Arachis accessions representing Arachis hypogaea botanical classes, A-, B-, and K- genome diploids, a synthetic amphidiploid, and a tetraploid wild species. RNASeq was performed on pools of three tissues, and de novo assembly was performed. Realignment of individual accession reads to transcripts of the cultivar OLin identified 306,820 biallelic SNPs. Among 10 naturally occurring tetraploid accessions, 40,382 unique homozygous SNPs were identified in 14,719 contigs. In eight diploid accessions, 291,115 unique SNPs were identified in 26,320 contigs. The average SNP rate among the 10 cultivated tetraploids was 0.5, and among eight diploids was 9.2 per 1000 bp. Diversity analysis indicated grouping of diploids according to genome classification, and cultivated tetraploids by subspecies. Cluster analysis of variants indicated that sequences of B genome species were the most similar to the tetraploids, and the next closest diploid accession belonged to the A genome species. A subset of 66 SNPs selected from the dataset was validated; of 782 SNP calls, 636 (81.32%) were confirmed using an allele-specific discrimination assay. We conclude that substantial genetic variability exists among wild species. Additionally, significant but lesser variability at the molecular level occurs among accessions of the cultivated species. This survey is the first to report significant SNP level diversity among transcripts, and may explain some of the phenotypic differences observed in germplasm surveys. Understanding SNP variants in the Arachis accessions will benefit in developing markers for selection.To test the hypothesis that the cultivated peanut species possesses almost no molecular variability, we sequenced a diverse panel of 22  Arachis hypogaea L.) is one of many polyploid species belonging to the genus Arachis, family Fabaceae, and is native to South America; many of these species come from a region including Brazil, Bolivia, and Paraguay , 831 (United States), and 582 (China) accessions  point toTechnology for DNA sequencing and SNP analysis has made great progress recently, both for high throughput and for low cost per sequence. Due to the ubiquity of SNPs, and the far greater power to identify polymorphisms than other types of marker analysis, sequencing is able to identify genetic diversity better than other marker types. RNASeq allows transcriptome profiling and SNP identification with high read depth at much lower cost than whole genome sequencing. Annotation of assembled RNASeq reads can help in understanding the function of a gene or a transcript. Annotations have been used in several crops for establishing genetic information where limited knowledge exists  to estimate number of genes in peanut, (c) to compare to the expected phylogenic relationships of the Arachis accessions, (d) to highlight SNPs specific to one accession or to a group of market types or to a group of different genome affiliations, and (e) to validate the SNPs obtained from the bioinformatics calls that can be used in a breeding program.The hypothesis of the current work is that, commensurate with the phenotypic differences observed in the field, there is actually a substantial amount of unrealized genetic diversity in peanut at the nucleotide level (both among the diploid and tetraploid species accessions). Although recent studies at the genome levels were carried out in two progenitor wild species . Accessions OLin, Tamrun OL07, Jupiter, and New Mexico Valencia C were sequenced on an Illumina GAIIx using 2 × 54 paired-end reads as reported previously . The GATK variant calling tool was used to identify SNPs and indels among these genotypes using default parameters for UnifiedGenotyper at a ploidy level of 2. Parameters of MQ  > 40.0, AF (Allele Frequency) > 0.10, and DP (Read Depth) > 50 were used to filter the SNP calls using vcftools  was performed using the File S3), including treatment with RNaseA during homogenization, and purified using the Qiagen DNAeasy miniprep kit . Sixty-six pairs of primers (File S4) were designed to perform validation of allele calls in the sequenced genotypes. For each putative SNP, two allele-specific forward primers, and one common reverse primer were designed .Genomic DNA was prepared from seed tissue of the 12 sequenced accessions  in a final volume of 10 µl containing 1× KASP Reaction Mix , 0.14 µl assay mix, and 10–20 ng of genomic DNA. The following cycling conditions were used: 15 min at 94°; 10 touchdown cycles of 20 sec at 94°, 60 sec at 65–57° (dropping 0.8° per cycle); and 26 cycles of 20 sec at 94°, 60 sec at 57°, and read at 37° for 5 sec. Fluorescence detection of the reaction was performed using a built-in scanner and the data were analyzed using the LightCycler 480 software .de novo the reads for each block and phase using parallelized runs. Haplotype information along with bam files were passed through readphaser to sort the reads within each block into phases based on HapCUT tables, and reassembled.To resolve homeologs in the peanut tetraploid reference sequence, we used the phasing approach. Genome-specific assemblies in tetraploid