen synthase kinase 3 beta (GSK3β) corrects impaired Celf1 activity, thus preventing the development of DM1 muscle pathology, including muscle atrophy21. However, independent studies have reported a lack of clear and consistent dystrophic and atrophic features in this model19. Conversely, researchers have discovered genetic and drug manipulations that can enhance muscle atrophy phenotypes in HSALR mice23. Selecting the disease-related phenotypes already described in DM1 patients, our study aimed to achieve MRI quantitation of muscle and fat volume, fat infiltration, and fat fraction (FF) in HSALR model mice.Nearly two dozen experimental treatments against DM1 are in developmentLR mice showed clear signs of fatty infiltration in the quadriceps and gastrocnemius muscles, as fatty clumps could easily be observed  mice were acquired under anesthesia in an MR scanner. Two individuals from each group had to be removed from the study because their breathing movement impeded the acquisition of images of sufficient quality for further quantitative analyses. Images of both hind limbs from each animal were acquired, and the leg compartment (LC) was segmented manually. The LC consists of all muscles, fat, and bones forming the leg, but the small size of the mice compared to the scanners used in clinical practice precluded us from differentiating small bones from muscle; therefore, the LC was stratified into macroscopic fat (fat LC) and muscle plus bone, defined as muscle LC. Note that, as the animals used in the experiment were of the same sex and age and did not express the transgenic construct in bone or bone precursors, bone structure size and density were assumed to be constant among the different individuals. A schematic workflow of the process is shown in Fig. Necropsies of 4.5-month-old HSAved Fig. . To quanU test). Individual analysis of both LC components showed that the difference was mainly due to a robust, almost fourfold increase in fat LC volume in the DM1 group relative to controls  in DM1 cells, which inhibits miR-7 biogenesis, leads to increases in autophagy and the activity of the ubiquitin‒proteasome system, two pathways with a strong contribution to the muscle impairment characteristic of DM132. Nonetheless, miR-7 levels in the diaphragm and gastrocnemius of HSALR mice were similar to those of control mice29. Therefore, the failure to reduce miR-7 in model mice may explain why no overt muscle atrophy was detected.Increased activity and stability of GSK3β have been proposed to explain the atrophic phenotype in DM1LR DM1 model mice35. Therefore, our finding that they reproduce some of the muscle-pathology-related phenotypes observed in patients, such as fat replacement, makes this model a valuable tool during preclinical evaluation. A particularly useful aspect of MRI in this context is that it is a noninvasive technique performed in vivo, enabling scientists to evaluate the effect of a candidate treatment on the same individual at different time points during the experiment. This brings two advantages: first, it allows researchers to study the phenotype's evolution over time; second, it allows them to identify the optimal treatment duration or point of maximum effect. Moreover, it allows the study of all muscles, rather than only a few, as with biopsy or electromyography.Preclinical testing of therapeutic effects in a mammalian animal model is a requirement of any drug development procedure. No studies have yet been published in which MRI determinations have been performed in the widely used HSA36. Our results are valuable for therapy research due to the vast difference between control and DM1 mice, allowing researchers to identify molecules with strong therapeutic potential.In conclusion, we observed evidence of fatty infiltration in the leg muscles of DM1 mice in necropsies, and quantitative MRI confirmed this phenotype, which has been reported to affect more than 70% of DM1 patientsMouse handling and experimental procedures were performed according to the European law regarding laboratory animal care and experimentation (2003/65/CE) and the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. All procedures were approved by the Consejeria de Agricultura, Generalidad Valenciana (Reference Number 2019/VSC/PEA/0198).LR mice (line 20b) were used as DM1 disease models; they were compared to 11 controls, which were mice with the same genetic background (FVB). Mice were provided by C. Thornton 18. All animals were 4.5 months old at the time of experimentation and were randomly selected for the experiment. The animals were bred in the animal facilities of the University of Valencia and then transferred to the facilities of the La Fe University Hospital. Once the acclimatization period had elapsed, images were acquired by MR while the animals were anesthetized with isoflurane and immobilized in the supine position; after acquisition was completed, the animals were sacrificed by cervical dislocation.Eleven male homozygous transgenic HSALR and six FVB mice were euthanized by cervical dislocation. All animals were 4.5-month-old males. The procedure was approved by the Consejería de Agricultura, Generalidad Valenciana (Reference Number 2018/VSC/PEA/0182).For the necropsy study, six HSAMR images were obtained using a Philips Achieva 3.0 TX  using an 8-Channel Sense Wrist Coil. Animals were placed in a prone position for transverse acquisition; the protocol was created with 22 slices to cover the full thigh. This plan was made on the basis of sagittal and coronal scout images. The acquisition protocol consisted of a T2-weighted multi-slice turbo spin‒echo (T2W MS TSE) sequence with a field of view (FOV) of 50 × 50 × 22 mm in the transverse plane (22 slices), voxel size = 0.14 × 0.14 × 1 mm; TE = 97 ms and TR = 2800 ms and an mDIXON sequence with the same FOV and voxel size; TE = 1.18/2.3 ms, TR = 6.4 ms and a flip angle of 10°. The mDIXON (modified Dixon) sequence employed in this study was developed by Philips researchers; this sequence employs two echoes with unrestricted TE values, a 7-peak fat model, and a B0 correction algorithm to improve fat-free imaging.The purpose of the image processing was to identify fat LC and muscle LC, from which fat infiltration, defined by the ratio between fat LC to the total LC volume and by the fat fraction over muscle LC, was determined.37; two individuals from each group were excluded during this process due to excessive movement.For this purpose, acquired images were converted from DICOM to NIFTI, and the LC was manually segmented in both muscles using open-access ITK-SNAP software38 (an image processing method to separate voxels into two classes) was implemented in MATLAB  and applied to the fat image provided by the mDIXON sequence to separate muscle LC from fat LC in the regions delineated as LC. The fat fraction mapping was obtained employing the water and fat images provided by mDIXON over muscle LC voxels according to the following formula: The Otsu methodU test.The Pingouin 0.5.1 library developed in Python for image processing results was used for statistical analysis. First, the Shapiro–Wilk test and Levene’s test, respectively, were applied to determine the normality and homoscedasticity of the distributions. ANOVA tests were performed using ANOVA for comparisons if both groups showed homoscedasticity and Welch’s ANOVA otherwise. In cases where the samples of either group did not follow a normal distribution, differences between control and DM1 model mice were assessed with a 2-tailed Mann‒Whitney


