Despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. This could be due to pathogens not tested for or novel divergent viruses of potential animal origin. Fecal virome analyses of Cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. This is the first attempt to describe the gut virome of humans from Cameroon. Therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. The studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. Hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area. Adenoviridae, Astroviridae, Caliciviridae, Picornaviridae, and Reoviridae. Interestingly, a mammalian orthoreovirus, picobirnaviruses, a smacovirus, and a pecovirus were also found. Although there was no evidence of interspecies transmission of the most common human gastroenteritis-related viruses , the phylogenies of the identified orthoreovirus, picobirnavirus, and smacovirus indicate a genetic relatedness of these viruses identified in stools of humans and those of bats and/or other animals. These findings points out the possibility of interspecies transmission or simply a shared host of these viruses  present in both animals (bats) and humans. Further screening of bat viruses in humans or vice versa will elucidate the epidemiological potential threats of animal viruses to human health. Furthermore, this study showed a huge diversity of highly divergent novel phages, thereby expanding the existing phageome considerably.Diarrhea remains one of the most common causes of deaths in children. A limited number of studies have investigated the prevalence of enteric pathogens in Cameroon, and as in many other African countries, the cause of many diarrheal episodes remains unexplained. A proportion of these unknown cases of diarrhea are likely caused by yet-unidentified viral agents, some of which could be the result of (recent) interspecies transmission from animal reservoirs, like bats. Using viral metagenomics, we screened fecal samples of 221 humans  between 0 and 89 years of age with different degrees of bat contact. We identified viruses belonging to families that are known to cause gastroenteritis such as IMPORTANCE Despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. This could be due to pathogens not tested for or novel divergent viruses of potential animal origin. Fecal virome analyses of Cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. This is the first attempt to describe the gut virome of humans from Cameroon. Therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. The studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. Hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area. Diarrhea is the second most common cause of death worldwide and accounts for about 8 to 9% of the 5.9 million yearly deaths in children under the age of 5 , 2. MostTransmission of these enteric viruses is predominantly fecal-oral, and humans are constantly exposed to these viruses through various routes . One of 13–In the Southwest region of Cameroon, bats are hunted and eaten. Such close interactions provide ample opportunity for zoonotic events to occur .Previously, we identified a plethora of known and novel eukaryotic viruses in Cameroonian fruit bats using a viral metagenomics approach, including viruses known to cause gastroenteritis in humans  and those not yet associated with gastroenteritis (bastrovirus and picobirna-like viruses) –23. In t–A total of 221 human fecal samples (131 from Kumba and 90 from Lysoka) were collected from two hospitals in the Southwest region of Cameroon, for viral metagenomics screening. From these fecal samples, a total of 63 pools were constituted in categories based on age, bat contact status, and location  Proportion of viral reads in different age categories. (B) Proportion of viral reads in different contact statuses. (C) Proportion of viral reads in different locations. Download Copyright © 2019 Yinda et al.2019Yinda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Astroviridae, Reoviridae, and Anelloviridae , and Picornaviridae (of which about 60% were enteroviruses  strains. Red, Cameroonian human RVA strain identified in this study; blue, Cameroonian bat RVA strains. Trees were constructed using the GTR+G+I nucleotide substitution model using RAxML, with the autoMRE flag, which enables Copyright © 2019 Yinda et al.2019Yinda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Picornaviridae represent a large family of small, cytoplasmic, nonenveloped icosahedral ssRNA viruses consisting of 80 species, grouped into 35 genera. They have a genome of 7.1 to 8.9 kb in size and are most often composed of a single ORF encoding a polyprotein flanked by a 5′ and 3′ UTR , myocarditis, hepatitis, upper respiratory tract infections, and diabetes  consists of 15 species: Enterovirus A to L and Rhinovirus A to C. EV A, B, C, and D are found in humans; E and F in cattle; G in pigs; H, J, and L in monkeys; K in rodents; and species I in dromedary camels (http://www.picornaviridae.com). In this study, eighteen (nearly) complete genomes of EVs were obtained. The strains were named EV/Human/CMRHPxx/CMR/2014, here referred to as EV-CMRHPxx. All eighteen genomes were found in pools of age groups A and B . Eight of these were identified in age group A, three  of which were pools consisting of samples of infants who had indirect contact with bats while the rest  were those that had no contact with bats. The ten other strains were identified in pools belonging to age group B, three of which had direct contact with bats , 5 indirect contact  and two with no contact (EV-CMRHP18 and 58). Based on the phylogenetic analysis of the VP1 nucleotide sequences, the EVs found in this study were quite divergent from each other, belonging to three different species of Enterovirus, A, B, and C , while EV-CMRHP35B, 39, and 45 clustered within the Enterovirus B genotype, and EVCMRHP5A, 5B, 52B, and 58 in the genogroup Enterovirus A. Some pools had multiple strains of EV present, and some of these clustered together (CMRHP8A and 8B: vaccine type PV-3), whereas other pools contained distinct EV species . The presence of vaccine strains (PV-3) in pool HP8 probably indicates recent vaccination events of the infants in this pool. Apart from EV-CMRHP39 (which clustered with 11C52_CMR), all the EV strains identified here were distantly related to those previously identified in the Far North region of Cameroon  and Parechovirus B  (–The genus k voles) . HPeV isk voles) –36. Here voles) –. The HPe voles) –, 38, thiCosavirus consists of five species , which have been associated with gastroenteritis in children  infecting humans. It has been found in cases with acute flaccid paralysis, respiratory tract infections, and diarrhea in China . The Hepatovirus A species is comprised of a single serotype, HAV, subdivided into human and simian viruses (Hepatitis A virus (HAV), now  viruses . It caus viruses . There w viruses . On the  viruses , increasAstroviridae is a family of nonenveloped, spherical viruses with a linear ssRNA(+) genome of 6.8 to 7kb, containing three overlapping ORFs. The family is divided into two genera: genus Mamastrovirus (MAstVs) and genus Avastrovirus (AAstVs). The genera are further divided into 33 and 7 species, respectively . Additionally, these pools were either from children less than 3 years old , age group 3 to <20 , or between 20 and <60 (HP34 and HP43). Phylogenetic analysis of the RdRp and capsid regions (AF260508) clustered with the novel strains CMRHP2, 3, and 35D in the capsid tree, while in the RdRp tree it clustered with the Chinese strain V4-Guangzhou, suggesting a recombination event between these strains in the past. Bat astrovirus identified in Cameroon  genome of 7.3 to 8.3 kb, containing two or three ORFs. The family contains five genera , from people who had indirect (HP1 and HP59) or no (HP18) contact with bats, and from age group A (HP1), B (HP18), or C (HP59). The phylogenetic tree (JF802509]) isolated from the Littoral Region of Cameroon in 2009, whereas strains of genogroup II from the same study  were distantly related to those identified here (II.12 and II.13) , divided into 5 genogroups. Genogroups I, II, and IV infect humans, whereas genogroup III infects bovine species and genogroup V has been isolated from mice . Three ntic tree  showed tSapovirus (SaV) consists of a single species, Sapporo virus. It has been detected in humans, pigs, minks, dogs, sea lions, bats, chimpanzees, rodents, and carnivores  remain(s) enigmatic. The disease association is unclear, but PBV infection has been associated with gastroenteritis in both animals and humans  RdRp sequences of PBVs from these 28 pools. Phylogenetic analysis based on RdRp (ANS53886) from genogroup I clustered together with human strains from Cameroon with an aa identity of 76% with strains CMRHP26A and CMRHP35. Likewise, in genogroup II, strains CMRHP34A, CMRHP63B, and CMRHP26C clustered closely (75 to 76% aa identity) with a Portuguese feline strain (AGZ93689). Intriguingly, the Cameroonian human picobirna-like viruses CMRHP9 and CMRHP10B were 99% identical to a Cameroonian bat strain picobirna-like virus, P11-300, suggesting a possible interspecies transmission. However, their true host has not yet been determined. It could be that the true hosts of PBVs are found in both humans and bats and that this therefore explains their presence in both.Picobirnaviruses (PBVs) belong to the family d humans , 53. Up  on RdRp  revealedKP233190) and human  strains from the United States, respectively. Based on the capsid region, these novel Cameroonian strains were 98 to 99% identical to the chimp strain and only 85% identical to the human strain HuSCV3. The close genetic relatedness of human strains to a strain found in a chimpanzee sample suggest that these viruses infect a host shared between chimps and humans, and if indeed smacovirus infects mammals, this could be a case of interspecies transmission (Smacovirus (SCV) is a relatively recently described virus with a small circular DNA genome with a size of about 2,529 bp. It belongs to the smacovirus group and is an unclassified eukaryotic virus of unknown origin . In thissmission . Phylogesmission  and the smission  indeed sPecoviruses (Peruvian stool-associated circo-like viruses [PeCVs]) are CRESS-DNA genomes that were first identified in the feces of a patient during an outbreak of acute gastroenteritis in the Netherlands and later in samples of Peruvian children , 56. Sub55–Myoviridae (236 contigs), Podoviridae (95 contigs), Siphoviridae (145 contigs), and Microviridae (36 contigs) families. To get insight into the differences in the bacteriophage communities, we compared the VirSorter-identified bacteriophage richness between the different age groups  different age groups, (B) different locations, and (C) bat contact status. Download Copyright © 2019 Yinda et al.2019Yinda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00585-18.9TABLE S2Table S2, PDF file, 0.1 MB.Results of CRISPR sequence BLASTn against the phage community revealing a diverse bacterial community. Download Copyright © 2019 Yinda et al.2019Yinda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In order to visualize the genetic relatedness between the human and bat gut phageome, a recently developed bioinformatics tool (vConTACT) was used. It groups phages based on their genome sequences into viral clusters which correlate rather well with viral genera as defined by the International Committee of Taxonomy of Viruses (ICTV) . A totaln = 221) from Cameroon and to further determine if bat viruses are possible causative agents of gastrointestinal infections in humans.Recently, we thoroughly investigated the gut virome of fruit bats from Cameroon –23, 63 a–Adenoviridae, Astroviridae, Caliciviridae, Picornaviridae, and Reoviridae), viruses that are uncommon causes of gastroenteritis (orthoreovirus), or those that have been identified in humans but not associated with disease .Twenty-four percent of the 471 million generated trimmed reads were assigned as viral. Most of these reads were bacteriophages, which is in accordance with previous studies . The eukPicornaviridae family were identified most frequently , a common viral gastroenteritis-causing agent, was identified only in a limited number of pools. This was previously observed in Cameroon, and possible reasons for the low prevalence could include the acute nature of rotavirus infections or seasonal changes in rotavirus infections . Of noteThis first MORV strain from Cameroon showed topological incongruence in its phylogeny, thereby pointing to possible reassortment events in the past. The phylogenetic clustering of some segments to strains from animals (rodents and bats) could be an indication of a zoonotic event or could also be due to the absence of related strains in databases from an unknown host. Given that this strain was from a pool of samples from two children suffering from severe diarrhea, it is not unlikely that this strain might have contributed to the disease. Therefore, MORV might be playing a greater role in diarrheal diseases in this region than was previously known. Hence, extensive epidemiological studies in different regions and in different hosts are required to fully delineate the prevalence, genomics, and interspecies transmissibility of MORV.Apart from the above-mentioned gastroenteritis-related viruses, several other viruses with unelucidated gastroenteric roles were also identified in this study. First, we observed fewer reads of picobirnaviruses (PBVs) in pools from children than in adults. Previous studies also detected a relatively low percentage of children with PBVs , 68. ThiSecond, for the first time, two strains of African smacovirus (SCV) were identified in Cameroonian samples. Their genetic relatedness to a chimpanzee strain (isolated from a captive chimp in a zoo in San Francisco) and a strain from a child from the United States , 55 indiAnelloviridae have an undefined implication in hosts’ health and are thought to be probably asymptomatic (harmless) or even beneficial. However, they have been associated with hepatitis, pulmonary diseases, hematologic disorders, myopathy, and lupus, but it is not clear if their presence is the cause or the result of disease progression , Caliciviridae (sapovirus), and Reoviridae (RVA) are genetically diverse from those identified in bats from the same region, indicating no evidence of recent interspecies transmissions between bats and humans  and their potential as emerging pathogens in the human population.Ethical authorization for the use of human samples was obtained from the Cameroon National Ethics Committee, Yaoundé. All human experiments were performed in accordance with the Ministry’s National Ethics Committee guidelines.Human fecal samples were collected between February and September 2014, after informed consent was obtained from patients in two different hospitals . This region was chosen because here bats are hunted, sold, and eaten. Diarrheic patients and/or people who came into contact with bats directly  or indirectly (if a family member was directly exposed to bats) were eligible for sampling. A total of 221 samples were collected from subjects between age 0 and <3 years , 3 and <20 , 20 and <60 , and 60 and older . All the samples were from people who had symptoms of gastroenteritis, except 2 from age group C who had contact with bats. Samples were then placed into labeled tubes containing universal transport medium (UTM), placed on dry ice, and stored at −20°C, until being shipped to the Laboratory of Viral Metagenomics, Leuven, Belgium. The samples were stored at −80°C until used . Each pool contained two to five samples, and for the different age groups (A to D) we had 22, 17, 20, and 4 pools, respectively. The pools were then treated according to the NetoVIR protocol . Brieflyde novo assembly or reads and annotation of reads were performed using SPAdes (with the meta flag) and Diamond (with the sensitive option using the GenBank nonredundant database), respectively  and midpoint rooted for purposes of clarity.NGS reads were analyzed as described in the work of Yinda et al. , 63. Briectively , 87, 88.ectively . Nucleotectively  or MAFFTectively . Substitectively , and phyectively , with thhttp://ecogenomics.github.io/BamM/). Abundance tables were obtained and normalized for total number of reads of each sample. For the richness comparison, Mann-Whitney tests were used, and for the clustering, an Adonis test was performed. All downstream analyses were done in R . Only sone in R  using thone in R . Furtherone in R  as queryTo see if the phage community of these humans is related to those of the bats from the same locality, a visualization of the network of both human and bat phageomes was performed using vConTACT . InitialMH608285 to MH608287 and MH933752 to MH933860  Accession numbers of all viruses described in the study. (B) Accession numbers of raw reads from individual pools submitted to Short Read Archive under the BioProject number PRJNA491626 and Tax ID 1861841. For ethical reasons, reads mapping to the human genome were removed. Download Copyright © 2019 Yinda et al.2019Yinda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


A severity of tissue alteration, expressed as different tissue damage scores were evaluated. Morphological structure of examinated tissues treated with of 0.1LD50 of all reactivators was comparable with the control group of rats. Moderate injuries were seen in visceral tissues treated with 0.5LD50 of asoxime, obidoxime and K027. Acute damages were enlarged after treatment with 0.5LD50 and 1.0LD50 of all reactivators during the next 7 days. The most prominent changes were seen in rats treated with 1.0LD50 of K048 and K075 (P < 0.001 vs. control and asoxime-treated group). All reactivators given by a single, high, unitary dose regimen, have an adverse effect not only on the main visceral tissue, but on the whole rat as well, but the exact mechanism of cellular injury remains to be confirmed in further investigation.Certain AChE reactivators, asoxime, obidoxime, K027, K048, and K075, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the different organs. As a continuation of previously published data, in this study, Wistar rats have sacrificed 24 hrs and 7 days after single  With the growing threat of terrorism, the possible intoxication caused by these agents is relatively high. Due to this, many countries are focused on the development of novel more efficient antidotes against these agents5.Organophosphorus compounds are broadly used as pesticides in agriculture or as chemical warfare agents in the military attacks. In the last decades, they were misused during a terrorist attack in Japan, Syria or most recently in MalaysiaAmong the most well-known representatives of nerve agents are sarin , soman , tabun  and VX (O-ethyl-S- (2-diisopropylaminoethyl) methylthiophosphonate) Fig. .Figure 17. Standard antidotal treatment of organophosphororus poisoning (OP) implies the administration of anticholinergic drugs to counteract muscarinic over-stimulation, an oxime to reactivate OP-inhibited AChE, and anticonvulsants to protect against central nervous system seizures10.Their toxic effect is based on the inhibition of enzyme acetylcholinesterase  through its phosphorylation. As a result of this inhibition, this enzyme can not fulfil its physiological function in the organism (cleavage of a neuromediator acetylcholine at nerve synapses). Afterwards, its accumulated amount overstimulates nicotinic and muscarinic receptors. Clinically, it is manifested as a cholinergic crisis. The intoxicated organism then dies without treatment as a result of respiratory and heart failureAChE reactivators are a group of drugs designed to restore AChE function. Among the most well-known representatives of this family are pralidoxime , obidoxime -2-oxo-propane dichloride) or asoxime (1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)  or bisquaternary  pyridinium compounds with a functional aldoxime group. These substances must contain in their structure some important features without which their reactivation capacity would be low or zero. The most important are: the presence oxime groups (responsible for cleavage of nerve agent-enzyme complex), the presence of quaternary nitrogen (responsible for affinity to AChE and for very good solubility of reactivators), etc.19. If novel reactivators are investigated throughout the world generally their cytotoxicity or acute toxicity is tested21. Sometimes, their pharmacokinetics with a special focus on blood-brain barrier penetration is investigated24. However, what is the real cause of the toxicity of reactivators?Although the structure-activity relationship between reactivators’ structure and their biological activity is well known (both reactivation efficacy and inhibition potency), data describing their toxic effect are rare and not complexly investigated26, in this third study, we focused our attention on an investigation of morphological lesions of visceral tissue’s produced by increasing doses of selected AChE reactivators. For this purpose, standard oximes (i.e. asoxime and obidoxime) and K-oximes  were selected as experimental reactivators. The whole experiment was conducted on Wistar rats.Since all the toxicological data were provided in the earlier studies50 or 0.5LD50 dose of different oximes, the clinical signs of acute poisoning were not seen. The muscular pain, weakness, and tremors were perceived only in rats after treatment with 1.0LD50 dose of each oxime during the whole study.In rats treated with 0.1LD50 dose of all oximes were similar to the control values. A small number of alveoli were filled with desquamated epithelial cells and single alveolar macrophages only in rats than received K075  and in the K027-treated rats , respectively. On day 7 of the study, the type of histopathological alterations was similar to those established after 24 hrs. The time-dependent differences were seen in all animals treated with asoxime, obidoxime  (Table st day) and in the K027-treated rats , respectively. Noticeable pulmonary injury was established in tissue samples of rats, sacrificed 7 days after treatment with obidoxime, K048 or K075  were accumulated in the vicinity of the blood vessels or in all parts of the tunica mucosa. Mild, diffuse oedema and hyperaemia were seen in the tunica muscularis. These smooth muscular layers were infiltrated with oedema fluid. In some animals, sacrificed on the day 1, focal necrosis of the epithelial cells was caused a deep defect (gastric erosions) of the tunica mucosa, which was extending only into a superficial part of the mucosa. This superficial loss of normal mucosa accompanied by congestion and mild haemorrhages were the highest in rats treated by K048 (GDS = 2.50 ± 0.51), or K075 (GDS = 2.93 ± 0.78). The frequency and severity of these gastric lesions were significantly intensive in comparison to asoxime-treated rats or the control rats, sacrificed after day 1 (P < 0.001). The time-dependent differences were the higher in the K075-treated rats (P < 0.01), and the highest in the asoxime-treated, obidoxime-treated, K027-treated . Moreover, in these groups of animals, the presence of severe vacuolar or parenchymal degeneration, diffuse hyperaemia, large haemorrhagic foci and tissue infiltration with neutrophils, lymphocytes, plasma cells and macrophages were accompanied by central lobular necrosis. In addition, macronodular vacuolization of hepatocytes was expressed in majority hepatic sections. These morphological changes of the hepatic cytoplasm were commonly associated with the pyknotic nucleus. In some areas of the same