peanut using the phasing approach were generated by aligning OLin raw reads to the OLin reference transcriptome  using the BWA backtrack approach. Polymorphisms among the mapped reads were detected using the GATK software as it has been shown to perform well on RNAseq data  with defTo evaluate the resolution of the assemblies generated from each of these approaches, we mapped the raw reads back to each assembly. SNPs were called on each of these assemblies using GATK. Parameters used to evaluate assemblies were the number of heterozygous SNPs, homozygous SNPs, and number of SNPs per transcript.De novo assemblies and annotations described in this manuscript have been deposited at Figshare at the following URLs: https://dx.doi.org/10.6084/m9.figshare.3580650.v2, https://dx.doi.org/10.6084/m9.figshare.3580656.v2, https://dx.doi.org/10.6084/m9.figshare.3580659.v1, and https://dx.doi.org/10.6084/m9.figshare.3580662.v1.BAM files are deposited in SRA database under bioproject PRJNA248910: SRR1534396, SRR1535034, SRR1535035, SRR4039437, SRR4039359, SRR4039358, SRR4039357, SRR4039017, SRR4039016, SRR4039015, SRR4038966, SRR4038965, SRR4038285, SRR4037988, SRR4038281, SRR4038254, SRR4038252, SRR4038060, SRR4037987, SRR4037985, SRR4037986, and SRR4037959. A. hypogaea, including different subspecies, and originating from different continents . Annotation of the sequences from the consensus assembly identified numerous transcription factors. Transcription factors accounted for 4.4% of the 165,892 transcripts in the consensus assembly, and these were categorized based on their DNA-binding domain (File S2). Transcription factors were assigned to different groups; b-ZIP, MADS, MYB, AP2/ERF, and C2H2, were among the top five categories , New Mexico Valencia C (3451), and Tamrun OL07 (3923). Landraces had more SNPs relative to OLin, ranging from 7081 to 10,832 SNPs . SNP div to OLin .For the wild species, the number of SNPs relative to OLin was far higher: for A- genome diploid accessions, from 106,882 to 117,812 per accession; for B-genome diploids, from 96,241 to 109,797; for the K-genome diploids, 157,444 to 168,289; and for natural and synthetic amphidiploids, 14,340 and 87,574, respectively . OLin reA total of 40,382 unique SNPs was present among the cultivated accessions in 14,719 transcripts, and 291,115 unique SNPs were present among the diploid accessions in 26,320 transcripts . The aveA. magna GKSSc30097 compared to OLin was 32,430, more than the 18,092 unique SNPs between the tetraploid ancestor B- genome A. ipaënsis (GKPSSc30076) and OLin , and the synthetic amphidiploid A- and K- genome (TxAG-6), and the differences were reflected in the number of unique SNPs  was designed to validate the alleles called by the bioinformatic approach, and was tested on genomic DNA of 12 sequenced accessions (File S3). A total of 782 SNP calls (12 genotypes and 66 primers) was selected, including the calls from diploid and tetraploid accessions for validation using KASP technology. Genotyping of two diploid accessions with 66 primers confirmed 88.52% of the 122 SNP calls, while 10 tetraploid accessions confirmed 80.00% of the 660 SNP calls (File S3).Accuracy of SNP calls was demonstrated by validation of selected SNPs by KASP marker analysis. A subset of 66 primer pairs (Y axis) identified three groups: A-genome diploids as the first group; tetraploids, TxAG-6 (synthetic tetraploid) and K- genome as the second group; and B- genome diploids as the third group (X axis) separated TxAG-6, K-genome diploids, and the tetraploids from each other. Clustering of genotypes for tetraploids using PCA failed to reveal evidence of subspecies of hypogaea, even after the third PCA including 22 accessions. To distinguish subspecies of tetraploids, we employed the PCA approach on the same set of SNPs from the tetraploids separately, and it was possible to distinguish var hypogaea from var fastigiata  that contained >1 SNP, which were therefore good candidates for polymorphism phasing . Assuminde novo assembly approach, but had lesser representation of the transcriptome, as only 76–78% of the reads mapped back. Variant calls from phased assemblies indicated a sharp decrease in the number of heterozygous SNPs, and the number of contigs with multiple SNPs per transcript was lower compared to the de novo assembly . The raw reads aligning to the reference ranged from 84 to 89%, suggesting that the assembly used in this study had a good representation of the peanut transcriptomes. A total of 520 SNPs was identified by comparing the OLin transcriptome to the reference assembly; this may suggest the presence of gene copy number variation, call errors, or artifacts in the software used. The presence of SNPs when mapping reads back to the same reference has been cited previously (Medicago truncatula (1.96 SNPs per kilobase)  . Tetraploid accessions were used to confirm the SNP calls that could be eventually used in population studies. The overall validation success rate of 81.32% of 782 selected SNP sites was much higher than in a previous study by Another goal of this research was to identify