Porcine circovirus type 2 (PCV2) is one of the most relevant infectious agents affecting domestic pigs. Recently, a surprising PCV2 genetic heterogenicity has been reported in Africa. Nevertheless, the knowledge of the epidemiology of PCV2 in African countries, in both domestic and wild species, is limited and sparse. Having this in mind, in the present study, the PCV2 circulation and its molecular epidemiology in Southwestern Ethiopia have been investigated by collecting 64 samples from domestic pigs, wild boars, and warthogs. PCV2 genome presence was detected and quantified using qPCR and ORF2 sequencing was attempted on positive samples. Ten samples, 8 wild boars, 1 domestic pig, and 1 warthog, tested PCV2 positive. Complete ORF2 sequences were obtained from 5 wild boars; 4 of those were classified as PCV2d and 1 as PCV2b. Both PCV2b and PCV2d were related to strains of Asian origin, most commonly from China. The role of this country in the exportation of PCV2 strains in Ethiopia, and Africa in general, might be supported by the crescent economic relationship between the two continents. The obtained evidence also testifies to the inadequacy and/or poor application of biosecurity measures separating wild and domestic animals. Further, extensive and systematic studies should be performed to more deeply characterize the molecular epidemiology of PCV2 in this region, in order to improve our understanding of these ecological niches in the evolution and dispersal of PCV2. Porcine circovirus type 2 (PCV2) is one of the most relevant infectious agents affecting domestic pigs. It has been associated with several clinical conditions, collectively named porcine circovirus diseases (PCVD), ranging from systemic disease (PCV2-SD), which includes what was initially described as PCV2-associated pneumonia and PCV2-associated enteritis, reproductive disorders (PCV2-RD), and porcine dermatitis and nephropathy syndrome (PDNS). Subclinical infection (PCV2-SI) is also considered part of PCVD since, although not featured by overt clinical signs, it leads to a decrease in productive performance and often represents a source of economic losses for the farmer, even higher than the clinical forms –4.PCVD is a typical example of a multifactorial disease, requiring the interaction among the pathogen, host, and environment to hesitate in clinical or productive effects. The change and intensification in the swine production system, typical of modern farming in high-income countries, clearly played a pivotal role . In fact−3–10−4 substitution/site/year) and recombination rate, leading to remarkable genetic and phenotypic variability [in silico, just a few have been properly characterized; ORF1 and ORF2 in particular have been more extensively investigated. ORF1 encodes 2 proteins (Rep and Rep') involved in rolling circle replication, while ORF2 encodes Cap, the only constituent of the viral capsid, that is involved in the viral attachment and is the main target of host immune response [Sus scrofa), and warthogs (Phacochoerus africanus) in Southwestern Ethiopia.PCV2 is a single-stranded DNA virus (ssDNA) with a circular genome of about 1.76 kb. Similarly to other ssDNA viruses, it is characterized by a high mutation  in Southwestern Ethiopia. None of the herds reported health problems.Samples from free-living adult wild boars were collected from the Buno Bedele zone in Dega and Dabo Hana districts in Southwestern Ethiopia from freshly shouted wild boars by local hunters for human consumption or by farmers to protect their cropland. The post-mortem examination was carried out in the nearby regional veterinary clinic laboratory. The liver, kidney, heart, spleen, and lung were collected after an aseptic incision and applied to the Whatman® FTA® card technology (WhatmanTM) and allowed to dry for 30 minutes at room temperature.g for 5 min. 125 µL of each of the obtained solutions were applied to one of the four FTA card sample areas and dried for 30 minutes at room temperature. The FTA cards were delivered to the laboratory of infectious disease of the dept. MAPS, Padua University, for PCV2 infection diagnosis and molecular characterization.In the Dinsho national park, Bale zone, Southwestern, Ethiopia, healthy warthog's fresh feces samples (∼ 10 g) were collected from the top of a pile of freshly passed feces. Stool suspensions (10%) were made in PBS pH 7.2 and centrifuged at 8000 × µL of PBS and incubated at room temperature for 1 h while continuous vortexing. A volume of 100 µL of the supernatant was extracted using the kit Viral DNA/RNA (A&A Biotechnology) according to manufacturer instructions. An exogenous internal control, provided by the QuantiNova Pathogen+ IC kit (Qiagen), was added before extraction.From each of the 4 FTA card sample areas, a strip of 1 cm (height) × 0.3 mm (width) was trimmed. The 4 obtained strips were pooled and mixed in 500 https://www.geospiza.com) and consensus sequences were generated using ChromasPro . The occurrence of double peaks, which could be suggestive of co-infections with multiple genotypes, was assessed both by visual inspection and using the dedicated software Poly Peak Parser [The PCV2 genome presence was detected and quantified using the qPCR assay described in Franzo et al., . Completk Parser . Finallyk Parser . In morek Parser  and a phk Parser .Finally, to preliminary assess the relationship with other countries, a complete dataset of more than seven thousand and five hundred ORF2 sequences with known collection countries were downloaded from GenBank and aligned with the reference and Ethiopian