hepatic tissue section, central lobular necrosis could be seen. According to these facts, the highest HDS value was in the K075-treated animals sacrificed 24 hrs after treatment (3.06 ± 0.78). In the K048-treated group, HDS value was a little less, but also significantly different from those established in the asoxime-treated or control rats (P < 0.001). Similar differences were noticed in these groups of rats sacrificed at the end of the study , and noticeably higher in comparison to the asoxime-treated rats , so-called T lymphocytes areas. The germinal centres without changes were persisted in the PALS of the lymphatic nodules. In several tissue sections, moderate dilatations of the central arterioles were seen, too. In the outer part of the PALS, normal B lymphocytes and a larger number of the plasma cells were presented. These histological alterations were associated with focal, moderate vascular changes of the red pulp and trabeculae, such as oedema, hyperaemia and haemorrhage. Also, observed microscopic findings were the most intensive in the inner parts of the spleen tissue, especially in the K048 and in the K-075-treated animals, sacrificed on day 1 and day 7   either directly or indirectly, resulting in cell homeostasis disturbance32.In accordance with this challenge, the present study aimed to prove our hypothesis that exposure to a toxic dose of different oxime induced visceral tissues injury as well. Therefore, this study was also conducted entirely with male rats and followed our 7-day protocol, and gut, lung, liver and spleen injury was confirmed by histopathology and semiquantitative analyses. Our previous study has confirmed that exposure to low, injury-free oxime concentration, which was unable to cause muscle cells’ damage. Further increase in their doses leads to different muscular tissue damages due to their potent cytotoxic and pro-inflammatory effect50 of different oxime, i.e. large areas of gastric, pulmonary, hepatic and spleen tissue had various tiny vacuoles and pale cytoplasm shape , and discrete amounts of inflammatory cells were also identified. The most distinguished increase of GDS, PDS, HDS and SDS were detected in each tissue sample treated by 1.0LD50 of K048 and K075 . At the highest concentrations of K048 and K075, the initial event is tissue necrosis, which mainly results in the destruction of cell membranes, leading to enhanced autophagocytosis and cells’ disappearance33. Many reactive intermediates of toxic drug doses are electrophiles, free radicals, or free-radical generators, which may potentiate the toxicity of tissue oxygen, depleting intracellular glutathione and biological antioxidants34. Moreover, the intensive release of reactive oxygen species (ROS) and free radicals that damage both DNA the cytoplasmic organelles and endoplasmic reticulum. The endoplasmic reticulum plays an important role within the intracellular quality control and sensitivity to oxidative stress35, and its stress is mainly related to the activation of apoptosis36. Several authors confirmed that ROS may act as messengers between the oxidative stress and endoplasmic reticulum stress38. During oxidative stress, mitochondrial dysfunction leads to the impaired production of ATP and a further increase in ROS production39.The first signs of disruption of cell homeostasis in our experiments were verified by semiquantitative histopathological analysis 1 day after treatment with 0.5LD44. Immediately after drug-induced tissue injuries, pro-inflammatory macrophages conduce to cell lysis and stimulate tissue proliferation, then a few days after toxic injury anti-inflammatory macrophages diminish the inflammatory reaction and stimulate tissue repair process45. The tissue regeneration process activated by low doses of the drug, while their high doses suppress compensatory tissue repair leading to improvement of tissue toxic injury47. In our study, after a period of 1 week, the prominent toxic tissue alterations were noticed in all visceral tissues following treatment with 0.5LD50 of obidoxime, K027, K048 and K075 . Also, these tissue damage scores were the lowest at the end of the study only in the asoxime-treated group . Afterwards, estimated PDS, GDS, HDS and SDS were enhanced after treatment by 1.0LD50 of asoxime but these scores were significantly lower in comparison to other oxime-treated groups (P < 0.001). The visceral tissues injuries with the highest were noticed after a week period in rats treated by 1.0LD50 of K048 and K075. The maximal calculated values of PDS, GDS, HDS and SDS were in the range of 4.13 to 4.53. These data, as well as ours previously published results25, confirm that the toxicity of oximes is time-dependent. Then, depending on the applied dose, the acute toxic tissue injury is associated with oximes-induced general toxicity.On the other hand, neutrophils excrete a huge amount of proinflammatory molecules , and create a suitable microenvironment for monocytes and macrophages collecting25, we can assume that 10% LD50 of all tested oximes is safe. In the case of asoxime and K027, also 50% LD50 seems to be still safe. So that, dosage 10% LD50 could be in future considered as appropriate treatment dosage which will have no adverse effect on treated organism regardless of oxime choice. Higher dosage is a question of selected oxime reactivator. As resulted from the obtained data, asoxime seems to be the safest oxime reactivator tested within these studies. This fact is in very good agreement with its acute toxicity data.Based on the obtained data within this study, as well in the previous oneBriefly, our research confirmed that each oxime given by a single, high, unitary dose regimen, has an adverse effect not only on the main visceral tissues but on the whole rat as well. In addition, our results provided certain evidence of beyond the adverse effects on the target tissues and general health condition of rats after exposure to different oximes. Moreover, basic histopathology analyses can help to establish toxicological dose-response attitude in a preclinical safety assessing for newel oximes. Therefore, our data can subserve for the choice of dose in further subacute, subchronic or chronic evaluation. The application of these results can be used in non-clinical safety evaluation and the development of new oximes in order increasing the ability to test and improve the identification of the first signs of oxime-induced target tissue adverse effects. Finally, these results together with other preclinical data have a crucial contribution in the examination of general safety and risk assessment of newly developed oximes.in vivo experiments. All three experimental K-oximes were chosen for this evaluation according to previously published data48. Their detailed synthetic preparations have been described earlier in the literature51. Prior to the study, their purity was tested using the standard analytical methods53.Five AChE reactivators - two standard oximes  and three experimental K-oximes   breed at the Institute of Medical Research, Military Medical Academy, Belgrade, Serbia. Plastic cages  filled with sawdust  were used for the accommodation of rats. Ambient conditions, monitored by the central computerized system, were set up in the following range: temperature 22 ± 2 °C, relative humidity 55 ± 15%, 15–20 air change/h, and 12 hrs light/dark cycles. Animals had free access to filtered water and commercial pellets for rats, produced by Veterinary Institute Subotica, Serbia.The Ethics Committee for Experiments on Animals, of the Military Medical Academy, Belgrade, Serbia approved (i) animal welfare procedures and study design (No. 282-12/2002); (ii) experimental protocol was performed according to the Guidelines for Animal Welfare of the Ethics Committee for Experiments on Animals of the Military Medical Academy, Belgrade, Serbia (No. 323-07-04943/2014-05/1), and the National Guidelines for Animal Welfare, Belgrade, Serbia (No. 41/2009).54. Thereafter, each oxime was used in the present study at a single dose of 0.1LD50, 0.5LD50 and 1.0LD50.Each oxime was preliminarily tested in animals using standard experimental procedure published earlier in the literatureWistar rats were divided into sixteen experimental groups as shown in Table 55. The general health condition of animals was monitored daily throughout the study, while a standard post-mortem examination was performed for 7 days.Immediately before the intramuscular application, each oxime is dissolved in a freshly prepared solution of normal saline (0.9% sodium chloride in distilled water). A single injection of each oxime was applied in lateral thigh muscle of the right leg25. As we mentioned in those studies, the selection of dose levels for careful histopathology analysis was primarily based on the results of acute toxicity testing26. The following 4 dose levels were selected to evaluate dose-dependent acute toxicity for different tissues at two different time intervals: control group; low dose group (5–10% of LD50), medium-dose group (≈50% of LD50) and high dose group (≈1.0 LD50). Specifically, in line with the OECD GLP-compliant guidelines, at the end of the acute toxicity tests, a pathohistological analysis is required to assess the safety of the chemicals being investigated58.The selected doses for this histopathological study was described in our previously published studiesst and 7th after the applied treatments59.Five animals from each experimental group were sacrificed, using light ether anaesthesia, on the 1At autopsy, different tissue samples  of each rat were fixed  and prepared for further standard histopathological analysis. Tissue’s dehydration was carried out by immersing specimens in a series of ethanol solutions of increasing concentration until pure, water-free alcohol is reached. Ethanol is miscible with water in all proportions so that the water in the specimen was progressively replaced by the alcohol. A series of increasing concentrations was used to avoid excessive distortion of the tissue. A typical dehydration process for specimens, not more than 4 μm thick was: 70% ethanol for 15 min, 90% ethanol for 15 min, 100% ethanol two times for 15 min, 100% ethanol for 30 min and 100% ethanol for 45 min, respectively. At this point, all but a tiny residue of tightly bound molecular water were removed from the specimen.60.After dehydration, each tissue sample was embedded into melted paraffin wax, the block was mounted on a microtome and cut into thin slices (2 µm). The slices were affixed to microscope slides at which point the wax was removed with a solvent and the tissue slices attached to the slides are rehydrated and were ready for staining. Finally, after the application of hematoxylin, followed by a rinse in a weak acid solution to remove excess staining, each tissue slice was counterstained with eosinFor detail histopathological and semiquantitative analyses, Olympus BKS-43  with a digital camera and Cell D software  were used. Complete histopathological and semiquantitative analyses were performed by pathologists blind of the treatment groups.67.The intensity of degenerative and vascular lesions in the lungs, stomach, liver and spleen were scored and counted through a light microscope in 30 tissues’ slices per group  with a magnification of 200x according to previously published literatureAs shown in Table For statistical evaluation, commercial statistical software  was used. All results shown in tables were expressed as the mean  animal welfare procedures and study design (No. 282-12/2002); (ii) experimental protocol was performed according to the Guidelines for Animal Welfare of the Ethics Committee for Experiments on Animals of the Military Medical Academy, Belgrade, Serbia (No. 323-07-04943/2014-05/1), and the National Guidelines for Animal Welfare, Belgrade, Serbia (No. 41/2009).