SNP markers that could be useful in breeding programs. Of the 782 selected SNP calls tested, 81% were confirmed on the LightCycler 480, which is significantly better than results of previous studies in peanut . Collectively, these datasets have the potential for enabling SNP identification throughout the genome for any polymorphic gene of interest, thus making the tagging of specific genes with molecular markers a possibility.Annotations of the transcripts in the consensus assembly and the OLin assembly by de novo assembly suggest that there are about 25,000–31,500 unique transcripts in diploid and tetraploid Arachis roots, leaves, and developing pod tissues. This number could be a minimum value, as not all tissues were sampled. The number of transcripts in tetraploids could be twice that of diploids because of homeologous copies. Given the allo-tetraploid nature of the peanut, genes from both genomes are similar, and tools used in the study in general could not distinguish homeologous genes from each other. Consensus assemblies had more contigs than any other assembly by itself, which suggested that each genotype has a certain percentage of transcripts that could not be merged at a minimum sequence similarity threshold of 95%. This increase in the number of transcripts could be due to the larger differences among species/accessions used, or differences in expression of transcripts in samples. For tetraploids, the increase in number of transcripts in the consensus assembly can also be a result of the presence of homeologs. Consensus and individual assemblies from this study could benefit annotation of the genome for rare transcripts and gene expression studies.Results of de novo assembly parameters. Tetraploid SNP calls consisted of many apparently heterozygous state genotypes, and to reduce these heterozygous calls, we evaluated the phasing approach. We utilized the postassembly approach reported by de novo assembly. One way to estimate the reduction of homeolog collapse would be to observe the number of heterozygous variants upon mapping the reads back. Reduction in heterozygous SNP calls compared to the reference would be the first step for improving the assembly qualities.In the case of diploids, the success rate of SNP validation was greater than for tetraploid calls. Complexity of the tetraploid genome resulting from homeologous copies may have affected the outcomes of validation. Based on previous studies in complex polyploids, such as wheat  longer sequence reads, because shorter reads can affect the extension of contigs with the poor quality on the ends of reads; (b) higher read coverage to support the haplotypes, because lower read depth haplotypes could be dropped by the phasing based approach; and (c) using the genome sequence of diploid progenitor species to avoid collapse, and to obtain a better representation of transcripts.vs. homeologous SNPs for genetic mapping and QTL analysis, study of genome rearrangements after polyploidization, and expression bias among genes and genomes. Outcomes of this research will help increase of genomic resources in peanut, and benefit QTL mapping relevant for agronomic traits.We report the first survey of significant SNP level and transcript level diversity in a wide array of peanut germplasm. Highlighted untapped genetic variability among accessions may explain some of the phenotypic differences observed in germplasm surveys. Transcriptome analysis among cultivated accessions, and comparison to wild species will open many avenues for future research. Among these are relationships of cultivated tetraploid species to their diploid ancestors, evolutionary development of the cultigen, distinguishing of homologous


Methods: A randomized, double-blind, placebo-controlled trial was conducted in exclusively breastfed infants with colic, randomly assigned to receive a probiotic-mixture or a placebo for 21 days. A structured diary of gastrointestinal events of the infants was given to the parents to complete. Samples of feces were also collected to evaluate microbial content and metabolome using fecal real-time polymerase chain reaction (qPCR) and Nuclear magnetic resonance (NMR)-based analysis. Study registered at ClinicalTrials.gov (NCT01869426). Results: Fifty-three exclusively-breastfed infants completed three weeks of treatment with a probiotic-mixture (n = 27) or a placebo (n = 26). Infants receiving the probiotic-mixture had less minutes of crying per day throughout the study by the end of treatment period . A higher rate of infants from the probiotic-mixture group responded to treatment (defined by reduction of crying times of ≥50% from baseline), on day 14, 12 vs. 5 (p = 0.04) and on day 21, 26 vs. 17 (p = 0.001). A higher quality of life, assessed by a 10-cm visual analogue scale, was reported by parents of the probiotic-mixture group on day 14, 7.1 ± 1.2 vs. 7.7 ± 0.9 (p = 0.02); and on day 21, 6.7 ± 1.6 vs. 5.9 ± 1.0 (p = 0.001). No differences between groups were found regarding anthropometric data, bowel movements, stool consistency or microbiota composition. Probiotics were found to affect