ones. Poorly aligned reads, those with premature stop codons or unknown bases, were removed from the alignment. Thereafter, a phylogenetic tree was reconstructed using FastTree2 .Suidae located in Oromia regional state; in brief, 40 samples were collected from wild boars (n = 40) from Buno Bedele and 8 samples (n = 8) were collected from domestic pigs from the intensive farm of Jimma University, College of Agriculture and Veterinary Medicine in Southwestern Ethiopia; 16 samples were collected from warthogs from Bale zone, Dinsho park in Southwestern Ethiopia. All animals were in good clinical condition, and no macroscopic lesions were detected in wild boars. Ten samples tested PCV2 positive  were part of different clades. However, in all instances, they demonstrated a close relationship with Chinese sequences  or the less intensive farming condition compared to high-income countries removed most of the co-factors that are typically required for high viral replication and clinical sign development. If pathogen-related factors, e.g. a lower virulence, were also involved remains to be established. A previous study reported the detection of PCV2a and PCV2b genotypes in Ethiopia ; unfortuSince the previous sequences originated from samples collected in 2014, it could be speculated that the higher PCV2d frequency might reflect the worldwide genotype shift . HoweverWarthogs were also confirmed to be susceptible to the infection. In a recent Namibian study, a surprisingly high frequency of PCV2c was proven in this species . If a hoThe comparison of the obtained sequences with a broad international dataset revealed a quite complex scenario. The PCV2b strain clustered with strains reported in Europe and Asia. The wide spread and dispersal of this genotype at the global level impede the identification of the precise source of introduction.However, a previous study estimated a likely PCV2b introduction in Ethiopia from Asia . TherefoOverall, the present study confirms the relevant PCV2 infection frequency in different suid hosts in Africa, both domestic and wild ones, and further extends the knowledge of the genetic variability featuring Ethiopia, where all major PCV2 genotypes have been detected. A pivotal role of foreign strain importation, mainly from Asia, has also been proven and should suggest the implementation of more effective control measures to limit such a phenomenon. Furthermore, extensive and systematic studies should be performed to more deeply characterize the molecular epidemiology of PCV2 in this region and improve our understanding of these ecological niches in the evolution and dispersal of PCV2.


Populus canadensis was impaired when PHE concentration increased above 400 mg kg−1. Values of alpha-diversity indicators of fungal diversity and richness were not affected by the PHE gradient. The PHE contamination had a stronger impact on the fungal community composition in the soil and root compartments compared to that of the aboveground organs. Most of the indicator species whose relative abundance was correlated with PHE contamination decreased along the gradient indicating a toxic effect of PHE on these fungal OTUs . However, the relative abundance of some OTUs such as Cadophora, Alternaria and Aspergillus, potentially linked to PHE degradation or being plant-beneficial taxa, increased along the gradient. Finally, this study allowed a deeper understanding of the dual response of plant and fungal communities in the case of a soil PAH contamination gradient leading to new perspectives on fungal assisted phytoremediation.Microbial populations associated to poplar are well described in non-contaminated and metal-contaminated environments but more poorly in the context of polycyclic aromatic hydrocarbon (PAH) contamination. This study aimed to understand how a gradient of phenanthrene (PHE) contamination affects poplar growth and the fungal microbiome in both soil and plant endosphere . Plant growth and fitness parameters indicated that the growth of  Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants produced during incomplete combustion of organic material and generated from numerous anthropogenic industrial sources. Many PAHs have toxic, mutagenic and/or carcinogenic properties, and 16 PAH compounds are listed as priority pollutants by the United States Environmental Protection Agency (US-EPA) due to their high persistence and toxicity in the environment ,3.The combined use of plants and their associated microbes is an appealing method for the ecological remediation of PAH-contaminated sites. This process called phytoremediation, which is assisted by microorganisms, makes use of the naturally occurring processes by which plants and their associated microbiome degrade pollutants . Some stBecause of their toxicity, we could hypothesize that PAH contaminants will induce a selection of microorganisms present in the soil and of the ones colonizing plant tissues, leading to a modification of the microbiome diversity. Only few studies have assessed plant-associated microbial taxonomic diversity and community shift in hydrocarbon contaminated sites . Most ofEndophytic microorganisms, which are defined by any microbial genetic material found inside plant tissues , are alsPoplar has been extensively studied for its phytoremediation capability due to an important root surface and high tolerance to a wide range of pollutants . Poplar With the aim to study the impact of PAHs on plant growth and physiology and on associated fungal communities, an experimental design with a PAH contamination gradient was set up. We hypothesized that along a PAH gradient, the impact of the pollutant on the plant growth and fitness would be correlated to the level of the contaminant, and that PAH contamination would shape the soil microbiome by selecting most tolerant microbes at the expense of sensitive ones. Concerning the endophytic microbiome diversity, it would be shaped both by the plant fitness and by the level of pollutant translocated through the plant tissue. The main objective of our study was therefore to understand how an increasing range of PAH contamination  affects poplar growth parameters and the diversity of the fungal microbiome in soil and the plant endosphere .P. canadensis roots was explored in both young and aged roots subjected to a PHE gradient  and highly spiked ones (700–1500 mg PHE kg−1), respectively (−1), we observed a high diversity of fungal structures such as typical hyphae, vesicles, arbuscular and Olpidium-like structures (−1), a less important diversity of fungal structures was observed with mainly typical hyphae and sclerotia-like structures . The stem biomass was not significantly impacted by the PHE gradient, whereas stem height was significantly lower for concentrations above 700 mg kg−1 (−1 soil).After four weeks of growth, the impact of the PHE toxicity on poplar fitness and growth was assessed. A significant decrease in leaf biomass was observed for concentrations above 400 mg kg mg kg−1 a,b compa−1 soil, compared to the lower-spiked conditions (0–100 mg PHE kg−1 soil). No significant differences were detected for anthocyan and flavanol contents in the leaves (−1 soil conditions compared to low-spiked conditions (0–100 mg PHE kg−1 soil)  .