Pueraria phaseoloides is a legume cover crop, found chiefly in the wet zone of Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop, comparable to the nodule-dwelling bacteria of most other legume plants. We isolated a bacterium (Sub1) from Pueraria phaseoloides, of coccobacillus cell shape, that showed nodulation, when assessed by hydroponics, showing nodules as early as 3 weeks after reinfection. When a nifH fragment from the genome of this bacterium was amplified using a pair of nifH primers, it yielded an amplicon of 360 bp that, when sequenced, helped us identify the bacterium, as belonging to a species of Pseudacidovorax intermedius, at 99% sequence identity. When we constructed a phylogenetic tree with neighboring sequences, we encountered nifH sequences of Pseudacidovorax, forming a monophyletic cluster, which too contained a single Azospirillum species. The genus Pseudacidovorax is a bacterium that, so far, has not been associated with legume nodules. Sub1 secreted a pair of enzymes to the extracellular medium to degrade cellulose and milk proteins. The Sub1 bacterium showed biofilm formation and secreted into the extracellular medium, indole acetic acid. Sub1 also showed a “bulls eye” swarming pattern for the chemoattractant proline, while showing no significant chemotaxis movement, for naringenin, quercetin, and glutamate. Sub1 too possesses the basic genetic foundation (nifH and nifD) to produce a molybdenum-dependent nitrogenase enzyme. We finally show that this rare nonrhizobial bacterium is able to impact, positively, nodulation and shoot length of Pueraria plants, demonstrating that this beta-proteobacterium can abet the biological vigor of this legume cover crop. Pueraria phaseoloides is a widely seen practice, in rubber plantations. Pueraria is called “Pohora Wal” (Fertilizer Vines) in Sri Lanka and is a common occupant in rubber plantations, which makes this an invaluable cover crop of economic importance.Nitrogen is a macronutrient for plant growth. It plays mandatory roles inside plant cells for the synthesis of enzymes, proteins, chlorophyll, DNA, and RNA . Rubber  Rhizobium, which fosters an advantage from the nitrogen enrichment, converting a large reservoir of nitrogen gas, into amino acids [Legumes emerged 70-130 million years ago early in the Cretaceous period . Legumesno acids .Pueraria phaseoloides, which is also known by the names, Puero , tropical kudzu (most of the tropics), centro grande, and feuille, is a cover crop that grows well in wet land soils, and supplies the voracious needs of the root system of rubber plants. One of the predominant features of Pueraria phaseoloides is the presence of a wide range of nitrogen-fixing bacteria inside the root nodules [ Pueraria phaseoloides the ideal legume to investigate its compliment of nonrhizobial nitrogen fixers. nodules . This suα and β subgroups of Proteobacteria such as Methylobacterium, Blastobacter, Devosia, Phyllobacterium, Ochrobactrum, Agrobacterium, Cupriavidus, Herbaspirillum, and Burkholderia and some δ-Proteobacteria have been identified this far [nifH) and nodulation (nodD or nodA) gene sequences [Several nodulation-competent nonrhizobial species, belonging to this far . Althougequences . Pueraria phaseoloides using a combination of molecular biology, microbiology, and plant growth experiments.We describe here, a rare nonrhizobial nitrogen fixer, residing inside nodules ofSurface sterilized  root nodTo study cell morphology, single colonies from agar plates were used to prepare slides and the prepared slides were stained with simple staining and Gram staining  techniquFor cellulose, pectin, and protein utilization, media plates were made with carboxy methyl cellulose, pectin, and skim milk and the hydrolysis zones around the bacterial colonies were observed/measured to identify the respective hydrolysis-promoting bacteria –12.μl of culture with 5ml of medium) and incubated 3-5 days. Cultures were removed carefully using a pipette and the boiling tube was washed 3-5 times with two times autoclaved distilled water. Then, 2ml of 0.5% crystal violet was added to each tube. After incubation of 20 minutes, crystal violet was removed and tubes were washed with two times autoclaved distilled water, 5 times. Tubes were turned upside down and they were dried overnight. Then, 2ml of 30% acetic acid in water was added to each tube and the tubes were incubated at room temperature for 10-15 minutes. Finally, absorbance was measured at 550 nm using 30% acetic acid in water as a blank [Cultures in YMB medium were incubated overnight at 150 rpm shaker. 12 boiling tubes were autoclaved by setting cotton plugs and aluminum foil. Overnight cultures were diluted 1:50  and 10mM quercetin (negative flavonoid) .−4M proline as the chemoattractant and mannitol as the energy source and the chemotaxis-based migration (swarming patterns) of bacteria were recorded after 4 days of incubation at room temperature [Furthermore, culture media were prepared with 0.3% of bacto agar, with 10perature .3, 49 ml of water, and 49 ml of 70% perchloric acid were mixed together). Resulting solution was analyzed using spectrophotometer at 530 nm. IAA concentration of each sample was calculated using standard curve of commercial IAA.Bacterial isolates were cultivated in YEM broth supplemented with 5 mM L-Tryptophan at 30°C for 05 days. A control was also maintained without inoculating bacteria. Then 1ml aliquots of each media were centrifuged at 10,000 rpm for 12 minutes after 24, 48, and 72 hours of growth (until 5 days). Then 1ml of sample was treated with 2ml of Salkowski's reagent in a test tube . The primers used were as shown in  nifH gene fragments were purified and sequenced bidirectionally by Sanger sequencing method. Sequences were assembled using DNA baser (V 4.20) software. Open Reading Frames (ORFs) were obtained without stop codons for the contig sequences using NCBI ORF finder.PCR amplified products for Pseudomonas putida (FJ404470.1)Phylogenetic analysis using neighbor joining (NJ) trees were constructed using sequences retrieved from NCBI GenBank  after ClustalW multiple alignment . The treStrength seedlings were transferred to sterilized boiling tubes which were filled with 1/4 strength sterilized nitrogen-free Hoagland solution. Each plant was inoculated with 1ml of 2-week-old inocula. Four were replicated in total for each inoculum. Root length, shoot length, number of leaves, and presence and absence of the nodules, were recorded weekly throughout two and a half months.Pueraria phaseoloides has been termed as a promiscuous plant for rhizobial strains [ Pueraria phaseoloides. We have isolated here a nonrhizobial partner, which we have named as Sub1. Using BLASTn analysis of a nifH fragment, sequenced in sense and in antisense, the closest level of identity we can arrive at is 99%, which is adequate to assign this bacterium, a specific species name. The genus that the BLASTn search identified at 99% identity was from the species Pseudacidovorax intermedius, of which little information is available. There were no rhizobia species in the BLASTn listing, demonstrating that this was a nonrhizobial contender, living inside the nodule of Pueraria phaseoloides plants. A bacterial strain, GAU11T, isolated from Japanese soils, has been shown to possess a sequence identity of 96.4% in 16s rRNA coding sequence to Pseudacidovorax intermedius CC21T [ Pseudacidovorax genus belongs to the family, Comamonadaceae, all members of the class beta-proteobacteria. strains . Howeverus CC21T . The PseNifH sequences are divided into four clusters: cluster 1 containing Cyanobacteria, Frankia, Proteobacteria, and some clostridia, bacilli, and Nitrospirae; cluster 2 comprising vnfH nitrogenases; cluster 3 forming archaea and anaerobic bacteria; cluster 4 containing uncharacterized and nonfunctional nifH genes, which makes the nifH sequence of this study as belonging to cluster 1 [ nifH sequences in public databases contain sequence data in the range between positions 100 and 500 bases (positions relative to Azotobacter vinelandii), which makes nifH sequences between this range, a good source to find suitable sequences, using nucleotide BLAST analysis [luster 1 . The majanalysis . nifH against that of 16s rDNA, the authors found out that >97% similarity in 16S rRNA genes can be as much as 23% dissimilarity/divergence in nifH sequences, which tell us that nifH is less conserved than 16s rDNA [ nifH identity, and 43% are candidates for <85% identity, again verifying that nifH presents a window of opportunity to zoom into molecular identities, which cannot be resolved by 16s rDNA genes [ nifH, to strains of Pseudacidovorax, tells us that a 99% identity of nifH is a stronger validation than the same match of 16s rDNA sequence; i.e., 99% identity of an otherwise variable nifH is proof that this sequence indeed belongs to the genus Pseudacidovorax.In a comparative study that assessed sequence divergence levels of16s rDNA . FurtherNA genes . Our 99%α and β subgroups of Proteobacteria such as Methylobacterium, Blastobacter, Devosia, Phyllobacterium, Ochrobactrum, Agrobacterium, Cupriavidus, Herbaspirillum, and Burkholderia and some δ-Proteobacteria have been identified this far [nifH) and nodulation (nodD or nodA) gene sequences, between Rhizobia and nonrhizobial nodule inhabitants [Several nodulation-competent nonrhizobial species, belonging to this far . Althougabitants .The shape of Sub1 cells was not rod shaped, as found in Rhizobia, but showed curved edges and was of a coccobacillus cell shape . This waThe nodulation potential of Sub1 was tested in a hydroponics system, where 4 replicates were used to assess the ability of reinfection with Sub1, to induce nodule formation. There are many studies that have employed hydroponics to study legume nodulation –24 and,  Pueraria phaseoloides plants treated with only Sub1. By week 4, all single plant replicates had shown development of nodules, indicating that this bacterium was capable of nodule formation in plants of Pueraria phaseoloides  from among four distinct isolates oft length . It has t length . The conSub1 infected hydroponic plants show elongation of shoots, significantly stronger than the control plants .The nodular inhabitants ofhizobium . The exaThe ability of Sub1 to produce IAA to the extracellular medium makes the bacterium a good candidate for plant growth promotion, of which the observable feature was the higher induced shoot length compared to the negative uninfected control. Other benefits from IAA include, seed germination, development of xylem and root systems, mediation