the fecal molecular profile. No adverse events were reported. Conclusions: Administration of a probiotic-mixture appears safe and reduces inconsolable crying in exclusively breastfed infants.Introduction: To investigate the effectiveness and the safety of a probiotic-mixture (Vivomixx Infantile colic is a benign, self-limited process characterized by paroxysms of inconsolable crying. It affects approximately 10% to 40% of infants worldwide .Infantile colic has been classified as a functional gastrointestinal disorder whose pathogenesis remains unknown, despite decades of research . ConsequRecent studies and preclinical data have suggested that changes of newborn intestinal microbiota can affect brain signaling systems related to pain, playing a pathogenetic role of infantile colic . For thiLactobacillus reuteri DSM 17938 (L. reuteri) seems to increase colic treatment success in meta-analysis [L. reuteri did not cause an increase in d-lactic acid beyond two weeks but d-lactic acidosis in infants who were fed lactic acid bacteria-containing products is still a concern in prolonged supplementation [Despite data not being conclusive, at present, only analysis ,11 and sanalysis ,13. L. rOther probiotics with a good safety profile have been evaluated for functional gastrointestinal disorders in pediatric patients. The high-concentrated multi-strain probiotic mixture, as specified below, has been proposed for the management of symptoms in children with irritable bowel syndrome (IBS) .Our research group has recently suggested a possible preventive role in infantile colic by using this multi-strain probiotic mixture through maternal supplementation during late pregnancy and lactation . This prClinicalTrials.gov: NCT01869426), performed to assess the efficacy and safety of a lyophilized high-concentration multi-strain probiotic mixture in reducing colic symptoms in exclusively breastfed infants.This was a double-blind, randomized, placebo-controlled clinical trial  section of the Department of Biomedical Science and Human Oncology, of “Aldo Moro” University of Bari, Italy.To be eligible for the study protocol, infants had to be healthy and well-fed, born at term, aged between 30 and 90 days, exclusively breastfed before and throughout all the study period, with a diagnosis of infantile colic  major acute or chronic diseases; (b) gastrointestinal diseases and gastro-esophageal reflux; (c) use of probiotics/antibiotics the week before or during randomization; (d) gastrointestinal malformations; and (e) concurrent enrolment in other clinical trials.Written informed consent was obtained from a parent/guardian, in accordance with the local ethics committee, which reviewed and approved the study protocol.All infants enroled were randomized to receive, either oral probiotics or the placebo. Randomization was performed using a computer-generated two-treatment allocation sequence . To avoid disproportionate numbers of patients in each group, a randomization scheme was performed in blocks of four participants. Randomization codes were secured until all data were analyzed.All participants, as well as scientific and medical personnel dedicated to the study, were blind to the group assignment. An independent person prepared the randomization schedule and took care of the packaging and labelling of the products.Parents were requested to administer to their infants 10 drops of the probiotics, once a day, for 21 days directly in the mouth, preferably in the morning before feeding. Instructions for keeping and maintaining the product were also provided according to manufacturer indications. Parental compliance was assessed by a diary. The parents were also asked to return used bottles.Even though maternal diet can influence colic frequency, no specific dietary restrictions during lactation were recommended, except to avoid any commercial products containing probiotics . ParentsL. paracasei DSM 24733, L. plantarum DSM 24730, L. acidophilus DSM 24735, and L. delbrueckii subsp. bulgaricus DSM 24734), three strains of bifidobacteria , and one strain of Streptococcus thermophilus DSM 24731, produced at Danisco-Dupont, WI, USA and currently sold in Continental Europe, USA and Korea under the brand Vivomixx®, Visbiome® and DeSimone Formulation respectively.The active study product was a medium chain triglycerides oil suspension containing a lyophilized high-concentration multi-strain probiotic mixture of four different strains of lactobacilli  of The placebo was characterized by an identical formulation, but without any probiotic. The active and placebo products were identical and supplied in the same bottle, in order to maintain a double-blind status. All study products were kept refrigerated until use. The randomization codes were secured during each phase of the trial and opened only after final data analysis.Both the active and placebo study products were donated by Prof De Simone/CD Investments who had no role in any phase of the study.According to the study protocol, when an infant was referred with a diagnosis of infantile colic and fulfilled all criteria for enrolment, parents