−1 soil. The initial PHE concentrations (T0) before the plant installation and also the final PHE concentrations after the four weeks of plant growth (T1) were measured, and the percentage of PHE dissipated calculated  were significantly higher  than the two highest (1500 and 2000 mg PHE kg−1) concentrations with a dissipation percentage of 70.5% and 71.1%, respectively. The remaining PHE concentration at T1 was significantly different in all PHE conditions still representing a gradient. The remaining PHE was also tested for its bioavailability (data not shown). The bioavailable fraction represented 12.1%, 42.8% and 51.9% of the PHE in the conditions spiked with 1000, 1500 and 2000 mg PHE kg−1, respectively, while it represented only 2.0 to 2.6% of the remaining PHE in the low-spiked conditions (0–200 mg PHE kg−1). The PHE concentration recovered in the roots and leaves was also measured. PHE concentration in the plant was proportional to that of the soil. For the two highest PHE soil concentrations (1500 and 2000 mg PHE kg−1), we found up to 25.4 and 52.5 mg PHE kg−1 plant FW in the roots and 1.9 and 2.3 mg PHE kg−1 plant FW in the leaves, respectively. At these two concentrations it represented 2.2% and 3.4% of the initial measured PHE concentration present in the soil transferred to the roots, respectively, whereas for the leaves it represented only 0.16% and 0.15% of the initial measured spiked concentration, respectively. For every PHE condition there was between 15 to 20 times more PHE found in the roots compared to the leaves.The dissipation percentage of three intermediate PHE conditions . After quality filtering and plant reads discarding, a total of 633,043, 669,654, 406,252 and 22,205 fungal ITS sequences from soil, roots, stems and leaves were kept for analysis, respectively. To compare the fungal richness and diversity among samples, data were rarefied to 22,188, 23,245, 3931 and 138 reads per sample for soil, roots, stems and leaves, respectively. Rarefaction curves indicated that the sequencing depths were sufficient to reliably describe the fungal microbiome for each compartment . A total−1) and high (1000 and 2000 mg PHE kg−1) PHE conditions. The Shannon diversity indices  was different among plant compartments , and was indices  were sig indices . Similar indices .p = 0.001, R2 = 0.364) and root  compartments. The PHE concentration explained 36 and 34% of the variance in the fungal communities from the soil and roots, respectively. No significant effect of the PHE concentration was observed on the community composition from the stem and leaf compartments .Three-dimensional non-metric multidimensional scaling (NMDS) analysis from the Bray-Curtis dissimilarity matrix was performed to compare the fungal community composition of the four different compartments  as a function of the PHE concentration . PERMANOBasidiomycota and Ascomycota were negatively correlated to each other . In the stem and leaf compartments, Pleosporales was the most abundant order  and in stems it was positively correlated to the PHE concentration (Pearson correlation r = 0.43), contrary to the root compartment. A higher proportion of Basidiomycota was detected in leaves compared to other compartments. The proportion of unclassified fungi was positively correlated to the PHE concentration (Pearson correlation r = 0.37).Fungal community diversity was explored at the phylum, class, order and genera levels. When considering the top 15 most abundant orders for each compartment, they belonged to six different phyla, 15 classes and 26 orders . The remch other . The relchoderma . For theFungal communities were explored concerning their trophic mode and how it was affected throughout the PHE contamination gradient. A trophic mode could be attributed to 14.9% to 52.6% of the fungal diversity depending on the condition . No significant difference could be observed for any trophic mode across the four compartments studied and the PHE gradient .−1 except for one decreasing from 1000 mg PHE kg−1. These OTUs belonged to the Sordariales , to unidentified fungi, and the last to Pezizales (Trichophaea flavobrunnea). The two last soil IS belonged to Chytridiales and Chaetothyriales, with RAs increasing along the gradient from 300–400 mg PHE kg−1. Eight of the IS for the root compartment had their RA decreasing along the PHE gradient at diverse change points from 150 to 1250 mg PHE kg−1 and half belonged to the Pleosporales, one to Hypocreales, and one to Atheliales. Only one IS for the roots belonging to Hypocreales (Clonostachys sp.) had its RA increasing with the PHE concentration (change point at 700 mg PHE kg−1). In the stems, one OTU was identified as IS; it belonged to the Dothideales and its RA, representing 0.3% at 0 mg PHE kg−1, increased up to 54% from 0 to 1000 mg PHE kg−1 and then decreased (change point at 1250 mg PHE kg−1) to 6% at 2000 mg PHE kg−1.We searched for indicator species (IS) having their relative abundance increasing or decreasing along the PHE gradient in the four compartments studied . We idenWe explored the impact of a PAH contamination gradient on poplar endophyte fungal diversity. Two hypotheses were made. First, the soil community diversity directly exposed to PAH will be modified and as soil microbial communities is the first reservoir of endophyte communities , the PAH−1), but it did not increase any further with the gradient. We deliberately limited the PHE concentration to 2000 mg kg−1 to prevent an excessive initial toxicity that would be lethal to the plants (preliminary data not shown). PAH contamination reduced root and leaf biomass, a finding already reported in several studies performed on various plant species in controlled conditions [Arabidopsis and wheat seedlings in controlled and sterile environments [In our study, we observed a significant impact of the PHE contamination on