of responses to abiotic factors, photosynthesis, pigment formation, and resistance to stressful conditions . The impIt is known that the rhizosphere of a plant is a conducive environment for biofilm formation, with both moisture and nutrition for such changes. Rhizobia form biofilms in their capacities to infect the plant through infection threads and to form nodules . The rol nif genes, nifH, and nifD, in their genomes. The primers relevant to the two genes, both gave amplicons of the correct size, demonstrating that the primary nif gene architecture for nitrogen fixation, i.e., nifD and nifH, was present in the genome of Sub1  that has been inferred to be receptive to horizontal gene transfer as well being lost from selective bacterial lineages [We tested for the genomic DNA of Sub1 to possess the main of Sub1 . The banlineages . Distinclineages .Neighborhood Joining,  Pseudacidovorax nifH sequences clustering together with Sub1, in the phylogenetic tree, which too included a single Azospirillum. The fact that only one Azospirillum clusters with four Pseudacidovorax, tells us that the Azospirillum bacterium is either a unique anomaly or is a misclassification. Azospirilla bacteria are found as associative symbiotic rhizosphere inhabitants, belonging to the division, alpha proteobacteria and are known for their plant growth promotion through the production of phytohormones. As far as we know, there are no Azospirilla belonging to beta proteobacteria, while their presence is limited to the rhizosphere and the soil and are not known to form nodules or live inside them. We state that Sub1 belongs to the genus Pseudacidovorax and is likely a species of Pseudacidovorax intermedius, from sequencing of the nifH fragment, which was further confirmed through phylogenetic inferences.When we constructed a phylogenetic tree ( Pueraria phaseoloides, which is of a rare nonrhizobial identity, for a nodular inhabitant.We have isolated here a nodulation-proficient bacterium, from the cover cropThe most significant finding of our study is the nodulation inducing potential of this nonrhizobial bacterium and its impact on shoot length. Pseudacidovorax intermedius, from the sequencing of a fragment of the nifH gene and its alignment, at 99% sequence identity, to Pseudacidovorax intermedius NH-1 nifH.We state that this bacterium belongs to the species Pseudacidovorax, with one exception, an Azospirillum species.The phylogenetic tree constructed with the nearest neighbors of Sub1 provided a monophyletic cluster for the genusThe bacterium was capable of secreting IAA, cellulases, and proteinases to the extracellular medium and contained the genetic foundation to produce the nitrogenase enzyme.Sub1 also showed some degree of biofilm formation, which could be helpful in the colonization of the nodules.


The unique foot morphology and distinctive functions facilitate complex tasks and strategies such as standing, walking, and running. In those weight-bearing activities, postural stability (PS) plays an important role. Correlations among foot type, PS, and other musculoskeletal problems that increase sport injury risk are known. However, long-term associations among the foot type, the PS, and body weight (BW) distribution are lacking. Thus, the purpose of this study was to longitudinally identify changes in foot morphology, PS, and symmetry in BW distribution during adolescence among elite male soccer players.p2) coefficient were used for investigating the effect of time on selected variables and effect size evaluation.Thirty-five Czech elite male soccer players  underwent foot type, PS, and BW distribution measurements during 3 consecutive years . The Chippaux-Smirak index (CSI), BW distribution, and centre of pressure (COP) displacement  of each player for the preferred (PL) and non-preferred leg (NL) were acquired. Repeated-measures analysis of variance (RM ANOVA), Bonferroni´s post hoc tests, and partial eta-squared  and COP displacement values  for both legs over 3-years period was identified. Furthermore, the Bonferroni´s post hoc analysis revealed a significant improvement of PS values in the PL . Only BW distribution parameter showed no significant differences, although slightly improved over time.Statistically significant effect of time on CSI values (PL: FWe observed changes in foot typology, PS, and BW distribution in young elite male soccer players during 3 consecutive years. Results demonstrated that changes in PS and body weight distribution under the high-load sport conditions during adolescence may improve with aging, except for foot morphology. Therefore, foot morphology should be carefully monitored to minimize sport injury risk in professional young soccer players during adolescence. Further research is necessary to determine more clear associations between these parameters, soccer-related injuries, and sport performances. The foot is a unique anatomic, neurophysiologic, and functional structure, which facilitate complex tasks and strategies such as standing, walking, and running in human movements . The fooSoccer players are exposed to high risks of musculoskeletal injuries. In professional soccer, the incidence of injury is 8 cases per 1000 hours . PreviouHence, the aim of our longitudinal study was to register longitudinal changes in foot typology, PS, and symmetry in BW distribution among adolescent elite soccer players during aging to adulthood within 3 consecutive years.A prospective, longitudinal study design was used to attain the purpose this study. The research was approved by the ethics committee of the Faculty of Physical Education and Sports at Charles University in Prague prior to commencement of this project. Each participant or his legal guardian was fully informed about the study design and signed an informed consent form.Seventy-five elite male soccer players from the national teams of Czech Republic initially agreed to voluntary participate in the study. However, in the first year, we lost 13 players (N = 62). In the second year, we further lost 13 players (N = 49), and during the third year, additional 14 players did not participate in this study (N = 35). Thus, at the end only 35 players completed the 3-year longitudinal study  and each lower limb of participant was investigated separately. The reason of dropout was mostly because of the exclusion criteria including lower limb, pelvic, or spinal injury within 6 months before the study started or at any time during the study period, and any acute musculoskeletal pain. Also, several players changed their soccer team affiliations during the study period or relocated to another geographic region. All data were collected one time per a year for 3 consecutive years. The timing of data collection was always during a pre-season in each year. Thus, each player followed measurements three times in total . Measurements were conducted in accordance with the ethical standards of the Declaration of Helsinki and sport and exercise science research .http://dx.doi.org/10.17504/protocols.io.QP8DVRWThe laboratory protocol for the methodology for postural stability, body weight distribution, and foot typology assessment is described in full at −2, a sampling rate of 10 Hz. The measuring plate was placed 2 m from the wall, where a visible point was placed exactly in the perpendicular vertical axis of the body of each player standing barefoot on the plate and at the level of the height of the eyes.This prospective, a longitudinal study of foot morphology, symmetry of BW distribution, and PS characteristics of elite soccer players was performed using the RS Footscan Balance 7.6 second-generation tensometric plate . The PS and BW distribution between the lower limbs during quiet standing were tested using the Footscan plate with a dimension of 50 x 40 cm and 4 100 sensors with a precision of 0.1 N.cmFor analysis of PS, we chose the centre of pressure (COP) displacement parameter expressed as total traveled way (TTW) in millimetres. We used the standard procedure described in previous studies . FirstlyThe NS test lasted for 30 s, and the one-leg stance tests took 60 s, with a 60-s rest between tests. In a standard protocol, a researcher informed the player when each test started and finished. In the beginning of NS test, the player stood in the middle of the measuring plate, with his feet as close as possible, but not touching each other, and arms alongside the body while he was instructed watching the point directly and being motionless during the testing. Then, the researcher started the 30s data collection. After finishing the test and 60s resting, the one-leg stance test was conducted for each leg. Before the test had started, the researcher instructed the player to watch the point and be motionless during the one-leg test. Then, the player could choose the leg he wanted to start with, and he stood on that leg. After that, he bended the non-stance leg knee up to 90° of flexion with a concurrent neutral position in the hip joint and held arms alongside the body. When the preparation was completed, the 60s one-leg stance test was conducted. After the first test, a 60s rest and a new process of the preparation followed for the second test. The player changed his legs and repeated the equal one-leg stance test with the same instructions but for the second leg.