were asked to record colic symptoms for one week. If symptoms fulfilled the “infant colic” definition during that period, infants were enrolled in the study protocol.A structured diary to record gastrointestinal events, such as a feeding schedule, daily episodes of fussing/crying, the number of minutes of inconsolable crying per day, the number of bowel movements per day, and stool consistency   was giveParents also recorded the times of administration of the study products, family quality of life, both at enrolment and at the end of the study, as well as any adverse events, such as constipation, vomiting, and skin reactions.Quality of life was assessed by a 10-cm visual analogue scale with a numerical rating scale from 0 (worst possible well-being) to 10 (perfect well-being), as already described previously . ParentsOn enrolment (day 0), a pediatric medical examination was performed and the following information was collected: (1) gestational age; (2) type of delivery; (3) birth weight; (4) anthropometric data at entry; (5) family history of gastrointestinal disease; and (6) family history of atopy.Follow-up visits were scheduled at 7, 14, and 21 days after the beginning of the administration of study products, performed by the same referring pediatrician.At the end of the study all the diaries were collected. Diary analysis and data entry were performed independently by two experienced investigators, both blinded from treatment allocation.To evaluate if probiotic supplementation showed a real impact on intestinal microbiota composition, two stool samples of infants were collected, once at enrolment and then 21 days after the beginning of the administration of study products. All samples were collected from diapers in sterile plastic tubes and stored at −80 °C until analysis. qPCR was used to quantify bifidobacteria and lactobacilli using genus-specific primers and conditions, as already described previously .1H-NMR) by mixing 80 mg of stool for 5 min with 1 mL of bi-distilled water and centrifuged at 4 °C and 18,630 g for 15 min. The supernatant (700 μL) was added to 100 μL of a D2O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mM, used as a reference for NMR chemical-shift, buffered at pH 7.00 by means of 1M phosphate buffer.Each sample of feces was also prepared for proton nuclear magnetic resonance  operating at a frequency of 600.13 MHz. According to Ventrella et al. [Each a et al. , a CPMG-1H-NMR spectra were baseline-adjusted by means of peak detection according to the “rolling ball” principle implemented in the baseline R package [To obtain quantitative values by NMR, 5 s of recycle were chosen, in agreement with the relaxation time of the protons under investigation . 1H-NMR  package ,24.Each spectrum was then linearly corrected, so to make the baseline points randomly spread around zero. Signals were not manually aligned, which was different from previous investigations .Probabilistic quotient normalization  was applThe signals were assigned by comparing their chemical shift and multiplicity with the Human Metabolome Database  and ChenThe primary outcome measures was the rate of responders and non-responders in improvement of colic symptoms. Success rate was defined by a reduction of the daily average crying time ≥50%, expressed in minutes.1H-NMR.Secondary outcome measures were: (1) average crying time per day; (2) parental quality of life; (3) any other gastrointestinal events; (4) anthropometrical evaluations; (5) side effects; (6) amount of lactobacilli and bifidobacteria in the stool samples of infants; (7) metabolomics evaluation of feces by means of A reduction of the daily average crying time ≥50% was used to calculate the sample size that was 26 patients per group. For α = 0.05, β = 0.10, and an estimated SD within groups of 55%, 26 patients were needed per group . ConsideData from the first visit forms, diaries, and the results of the analysis of stool samples were reported in a database created by Google Drive software .Statistical analyses were performed using Stata12MP  and a per-protocol approach.t-test. The Mann-Whitney U Test was used for non-normally distributed variables. Proportions were compared using the χ2 test or Fisher Exact Test, as appropriate.Quantitative variables with normal distribution were compared using the Student’s t values were calculated for linear regression. For all tests, a p value of <0.05 was considered as significant.Linear regression and logistic regression were performed to determine the effects of probiotic supplementation, vaginal delivery, family history of atopy and family history of gastrointestinal disease (determinants) on the duration of crying at 21 days, the total average crying minutes and the treatment response at day 14 and at day 21 (outcomes). In the logistic regression, the adjusted odds ratio, with 95% CI, were calculated and a z-score test was performed. Coefficiency with 95% CI and 21–T0 differences and then they were compared by means of Mann-Whitney U test.In the fecal metabolomics evaluation, molecules whose concentration varied in relation to the investigated