the measured plant parameters after a certain concentration  as they were already described as potential hydrocarbon degraders [The overall abundance of Sordariales in the soil was positively correlated to the contamination gradient (even though the abundance of some IS decreased). This order was already described in a PAH-contaminated soil and its abundance was also positively correlated to the contamination level . Moreovestresses ,43,44,45egraders ,47,48.Colletotrichum, composed of mainly opportunistic phytopathogens species [Glomerellales and its associated genus  species , were po species .Paraphoma sp. were identified in our study [We observed a majority of indicator species belonging to the soil and root compartments, confirming that the impact of the PHE contamination was mainly found in the belowground habitats. Moreover, we observed that most of the indicator species had their relative abundance decreasing when the contamination increased. This finding could indicate that the contamination had a toxic effect on fungal communities at the OTU level. In the root compartment, several IS belonging to the Pleosporales for which some species were evidenced as PGP fungi like ur study . If PHE We also investigated the fungal communities in the aboveground compartments (stems and leaves). Indeed, this was reported in the present study for poplar and previously in 12 other plant species . PHE canOculimacula was negatively correlated in both stems and leaves. Pleosporales have often been reported in environments with high hydrocarbon contamination [Oculimacula species, belonging to dark septate endophytes, have been isolated from extreme environments . Species belonging to Dothideales have not usually been found in hydrocarbon contaminated sites, even if they were found in environments under other abiotic stresses like high salinity or extreme temperatures. These heavily melanized fungi are usually highly resistant to abiotic stresses [−1 of PHE, its impact is very limited on the fungal communities in the aboveground habitats.A single indicator species (OTU7), belonging to Dothideales, was significantly correlated to the PHE gradient in the stems and was the major fungal taxa in several PHE conditions  concentration was 11.7 ± 1.6 mg kg−1 DW.The soil was sampled at 2–20 cm depth in a natural attenuation area of a brownfield site that formerly hosted a coking plant during the 20th century . Soil was air-dried and sieved to 2.4 mm and stored at room temperature until experimental set up. Physico-chemical analyses were performed at the Laboratoire d’Analyses des Sols . It is a sandy soil , with a pHw/w) sand to increase its porosity and spiked with increasing phenanthrene (PHE) concentrations. The soil was spiked with PHE to reach final nominal concentrations of 0, 100, 200, 400, 700, 1000, 1500 and 2000 mg kg−1 DW soil. Aliquots of 100 g DW soil were prepared and a PHE-hexane solution was added to 1:10 of the soil aliquots for spiking (for 0 PHE condition soil was spiked with hexane without PHE). Hexane was evaporated under a fume hood for 48 h and then the spiked soil was homogenized with the remaining 9:10 of the soil aliquot. The mixed spiked soil was used for microcosm experiments.To set up microcosms, the soil was mixed with 10% , with equal size (1 cm diameter and 10 cm height), were grown hydroponically in perlite and tap water for four weeks until rooting (3 to 10 cm long roots) was observed. The cuttings were then transplanted into the soil of the microcosms. A first experiment was conducted to explore the fungal colonization of roots through microscopy. In this experiment, only five PHE concentrations were used to reach final nominal concentrations of 0, 200, 700, 1000 and 1500 mg kg−1 DW soil. Plants were grown in triplicate in pots containing three cuttings per pot. The second experiment consisted of microcosms conducted with the eight different PHE concentrations. Soil was added to 100 mL glass beakers and wrapped with aluminum foil. Four replicates per PHE concentration were used, representing a total of 32 microcosms. One poplar cutting per microcosm was planted, and soil was watered to 100% of its water retention capacity (WRC) for the first three days and then maintained at 70% WRC using tap water. The experiments were conducted for four weeks in a growth chamber under controlled conditions . After four weeks of growth, before plant harvest, the flavanol, anthocyanin and chlorophyll contents were measured using a Dualex FORCE-A  on the second row of leaves.The plant cuttings of ®488  solution in 1X PBS and incubated at room temperature for 30 min. Finally, the roots were washed in distilled water and mounted between slides and cover slips with a drop of 20% glycerol for observation under an optical/epifluorescence microscope (Nikon Eclipse 80i). Light microscopy was used to investigate for Trypan blue-stained mycorrhizal and melanized fungi and fluorescent observation was done to visualize fungal cell walls stained with WGA . The fungal colonization percentage was calculated using Trouvelot’s method [Fungal root colonization was assessed through two different dyes (Trypan blue and WGA) on poplar roots from the first experiment. After microcosm harvesting, the root samples were washed three times with distilled water and stored in 70% ethanol. Apex (<1 mm diameter) and aged roots (1–2 mm diameter) were incubated at 95 °C in 10% (m/v) KOH for 20 min. After three washes in distilled water, root systems were incubated for 4 h with 1% HCl. The HCl was removed after three washes with distilled water. The root system was soaked in Trypan Blue (0.1%) and incubated for 20 min at 90 °C. After three washes with distilled water, the roots were incubated overnight in 20% acidified glycerol (1% HCl). After solution removal, the roots were then stained with a second dye using 50 µg/mL WGA-AFs method  on an avAfter four weeks of growth, the number of leaves and the size of the plants of each microcosm were measured. The plants were removed from the pots and the rhizospheric soil was recovered by slightly shaking the roots. Rhizospheric soil was stored at −80 °C in glassware. Roots, stems and leaves were collected separately, the remaining soil adhering to the roots was gently removed with a fine paintbrush and plant materials were surface sterilized . Plant material was gently soaked with paper towels, weighted and directly flash-frozen and kept at −80 °C until DNA extraction and PHE analysis. Root, stem and leaf samples were crushed in liquid nitrogen for homogenization before use