Δ) and set the normative value of asymmetry for the 5% difference [From the NS test, we found the percentage values of BW distribution between the legs , which is a validated index for foot typology description , we usedp2). The foot type differences over three observed years were analysed by non-parametric χ2 (chi square test) and Cramer’s V. For all the analyses, the statistical significance level was set as a p value of 0.05 to reject the null hypotheses. Statistical analysis was performed using IBM SPSS v21 .For statistical processing of the data, we used descriptive and inductive statistics. Measurements were expressed using the arithmetic mean, and the measure of variability was expressed using standard deviation. Data normality was evaluated using the Shapiro-Wilk test. To assess the significance of the independent variables (time of observation) based on the dependent variables a repeated-measures analysis of variance (RM ANOVA) was used. To evaluate sphericity, an assumption of RM ANOVA, the Mauchly test was performed. Multiple comparisons of the means of the monitored variables were performed using the Bonferroni´s post hoc test. Moreover, the effect size coefficient was assessed using partial eta squared . The Bonferroni post hoc analysis revealed a significantly higher level of PS in favour of the observed time in PL .The COP displacement values of the group at time T1, T2, and T3 are depicted in 2,68 = 5.08, p < 0.01, ηp2 = 0.13 and NL: F2,68 = 10.87, p < 0.01, ηp2 = 0.24) (PL) during the whole period in comparison with the NL (CSINL). Significant differences were found between the CSIPL values in years T2 (38.99 ± 9.10%) and T3  and CSINL values between years T1 (38.01 ± 7.85%) and T3 , and between T2 (37.04 ± 9.36%) and T3 .Repeated measurement ANOVA revealed a significant effect of the main factor (Time) on the CSI values for both legs (PL: F = 0.24) . The parT1 = 43%), followed by the lowered (LT1 = 29%) and flatfoot (FT1 = 17%) types. Only 11% of the participants had a normal arch foot type. Intermediate and normal FTs had slightly increasing level of distribution at T2 but showed a prominent decreasing tendency at T3 in the sample representation. On the other hand, the lowered FT initially showed a decreasing trend in the distribution but, at T3, increased back to the same value as at T1. Only the flatfoot type had a purely increasing tendency in the sample representation throughout the whole 3-year period .Foot types distribution and time progression are shown in Figs The purpose of this study was to observe progression in foot morphology, BW distribution symmetry, and PS changes during the adolescence period of elite male soccer players. In our research sample, we found that the BW distribution asymmetry in between legs became higher than the previously reported value of 5% . ConcretDespite the maturation of the medial longitudinal foot arch during childhood period (4–10 years) , some chHowever, the future research is necessary to identify these factors influencing the sport performance and injury risks in adolescent soccer players more deeply with reflection to lower limbs laterality, age at professional sport training, strength asymmetry, and possible correlations between these parameters.Several limitations need to be stated. This study did not take in account the age at professional sport training of the players and the playing position on the pitch. Additionally, generalizability is another limitation. The study participants consist of elite male soccer players. Thus, the current study results may not be applicable to non-elite and female soccer players.We observed changes in foot typology, BW distribution, and PS in young elite male soccer players during 3 consecutive years. We found that lower limbs difference in BW distribution tended to improve during the adolescence; however, statistically significant differences were not detected. We found significant changes in foot typology. There was an evidence of constant increasing in flatfoot type distribution during the study period. In addition, the PS parameter, TTW of COP, showed a significant improvement by the decreasing value in both one-leg stance tests and in the TTW difference between the legs. These results indicated that selected PS parameters and BW distribution could improve with aging. However, a caution needs to be made for an interpretation. Foot morphology and function should be carefully monitored to avoid further problems related with overuse injury in professional young soccer players during maturation. Further research is needed to determine more clear association between these parameters, soccer-related injuries, and sport performances.S1 Table(XLSX)Click here for additional data file.


Int J Epidemiol, 2018, 47(3), 942–952, doi: https://doi.org/10.1093/ije/dyx263.First published online: 14 December 2017, International Journal of Epidemiology ‘Rapidly increasing body mass index among children, adolescents and young adults in a transitioning population, South Africa, 2008–15’ by B Sartorius, K Sartorius, M Taylor, J Aagaard-Hansen, N Dukhi, C Day, N Ndlovu, R Slotow and K Hofman published online on 14 December 2017 and in the International Journal of Epidemiology, Volume 47, Issue 3, June 2018, Pages 942–952 by Oxford University Press has been retracted by agreement between the journal Editor, Professor Stephen Leeder, the lead author Professor Benn Sartorius and all co-authors, the International Epidemiological Association and Oxford University Press.The above article from The retraction follows the discovery of a mistake relating to the analysis and interpretation of the South African National Income Dynamics Study (SA-NIDS) data, especially that from Wave 4 presented in Figure 1. This error resulted from not incrementing age between Waves 1 and 4 and so not accounting for the normal increase with age in BMI among young people. Recalculation of the estimates shows that there is little change in mean BMI by age over the period between Waves 1 and 4 and thus no evidence of the large increase in the prevalence of overweight and obesity in young people shown in Figure 3 that is the main finding of the study. All authors accept responsibility and apologise for this error.


Background: Prepulse inhibition (PPI) of the startle response is a highly conserved form of sensorimotor gating, disruption of which is found in schizophrenia patients and their unaffected first-degree relatives. PPI can be measured in many species, and shows considerable phenotypic variation between and within rodent models. This makes PPI a useful endophenotype. Genome-wide association studies (GWAS) have been carried out to identify genetic variants underlying schizophrenia, and these suggest that schizophrenia is highly polygenic. GWAS have been unable to account for the high heritability of schizophrenia seen in family studies, partly because of the low power of GWAS due to multiple comparisons. By contrast, complementary mouse model linkage studies often have high statistical power to detect variants for behavioral traits but lower resolution, producing loci that include tens or hundreds of genes. To capitalize on the advantages of both GWAS and genetic mouse models, our study uses a cross-species approach to identify novel genes associated with PPI regulation, which thus may contribute to the PPI deficits seen in schizophrenia.Results: Using experimental data from the recombinant inbred (RI) mouse panel BXD, we identified two significant loci affecting PPI. These genomic regions contain genetic variants which influence PPI in mice and are therefore candidates that may be influencing aspects of schizophrenia in humans. We next investigated these regions in whole-genome data from the Psychiatric Genomics Consortium (PGC) schizophrenia GWAS and identify one novel candidate gene (ABPP1IP) that was significantly associated with PPI in mice and risk of schizophrenia in humans. A systems genetics approach demonstrates that APBB1IP coexpresses with several other genes related to schizophrenia in several brain regions. Gene coexpression and enrichment analysis shows clear links between APBB1IP and the immune system.Conclusion: The combination of human GWAS and mouse quantitative trait loci (QTL) from some of the largest study systems available has enabled us to identify a novel gene, APBB1IP, which influences schizophrenia in humans and PPI in mice. Our systems genetics approach demonstrates that APBB1IP coexpresses with several other genes related to schizophrenia in several brain regions, providing a potential mechanism of action.Animal models have proven invaluable in genetics research and previous research has demonstrated the effectiveness of using a cross-species approach to identify genes underlying specific traits  at the genome-wide level. Significance was calculated within p-value of 0.05 is defined as a likelihood ratio statistic (LRS) score greater than 95% of the permutated datasets .We began by identifying QTL for PPI in the BXD RI family. Next, we investigated the homologous genomic regions in human schizophrenia GWAS data and were able to identify one novel candidate gene, PPI deficits are associated with schizophrenia  value of 6.16, compared to the surrounding marker values of 4.13. This marker lies within the Apbb1ip gene. For GN11428, the most significant marker was rs6197068 at chr19: 32.74 Mb. This marker has a −log(p) value of 5.69. This lies between the genes Atad1 and Pten.To further characterize the two QTL we used Genome-wide Efficient Mixed Model Association (GEMMA) software .The QTL on proximal chromosome 2 (22.62–22.86 Mbp) contains four genes with human homologs, and the QTL on proximal chromosome 19 (31.95–32.75 Mbp) contains eight genes with human homologs, all of which are on chromosome 10 . We thenAPBB1IP are significantly associated with self-reported schizophrenia (UKBB ID 20002_1289). The first is rs570949810 at chr10: 26,823,566  and the second is rs554788824 at chr10:26,818,850 . Both variants are also associated with usage of two antipsychotics used to treat schizophrenia, trifluoperazine  and amisulpride . This further supports APBB1IP as a schizophrenia associated gene.Next, we checked for associations in an independent dataset, the UKBiobank collection using PheWeb  portale spleen . This liAPBB1IP expression increases postnatally in a number of brain areas  to identify regions of the genome that are regulated by our gene of interest. Second, we can examine gene co-expression to find groups of genes which are under similar regulatory control and are therefore likely to be involved in the same biological processes. Thirdly, we can detect low levels of gene expression, or gene expression that only occurs under specific conditions.APBB1IP in human lymphocytes, we first used two lymphocyte cell populations from the BXD family, T-helper cells and T-regulatory cells. In both cell types there is a cis-eQTL, indicating that a variant in Apbb1ip regulates expression of this gene, although this only reaches genome-wide significance in the T-helper cells , and reaches chromosome-wide significance in T-regulatory cells. We next looked for the probes that correlated significantly with the expression of Apbb1ip (p < 0.05) in both of these datasets, to find a co-regulated network. In T-helper cells, 3,623 probes , showing that a variant near Apbb1ip influences its expression across tissues. This does not mean that this variant is not expressed in tissues other than those given above, but simply that no effect could be detected in that particular dataset. For each of these issues with a confirmed Apbb1ip cis-eQTL, we carried out a coexpression analysis. If the tissue contained multiple probes for Apbb1ip then these were combined together using a PCA to produce an eigengene  had a very strong cis-eQTL . As this clearly showed that ed genes . These ged genes . This