treatments were looked for by calculating the TTo highlight the underlying trends characterizing the samples, a robust Principal Component Analysis (rPCA) was built on the molecules concentration, centered and scaled to unity variance, according to Hubert .0 were subtracted from every timepoint, as suggested by Ndagijimana [For each PCA model, we calculated the scoreplot and the projection of the samples in the PC space, tailored to highlight the underlying structure of the data. We calculated the correlation plot, relating the concentration of each variable to the components of the PCA model, therefore, tailoring the samples to highlight the most important molecules in determining the trends emphasized by the scoreplot. The same subjects were followed along the entire experiment, giving rise to a repeated measurement data structure. Molecule concentrations at timepoint Tgijimana .n = 33) or probiotics (n = 33). After 21 days of intervention, 13 infants failed the follow-up, so that 53 (26 in the placebo group and 27 in the probiotics group) completed the study  were evaluated with qPCR and metabolomics analysis. Characteristics of the infants at baseline are detailed in No significant differences were observed for age, sex, birth weight, and anthropometrical data. Vaginal birth, family history of gastrointestinal diseases, and atopy were significantly higher in probiotic group. At baseline, the average duration of crying was similar for the two groups.A higher and statistically significant rate of treatment success was found in the probiotic group, compared to the placebo group on day 14 and on day 21 .The total average crying minutes throughout the 21 days of the study were significantly less in the probiotic group compared to the placebo group .Parents of the infants of the probiotic group reported an improved family quality of life compared to the placebo group on day 14 and on day 21. Bowel movements and stool consistency did not differ between the two groups, as well as there being no differences of growth. Both the probiotic and the placebo were well tolerated and no side effects were reported.t = −3.92; 95%CI: −63.2–−20.3; p = 0.00) and a reduction also of the total average crying minutes throughout the study period . The treatment response on day 14  and on day 21  were associated with probiotic administration, and with no confounding effects from vaginal delivery, family history of gastrointestinal disease, and a family history of atopy (p > 0.05).Multivariate analyses confirmed that the probiotic group showed a reduction of crying on day 21  of lactobacilli in stool samples of the probiotic supplemented infants was observed  of the intragroup/intergroup samples distance in the 12 dimensions space.Interestingly, 10 out of 12 molecules showed opposite trends for the two groups, with acetate and methylamine representing the only exceptions. Consequently, samples at TTo have an overview of such findings, we calculated an rPCA model on the centered and scaled concentrations of these molecules .0 from every timepoint. This is why, in the scoreplot , with negative scores. PC 2, therefore, gives a holistic view of the different responses of infants, to the two treatments, evidenced by 21 appear differently from samples at T0 only for infants treated with probiotics (p < 0.01). The molecules that mostly contributed to the trends  produced during microbiota fermentation , whose fIn the present investigation, propylene glycol appeared as the clearest biomarker of subjects supplemented with probiotics. Propylene glycol is normally found in newborn feces and, interestingly, a higher concentration has been found in the feces of breast-fed infants in comparison with formula-fed ones, which suggesting a beneficial effect for this molecule .According to Gonzalez et al. , this isWe are awake of some limitations. First, we used a nonvalidated diary of infant crying duration. Thus, we fully relied on parents’ reports on the duration of crying recorded, and mothers filled in every hour throughout the day to minimize recall bias. Second, we are not able to report on potential counfounders, such as maternal depression, maternal diets and early use of probiotic/antibiotic.In conclusion, we speculate that the probiotic biological effects in this study are linked to microbial metabolomics with respect to the ability to colonize the gastrointestinal tract. Despite further studies being needed to fully understand these aspects, fucosylated milk oligosaccharides and their microbial degradation products seem to play a key role.Daily supplementation with a specific, high-concentration probiotic preparation to breastfed infants modulates infantile colic symptoms by the end of treatment (21 days). Although colic is generally a self-limiting condition, a variety of management modalities have been proposed to decrease parental anxiety, frustration, and stress, as well as medical interventions and increased risk for developing recurrent abdominal pain. Further studies are needed to confirm that this specific probiotic mixture may be used for infantile colic treatment.