for further analyses.0 phenanthrene spiking right after mixed spiked soil preparation and served as controls of the measured initial PHE concentration. Moreover, rhizosphere soil samples (T1 after 4 weeks) were also analyzed. All samples were freeze-dried and ground. PHE was extracted from 2 g DW soil using dichloromethane (DCM) and a high-pressure (100 bars) and high-temperature (130 °C) DIONEX® 200 ASE (Accelerated Solvent Extractor) automated extractor. DCM was evaporated from the PAH extracts under nitrogen flow and replaced by acetonitrile for PAH analysis. PAH extracts were analyzed using a reverse-phase chromatography UHPLC Ultimate 3000 RSLC system (Dionex) equipped with a Diode Array Detector and a Zorbax Eclipse PAH RRHD column . The mobile phase was a mixture of water/acetonitrile in gradient mode, with a flow rate of 0.42 mL min−1. The wavelength used for detection was 254 nm.For each PHE condition of the second experiment, three replicates were directly stored at −80 °C to analyze TThe bioavailable soil PHE concentration was measured from 1 g of soil (fresh weight) with 50 mM hydroxypropyl-β-cyclodextrin . After two liquid:liquid extractions with dichloromethane, the extracts were pooled and evaporated under nitrogen flux and resuspended in acetonitrile. The extracts were filtered at 0.45 µm  and analysed by UHPLC as described above.PHE concentration was also measured in plant tissues. Two hundred mg of frozen powder of the roots and leaves were extracted with 1 mL of methanol at −20 °C over 16 h. After centrifugation , supernatants were dried in a speed vacuum system. A second extraction with 1 mL of ethyl acetate was performed and the supernatant was added to the first pellet, dried and resuspended in 300 µL of methanol. Samples (1 µL) were analyzed by GC-MS  with He carrier gas inlet pressure programmed for constant flow of 1 mL/min and mass spectrometric detector . The injector temperature was set at 310 °C with a pressure pulse . GC was carried out with temperature programmed injection at 70 °C, oven 0.7 min at 70 °C, raised by 30 °C/min to 180 °C and then 10 °C/min to 300 °C and held for 9 min at 300 °C. Absolute quantification was achieved by comparison of sample signals with a dose–response curve established with pure phenanthrene. Compound specific detection was performed in Multiple Reaction Monitoring mode using transitions 178 > 152 and 178 > 176, with collision energy 30 V. The data analysis was performed with the Bruker MS Data Review software (ver 8.2.1).Populus species [DNA was extracted from approximatively 50 mg of powder of roots, stems and leaves using the DNeasy Plant Mini Kit . DNA from approximatively 300 mg fresh weight of rhizosphere soil was extracted using the FastDNA Spin Kit for soil . DNA concentration and quality were estimated using a NanoDrop™ One spectrophotometer . A two-step PCR approach was used to barcode-tag templates. For the first PCR, ITS3 and ITS4 primers (0.2 µM) targeting the fungal ITS2 rRNA region and merged to specific tags for the second PCR, were used . To inhi species . A high-rarecurve in the package Vegan v2.5-7 in R (version 4.1.0) [−1 DW soil, two for roots at 700 and 2000 mg PHE kg−1 DW soil, three for stems at 100, 1500 and 2000 mg PHE kg−1 DW soil, and one for leaves at 1000 mg PHE kg−1 DW soil). The Alpha diversity indices  were calculated using the Phyloseq package, which was also used for fungal community composition and structure analysis. Each fungal OTU was further assigned to trophic groups of fungi using the FungalTraits database [Reads were processed using FROGS (Bioinfo Genotoul) . The Fasn 4.1.0) .Taxonomin 4.1.0)  to achiedatabase . For eacp < 0.05) followed by Dunn’s post-hoc test or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test when non-parametric or parametric distribution (after Shapiro-Wilk test) were observed, respectively.Statistical analyses and data representations were performed using R software . Plant growth parameters, PHE concentration and fungal colonization data were compared by using a Kruskal-Wallis test  was compared among samples using nonmetric multidimensional scaling analysis (NMDS) based on Bray-Curtis dissimilarity matrices, analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA) using the pairwise Adonis package .corrplot package on R [p < 0.05 for significant correlation. A Venn diagram was made using the Jvenn plug-in from the INRAE Toulouse [Average relative abundances (RAs) of taxonomic groups were achieved by averaging three to four replicates. These mean RA values were used to estimate differential abundance of taxonomic groups depending on PHE concentration using Kruskal-Wallis followed by the Benjamini-Hochberg false discovery rate (FDR) correction (non-parametric distribution of the data was checked with a Shapiro-Wilk test). Correlation matrices were made using the age on R  and usinToulouse . RAs of Toulouse  package This study, thanks to an innovative experimental design (multiple PHE concentration and thee plant organs studied), has provided a singular view into poplar microbiome in a PAH-contaminated environment. We found out that the impact of the phenanthrene contamination on the fungal communities was different depending on the compartment studied. We observed a toxic effect of the contamination on the belowground habitats,  which resulted in a modification of the community structure, and sensitive species’ abundance was reduced while other species more tolerant and potentially involved in PHE degradation saw their abundance increase. The impact of the PAH contamination was negligible on the aboveground compartments. The endophytes present in the stems and leaves are probably less affected by the direct effect of PHE (limited concentration of PHE in leaves) but could be affected by a shift in the plant physiology due to the response to the contamination. This study highlighted a global overview of a complex fungal-plant system in the case of a soil PAH contamination for the first time. The findings could represent potential advancements for field applications using poplar associated fungi in PAH bioremediation systems. To have a better understanding, it would be interesting to go deeper in the physiological response of the plant to the contamination by investigating the oxidative stress response, as well as by using a multi-omics approach targeting the plant transcriptome, proteome and metabolome. A focus on other microorganisms like bacteria that potentially play a major role in a phytoremediation context could also be of great interest.