shp = 2.06e-05, N = 17). This is of interest, as reaction time is known to be disrupted in schizophrenia patients and their relatives . There was significant coexpression with 17,960 probes, mapping to 9,160 genes, and once again we found immune system processes in the enrichment analysis .In the hypothalamus data (GN159) we saw a significant Pituitary (GN335) had nine probes with strong cis-eQTLs, so these were combined into an eigengene, and remapped to produce a cis-eQTL with a max LOD of 14.55 running from 19.02 to 23.77Mb. Once again, amongst the coexpressed genes  we saw eApbb1ip (In the spleen (GN227), 3,700 probes, mappable to 2,846 genes, were significantly correlated with Apbb1ip . Unlike Apbb1ip .Apbb1ip using a transcriptome dataset for postnatal day 14 mice. Again, we used Apbb1ip, which show a strong QTL on chromosome 2, between 88 and 98 Mbps. We computed the 1st principal component of these three probe sets , and the expression of which correlated with Apbb1ip. These genes were Morf4l1, Mthfs, 1600029I14Rik, Plscr4, 1190002N15Rik, Armc8, Zbtb38, Faim, Rbp1, C730029A08Rik, Zic1, Trpc1, Adamts7, and 2810026P18Rik. Of these, Plscr4, Armc8, Zbtb38, Rbp1, Zic1, and Trpc1 have been linked to schizophrenia or schizophrenia-related disorders in previous studies , chromosome 2:81.02–93.32, i.e., overlapping with the eQTL which we observed in the striatum. There are 5,830 probes , represeApbb1ip and are enriched for annotations linked to schizophrenia, this does not directly link them with schizophrenia. We thus performed a permutation analysis to look specifically for enrichment for genes that have been previously associated with schizophrenia.Although we have identified genes that are coexpressed with n = 708) and for many, several potential genes were reported . These associations represented 1326 unique human genes, of which 959 had murine homologs. For each of the above tissues, we produced a full list of genes examined in the entire dataset . For each tissue, we recorded the overlap in genes coexpressed with Apbb1ip and with genes associated with schizophrenia. We then performed 100,000 permutations, taking random subsets of the same length as the coexpressed genes, and recording their overlap with the schizophrenia genes. The number of times that the random genes had a higher overlap than the co-expressed genes, divided by 100,000, produced an empirical p-value.We used the GWAS catalog for schizophrenia  to identApbb1ip coexpression genes from mouse and human tissue. The species and tissue used for coexpression are shown, the significance of the overlap between genes associated with schizophrenia and genes coexpressed with Apbb1ip, the number of genes which overlap between genes associated with schizophrenia and genes coexpressed with Apbb1ip, the total number of genes which coexpressed with Apbb1ip and the total number of schizophrenia-associated genes included in the analysis .APBB1IP, on chromosome 10 in humans and chromosome 2 in mice, as a novel gene that influences schizophrenia in humans and PPI in mice, and identified a number of genes which may be involved in this association. APBB1IP is found to have high expression in the whole blood, lymphocytes and the spleen, linking it to the immune system. Coexpression and enrichment analyses in the whole blood, cerebella and hippocampus showed enrichment for immune-related genes. Similar enrichment in immune-related processes showed that genes commonly coexpressing with Apbb1ip are also involved in immune responses in the mouse.We have identified APBB1IP is involved in cell motility, the immune response and neuronal development, all of which have been linked to schizophrenia. Apbb1ip is found to be expressed in homologous tissues to ABPP1IP, particularly the dentate gyrus, previously linked to schizophrenia , https://www.ebi.ac.uk/gwas/efotraits/EFO_0000692. The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from www.gtexportal.org, the GTEx Portal on 04/06/2019.The datasets analyzed during the current study are available in the GeneNetwork repository, RH, SC and DA conceptualized and designed the study. SC and DA carried out analyses. DA and SC wrote the manuscript, with contributions from RH. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arundo donax L. represents one of most studied one characterized by very low genetic biodiversity. Although it is a perennial rhizomatous tall grass native to eastern and southern Asia, it spreads only asexually in the invaded range all over the world thriving very well in a large array of pedo-climatic conditions. This ability to morphologically or physiologically adapt to a broad array of conditions could be attributed to epigenetic mechanisms. To shade light on this relevant issue, 96 stems of A. donax from spontaneous populations distributed across the Italian invaded range  were analysed. Leaf DNAs were extracted and processed through AFLPs and MSAPs for defining either genetic and epigenetic profiles. Both analyses clearly showed that the A. donax populations of Sardinia island are genetically distinct from those of Italian mainland; AFLPs showed an extremely low genetic biodiversity due to vegetative reproduction, whilst, epi-biodiversity, estimated through MSAP marker, increased within the analyzed populations. These results suggest that the capability of A. donax to invade and thrive in diverse environmental conditions can be, at least, partially attributed to a higher epigenetic variability. Therefore, the different DNA methylation status may have significant and important biological meaning, in particular, in the case of invasive clonal plants such as A. donax, also for the biodiversity definition, and MSAP marker can be considered an useful and cost effective marker to reveal it.Among the most widespread plant species with clonal reproduction  Arundo donax L. , also known as giant reed, is a perennial rhizomatous tall grass, native to eastern and southern Asia  were calculated using Arlequin [st value following the methods of Excoffier, Smouse [One of our aims was to compare the genetic biodiversity, calculated considering the Arlequin  and GeneArlequin , 36. In , Smouse  and Peak, Smouse . The bin, Smouse . Clustere.g., two dimension scatterplots).The multivariate AFLP or MSAP datasets were further analysed by Principal Coordinate Analysis (PCoA) using a covariance matrix. The major axes of variation in each dataset were identified and the assessments of the main trends in the data were graphically represented in a reduced-dimension space  to belong, partially or fully, to one or more populations under investigation, it is possible to speculate if and how the DNA methylation status alters the population structure. The number of populations (K) was estimated by performing 10 runs for each population, from K = 1 to K = 10. Each run consisted of 100,000 MCMC (Markov Chain Monte Carlo) permutations with a burn period of 10,000, assuming no a priori information on population affiliation, the admixture and correlated allele frequency methods. The K values were estimated using the method of Evanno, Regnaut [A Bayesian approach was applied, using Structure ver. 2.2 , to infe Regnaut  with 20  Regnaut .MseI profile), and of the MSAP (MspI and HpaII profiles), the following subtractions between genetic distance (GD) matrices were performed: GDMSP-GDAFLP; GDHPA-GDMSP; GDHPA-GDAFLP. It might happen that two distances are roughly similar ; other cases where the AFLP or MspI distances are greater than those of MspI or HpaII, showing somewhat smaller values, or, if the opposite is true , positive values would be obtained.To quantify the differences between the genetic distances, calculated on the basis of the AFLP  reflected DNA methylation variability.The ninety-six specimens of A. donax was very low (i.e. for Pop9 and Pop13. The highest number of common bands (< = 50%) was found in the populations collected in mainland Italy (from Pop8 to Pop13); in contrast, in the other populations (from Pop1 to Pop7 and Pop14), they were absent or close to zero. When the number of common bands (< = 25%) was considered, a maximum value of four was reached within Pop10 and Pop13, three within Pop9, two within Pop12, and one within Pop8 and Pop14.The main biodiversity indices, calculated for the AFLP profiles, demonstrated that the genetic biodiversity of the 14 investigated populations of very low ; in partIn general, the Shannon index (I) was very low ; only in the case of Pop10 and Pop13, which were collected in Emilia Romagna , it was 0.065 and 0.056, respectively. However, the molecular variance calculated among populations, equal to 89%, was much higher than that estimated within populations, equal to 11%. This result was due to a higher number of polymorphic loci found in the Pop8, 10, 13, and 14  with respect to those obtained by analysing all the other populations, which showed a percentage of polymorphic loci equal or close to zero.A. donax, Bayesian analyses were performed on the AFLP data set using the Structure software [A. donax from Sardinia and Campania region (red), whilst, the second, the samples collected in Emilia Romagna region (green).To investigate the whole Italian population of software . The delA. donax, Jaccard Dissimilarity Coefficient (JDC) was calculated, and the output UPGMA bootstrap dendrogram  including: 1E) samples ADE10_3 and ADE10_4; 2E) samples from ADE13_1 to ADE13_4; 3E) sample from ADE9_1 to ADE9_4, ADE10_1, ADE10_2, and from ADE12_1 to ADE12_4; 4E) samples from ADE10_5 to 10_12, and ADE11_1 to ADE11_4. The highest JDC was reached in the case of the node that grouped samples ADE10_3 and ADE10_4, which can be considered, therefore, as an out-group. However, the AFLP analyses confirm that A. donax adopts vegetative reproduction and, in particular, the population are constituted by mono-clonal stands.In order to estimate the genetic similarity among the analysed specimens of ndrogram  confirmeA. donax to invade and thrive in diverse non-native habitats, and in Sardinia in particular, and the fact that it shows different phenotypes and phenology in clonal populations [The ability of ulations , 43, mayA. donax populations. Given that the specimens collecting from the 14 populations shown very similar AFLP genetic profiles and that the statistical analyses divided all the samples in just two groups (meta-populations), we fixed the a priori number of groups equal to two for the analyses performed on MSAP data, in agreement with the estimated number of groups obtained by the Structure software and UPGMA analyses for AFLP data.The “msap” algorithm, developed