Viability results showed a high percentage of viability  of human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVEC) after 24 h extrusion. Additionally, HUVEC and HASMCs were mainly localized in their respective layers, mimicking the native distribution. All in all, this approach allows the direct extrusion of collagen TEBVs in a one-step procedure with enough mechanical properties to be perfused.Cardiovascular diseases are considered one of the worldwide causes of death, with atherosclerosis being the most predominant. Nowadays, the gold standard treatment is blood vessel replacement by bypass surgery; however, autologous source is not always possible. Thereby, tissue-engineered blood vessels (TEBVs) are emerging as a potential alternative source. In terms of composition, collagen has been selected in many occasions to develop TEBVs as it is one of the main extracellular matrix components of arteries. However, it requires specific support or additional processing to maintain the tubular structure and appropriate mechanical properties. Here, we present a method to develop support-free collagen TEBVs with co-axial extrusion in a one-step procedure with high concentrated collagen. The highest concentration of collagen of 20 mg/mL presented a burst pressure of 619.55 ± 48.77 mmHg, being able to withstand perfusion of 10 dynes/cm Cardiovascular diseases (CVDs) are one of the leading causes of death according to the World Health Organization (WHO), representing 32% of all global deaths . One of An ideal TEBV should provide cells with an extracellular matrix (ECM) similar to that of native tissue, mainly collagen, while allowing cells to spread throughout the structure in low periods of time to provide the required functionality to the TEBV . GeneralHere, we aim to develop tissue-engineered blood vessel (TEBV)-like structures by using high concentrations of collagen both in the inner and outer layer through co-axial extrusion method to allow encapsulating SMC and human umbilical vein endothelial cells (HUVEC) in two distinct layers, both being collagen-based. To this purpose, we will assess the ability of the collagen concentrations to provide a self-supporting structure. The cell survival ability of both cell types after extrusion will be tested in their respective vascular layers while maintaining the initial allocation. To the best of our knowledge, the direct extrusion of solely collagen with vascular cells for TEBV development has not been reported before with the co-axial extrusion method.Both 10 and 20 mg/mL of collagen could be dissolved, obtaining a homogenous viscous solution with a gelling time of 10–15 min at 37 °C. As a first step, the choice of the extrusion bath for the extrusion of collagen TEBV was studied. Then, the effect of the injection speed on the size of TEBVS and which one facilitated the extrusion process was investigated.On the one hand, regarding the extrusion baths, gelatin microparticles size was around 100 µm, as illustrated in On the other hand, the results of TEBVs sizes  depending on the injection speed and collagen concentration are represented in As the triple co-axial nozzle had to be moved manually along the petri dish during extrusion, the 25 mL/h injection speed was selected for subsequent experiments. This injection speed allowed more uniform control of the movement of the triple co-axial nozzle, facilitating the formation of straight TEBVs, whereas with 50 mL/h, this fact was not possible, and for the 10 mL/h, the time needed to extrude the TEBV was significantly increased.2. These TEBVs demonstrated their ability to withstand physiological mechanical stimulation without rupture . Afterwards, lyophilized collagen was cut into small pieces and dissolved to the desired concentration with ice cold 50 mM acetic acid. The concentrations used were 10 and 20 mg/mL.For the extrusion of TEBV, we aimed to test two different support baths: (1) gelatin microparticles, and (2) cell culture media alone  ). The purpose of testing gelatin microparticles slurry as a support bath was to ensure structural support to the extruded collagen, allowing its gelification without spreading. We also wanted to test if collagen could maintain its structure once extruded into EGM-2 medium without the need for structural support, as there is previous evidence that this might be possible .w/v) gelatin Type B , 0.25% (w/v) Pluronic F-127  and 0.1% (w/v) gum arabic  in 1 L of 50% (v/v) ethanol  was heated to 45 °C. The pH was adjusted to 6.25 with 1 M hydrochloric acid (HCl). Then, the beaker was placed on stirrer , sealed with parafilm to avoid solution evaporation and cooled at room temperature while stirring overnight for microparticle formation. To compact the gelatin microparticles, the slurry was divided with 50 mL conical tubes and centrifuged at 300 g for 5 min. After removing the supernatant, the microparticles were resuspended with 1x phosphate buffered saline (1x PBS) at pH 7.4 (washing solution), and centrifuged at 1000 g for 2 min. This washing step was repeated three times. Then, the gelatin microparticle slurry was kept at 4 °C with 1x PBS in its uncompacted state. Prior to TEBV extrusion, gelatin microparticles were compacted by centrifuging them at 2000 g for 5 min. Supernatant was removed and gelatin slurry was transferred into a petri dish.Gelatin microparticle formation was developed following a previously published method . First, Schematic representation of the method