in the R environment , was useMspI  was the most abundant methylation status.HpaII and MspI profiles. The results obtained were 0.51 and 0.35, respectively. A Wilcoxon rank sum test with continuity correction was significant (P<0.01). The percentage of polymorphic methylation sensitive loci, revealed by MspI and HpaII digestions, was 15% and 90%, respectively.In order to estimate the biodiversity due to different DNA methylation status, the Shannon diversity index was calculated separately for MspI and HpaII) have been processed together, as we made above. However, in our opinion, in the case of clonal populations, it could be possible to consider the two profiles independent; in this way each difference in the biostatistical indices will be attributed to DNA methylation effect, or to error rate. The main biodiversity indices are reported in Tables A new approach was applied to analyse the MSAP data; until now the two profiles obtained during the MSAP analysis  for MspI profile. In the case of HpaII, Pop2, Pop3, and populations from 9 to 13 showed the highest number of common bands (< = 50%); the highest number of common bands was detected in Pop13. Nevertheless, the percentage of polymorphic bands ranged between 0.0% (Pop11 and 12) and 39.0% (Pop3) in the case of the MspI profile, and between 0.0% (Pop11 and 12) and 51.0% (Pop3) in the case of HpaII, and, in general, it increased when the latter profile was considered. The inter- and intra-population molecular variance was 53% and 47% for MspI profile, and 58% and 42% for HpaII profile.The number of private bands in each of the 14 populations was zero in the case of the A. donax populations was analysed with no a priori information, using the Structure software [MspI or HpaII). The statistical model described by Evanno, Regnaut [MspI at the K value two and three , and the other cluster included all of the other A. donax specimens collected in mainland Italy (red bars). In contrast to the AFLP result, ten specimens, collected in Emilia Romagna region , were assigned both to red cluster  and to green cluster (with the probability of roughly 20%). When K = 4 was considered  were assigned to the yellow one; the specimens from ADE12_1 to ADE13_4 constituted the green cluster.The bar plot obtained for the se K = 2  was quitnsidered , the SarHpaII profile was considered. In this case, K was equal to three, and several specimens showed an ambiguous membership  to be assigned to the green one. Furthermore, the samples collected in Emilia Romagna were clearly assigned to the red cluster.The optimal population number and the cluster membership changed when the mbership . In the A. donax specimens, the JDC was calculated for both MspI and HpaII data sets, and the two UPGMA dendrograms are reported in Figs MspI and HpaII profiles showed an increased epigenetic diversity when compared to those determined by AFLP. In the case of MspI profile (A. donax collected in Sardinia Island plants are separated in 4 clusters with the highest JDC ≅ 2: 1S) sample ADS3_1 and ADS3_2; 2S) from ADS1_1 to ADS1_8, ADS6_3 and ADS6_4, ADS8_1 to ADS8_4, and ADS14_3 and 14_4. In the case of A. donax collected in Emilia Romagna, the specimens were subdivided into five subgroups (JDC≅ 3) including: 1E) samples ADE10_3 and ADE10_4; 2E) from ADE13_1 to ADE13_4; 3E) ADE9_1 to ADE9_4, ADE10_1 and ADE10_2, from ADE12_1 to ADE12_4; 4E) from ADE11_1 to ADE11_4; 5E) from ADE10_5 to ADE10_12.In order to visualize the level of similarity among the  profile , we founHpaII profile   matrices were performed: GDMSP-GDAFLP; GDHPA-GDMSP; GDHPA-GDAFLP (as reported in section 2.2). In our study, the GDHPA was greater than that calculated in the case of AFLP and n green) . A similn green) . In thisn green) . When GDn green) , the GDHA. donax in Italy, we analysed 96 individuals from 14 naturalized populations using AFLP (EcoRI/MseI–genetic diversity) and MSAP (EcoRI/MspI and EcoRI/HpaII–epigenetic diversity).Plants in general, and in particular the invasive riparian one, may have developed molecular processes to adapt themselves in order to colonize new and frequently changing habitats. Epigenetic mechanisms are, very likely, the trigger of a series of complex molecular mechanisms that allow plants to thrive in sometimes harsh conditions. In order to understand whether epigenetic biodiversity, rather than genetic diversity, might better explain the invasive success of the archaeophyte In general, plants use epigenetic mechanisms to counteract environmental variations and habitat changes , or copeDifferent epi-phenotypes, although related to the same genome, are hypothesized to promote significantly environmental adaptation, because they promote phenotype diversification and increase the probability to win the “war for life” in new or highly competitive ecosystems, or in relation to environment changes. The benefits of phenotype diversification induced by epigenetic variation are particularly noticeable in plant species that adopt vegetative reproduction, which may explain the “invasion paradox” , or the A. donax, and to compare it with genetic diversity, a set of genetic biodiversity indices, estimated on the basis of AFLP data (genetic profile), were compared with those obtained with MSAP markers (epigenetic profiles). The Jaccard Distance Coefficient (JDC) revealed a very low genetic biodiversity in the case of A. donax collected in Sardinia (ADS) and in Emilia Romagna (ADE). The AFLP analyses confirmed the particular tendency for vegetative reproduction and, at the same time, demonstrated that the specimens of A. donax collected in Sardinia are genetically isolated from those thriving in Emilia Romagna. A high similarity value was expected; in fact, Mariani, Cabrini [A. donax, also asserted that this species, originating in Asia, where it is not sterile [A. donax populations of the Mediterranean basin are characterized by a very low genetic diversity, and by a low incidence of rare polymorphic fragments. Despite the low genetic biodiversity of A. donax found in Sardinia and in southern Italy, Pilu, Cassani [etc.) and biomass production, the results reported by Pilu et al. [A. donax to invade and colonize different environments, although the Italian population of giant reed is characterized by a very limited genetic biodiversity due to primary asexual reproduction and seed sterility [In order to evaluate the epigenetic status of an invasive alien plant species such as  Cabrini , suggest sterile , spread  Cassani  and Cose Cassani , respectu et al.  and Coseu et al.  showed aterility . This abA. donax to diverse habitats, and might be responsible for the different phenotypes and physiological responses, as highlighted by Pilu, Cassani [A. donax is a good model organism for studies comparing biodiversity and population structure as obtained by AFLP and MSAP because of its vegetative reproduction. The differences observed in the populations studied here, using these two molecular approaches, are probably related to the different environments in which the populations are now thriving. Presumably, as plants cannot move around to find the best habitat, they had to adopt molecular mechanisms, such as epigenetic changes, to counteract diverse external stimuli  and, consequently, modify gene expression [A. donax is highly methylated (ca. 50% of the amplified loci were sensitive to methylation). It is known that epigenetic modifications are crucial for promoting phenotypic variation in living organisms [Ranunculus reptans L. and Rhamnus frangula L., respectively, to explain the late appearance of the invasive features with respect to the start of their habitat colonization. After each generation, the specimens would be better adapted to the local conditions, independently of their genetic profile [Our data are support the hypothesis that epigenetic mechanisms, and in particular DNA methylation status, may be involved in the adaptation of  Cassani . This st Cassani , 47–49;  Cassani , 50–52. pression , 56, 57.pression ), but thpression  and Haubpression  found eppression  demonstrrganisms –64, as arganisms . Epigenerganisms , 65, sugrganisms . Such a rganisms  and Fraprganisms  in the c profile .A. donax. Bayesian and non-Bayesian analyses showed consistent results for each profile analysed. In particular, when HpaII profiles were considered, the optimal number of populations and the recognized clusters present in the dendrogram were different compared to those obtained using AFLP and MspI profiles. Therefore, the different optimal population numbers underlie diverse memberships on the basis of DNA methylation status and a higher biodiversity. In general, when MspI and HpaII dendrograms were considered, the similarity coefficient decreased because of the different DNA methylation status of each A. donax specimen. Similar to the bar plot, the AFLP dendrogram well separated ADS and ADE specimens into clusters, although the similarity value and the cluster memberships varied when the two different molecular profiles were considered, revealing a variation of the clustering when compared with AFLP (genetic), or MspI (epigenetic) profiles.Our results, here reported, shed light on the effects of DNA methylation on biodiversity and on the invasive ability of This observation suggests that pedo-climatic conditions may generate variations in the DNA methylation status that drive the mechanisms of convergence and/or divergence of populations experiencing similar/dissimilar habitat; as suggested by Schulz et al. 2014 , this maA. donax, present in diverse regions in Italy, is very low. Moreover, the populations of Sardinia are genetically isolated and clearly differentiated from those growing in Emilia Romagna, suggesting different introduction routes of this plant species in the two Italian regions. The estimated indices and population structure show that the similarity decreases when MspI and HpaII profiles are considered, and, furthermore, that the population structure is altered by the different DNA methylation status. Our results suggest that the ability of A. donax to invade and thrive in diverse environmental conditions can be attributed, at least, in part to a higher epigenetic variability as previously hypothesized by Pilu, Cassani [In conclusion, our study confirms that the genetic biodiversity of the archaeophyte  Cassani  in the c Cassani  and by o Cassani , 26, Gua Cassani , 69, 70.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Document(PDF)Click here for additional data file.S2 Document(PDF)Click here for additional data file.S3 Document(PDF)Click here for additional data file.