is shown in Three injection speeds were tested  to allow the effect of the injection speed on the general parameters of the blood vessel-like structure, mainly shell thickness and inner and outer diameter, to be studied. Besides the effect on the size of the vessel, speed also played a significant role in allowing the deposition of the TEBV at an adequate rate that allows manipulation and proper movement see Bii–iii, Burst pressure is defined as the maximum pressure that a TEBV can withstand before rupture. In order to measure it, TEBVs with the highest concentration (20 mg/mL) of collagen were attached in the perfusion chambers with one end sealed and the other connected to a differential pressure gauge . PBS was injected through TEBVs until failure, and the maximum pressure achieved was recorded. Burst pressure was performed with cell-free TEBVs to record the basal mechanical properties. A total of three TEBVs were analyzed.2 to perform functional assays in the future. To this purpose, we calculated the appropriate flow rate to provide a shear stress of 10 dynes/cm2 according to our TEBVs size with Poiseuille formula: SS is the shear stress, µ is the viscosity of the fluid, Q is the fluid rate, and r is the radius of the vessel. As a proof of concept, we tested if they could withstand physiological arterial shear stress with cell free TEBV containing collagen at a concentration of 20 mg/mL and perfuse with endothelial cell growth medium-2 . The perfusion system consisted of a closed tubing loop which connected the cell culture media reservoir with the perfusion chamber containing the TEBVs. The flow rate was achieved by using a peristaltic pump  connected with a head pump .After measuring the burst pressure of the TEBVs, we wanted to check if these TEBVs were able to withstand physiological flow rate with an arterial shear stress of 10 dynes/cm2. HASMCs were seeded with vascular cell basal medium (ATCC) supplemented with vascular smooth muscle cell growth kit (ATCC) at a cell density of 2500 cells/cm2. Both cell types were passaged using 0.25% Trypsin-EDTA  when they reached 70–85% confluence. Both cell types were cultured at 37 °C and 5% CO2.For the encapsulation of vascular cells within TEBV structures, human umbilical vein endothelial cells (HUVECs) (Lonza) and human aortic smooth muscle cells (HASMCs) (ATCC) were used. For their expansion, HUVEC were seeded with endothelial growth medium-2 bulletkit (EGM-2) (Lonza) at a cell density of 2500 cells/cm6 cells/mL in the middle collagen syringe, and HASMCs at a concentration of 10 × 106 cells/mL in the outer collagen syringe.For cell encapsulation, the procedure described in Our initial hypothesis is that the increase of collagen concentration increases the stability of the TEBV. However, there is uncertainty whether encapsulated cells would be able to survive with higher concentrations and the effect of the shear stress produced by the extrusion method. For this reason, we aimed to assess the viability of HUVEC and HASMCs when encapsulated within TEBVs with two collagen concentrations: 10 and 20 mg/mL. To this purpose, a LIVE/DEAD cell imaging kit was used following the manufacturer’s instructions (Invitrogen). Briefly, live cells were stained with a non-fluorescent cell-permeant component  that was converted by intracellular enzymes to fluorescence Calcein AM, giving a green fluorescence signal at 488/515 nm (excitation/emission wavelengths), and dead cells were stained with a cell-impermeant component (BOBO-3 iodide) as it enters within cells with a compromised cell membrane, producing a red nuclear fluorescence at 570/602 nm (excitation/emission wavelengths). Both components were added at equal volumes in the cell culture medium with TEBVs, with a final concentration of 1x. Cells were imaged with a laser scanning confocal microscope  after 15 min of incubation in the dark. For the viability analysis, three samples per time point were analyzed, and three captures at different plans per sample were acquired. Live and dead cells were counted from each image using ImageJ software (ImageJ 1.52 a). Viability was calculated as a percentage, as the number of live cells divided per total number of live and dead cells. The viability results determined which concentration of collagen was used for the subsequent experiments.In order to check if HUVEC and HASMCs could remain in their respective layers once extruded, inner and outer layers, respectively, each cell type was stained with different colored cell tracker fluorescence probes prior to the development of TEBV structures. HUVEC cells were stained with Green CMFDA  and HASMC were stained with Red CMTPX (Invitrogen), emitting a green and red signal, respectively. The final concentration in the cell culture media of both cell trackers was 10 µM. After adding each cell tracker into its respective cell culture media, cells were incubated with them for 45 min at 37 °C, while protected from light. For blood vessel formation, cells were added at each specific syringe, as explained in n = 8. A p-value of less than 0.05 was considered statistically significant.Statistical analysis was performed using SPSS software . Kruskal–Wallis and Mann–Whitney U non-parametric tests were used to compare if there were differences between the outer diameter, inner diameter and wall thickness of TEBVs extruded with two different collagen concentrations for each injection speed. Data was represented as mean ± standard deviation;