The effect of cigarette smoking on the onset of nonalcoholic fatty liver disease (NAFLD) is unclear, especially that associated with drinking small amounts of alcohol. We conducted a longitudinal study to investigate the relationship between cigarette smoking and NAFLD onset, which was stratified according to the amount of alcohol consumed.We enrolled 7,905 Japanese subjects who had received annual health checkups more than twice between April 2003 and August 2013, 4,045 of whom met at least one of the following exclusion criteria and were excluded: (a) fatty liver at baseline; (b) hepatitis B or hepatitis C; (c) alcohol consumption ; (d) change in alcohol drinking status between baseline and the study’s endpoint; (e) change in cigarette smoking habits between baseline and the study’s endpoint; or (f) current treatment with antidiabetic agents, antihypertensive agents, and/or lipid-lowering agents. The remaining 3,860 subjects  were divided into two groups based on average alcohol consumption.p = 0.034). No association was found between smoking and fatty liver disease onset in the low alcohol consumption group . The fatty liver disease incidence increased significantly among the nondrinkers as the number of cigarettes smoked increased (p = 0.001).After adjusting for the variables associated with metabolic disease, smoking was associated with fatty liver disease onset compared with nonsmokers in nondrinkers (adjusted hazard ratio = 1.988, 95% confidence interval 1.057–3.595; Cigarette smoking may be a significant risk factor associated with NAFLD onset in nondrinkers. These results may help clinicians to identify patients who are at a high risk of developing NAFLD and to prevent the progression of NAFLD by promoting earlier interventions that help people discontinue unhealthy lifestyle habits. Nonalcoholic fatty liver disease (NAFLD) is a common lifestyle-related disease –3 that mNumerous studies have examined the relationships between cigarette smoking and lifestyle-related diseases, including type 2 diabetes, dyslipidemia, hypertension, and cardiovascular disease, and these studies’ findings have demonstrated that these pathological conditions are affected by cigarette smoking –10. SimiTo clarify the association between cigarette smoking and the onset of NAFLD, it may be appropriate to categorize subjects according to the amounts of alcohol they consume. Therefore, we conducted a longitudinal cohort study to assess the epidemiologic relationship between cigarette smoking and the development of NAFLD in subjects who consumed low amounts of or no alcohol at baseline, and to analyze this relationship in all of the subjects, regardless of the amount of alcohol consumed.We enrolled 7,905 subjects, comprising 3,863 men and 4,042 women whose ages ranged from 18 years to 80 years, who had undergone more than two annual health checkups between April 2003 and August 2013 at the Ehime General Health Care Association .Before their health checkups, all of the subjects were asked to complete a questionnaire that gathered baseline data about their medical histories, prescribed medications, exercise habits , snacking habits , sleep durations , alcohol consumption (frequencies and quantities), and the numbers of cigarettes smoked   was calculated based on a subject’s height and weight that were measured while the subjects wore light clothing and no shoes. Blood pressure was measured using an automated sphygmomanometer while the subjects were seated. Blood samples were collected after the subjects had fasted for ≥10 h, and the total cholesterol, triacylglycerol, fasting plasma glucose, uric acid, serum creatinine, hepatitis B surface antigen, and hepatitis C antibody levels were measured. Trained technicians who were blinded to each subject’s data, diagnosed fatty liver disease using abdominal ultrasonography (US) . Fatty liver disease was diagnosed based on the following established diagnostic criteria: hepatorenal echo contrast, liver brightness, deep attenuation, and vascular blurring .n = 1,773), they tested positive for the hepatitis B surface antigen (n = 115) or the hepatitis C antibody (n = 79), their average alcohol consumption was ≥210 g/week if they were male or ≥140 g/week if they were female (n = 633), their alcohol consumption status changed from baseline to the study’s endpoint , namely, from being a nondrinker to consuming low amounts of alcohol or vice versa, their cigarette smoking habit differed between baseline and the study’s endpoint (n = 493), that is, those who had newly stopped smoking or had newly started smoking, or because they were currently being treated with antidiabetic (n = 132), antihypertensive (n = 366), or lipid-lowering (n = 170) agents  agents  Tables.All of the participants were assigned a numerical code for the study period, and all of the data were stored on a secure database to maintain the subjects’ anonymity. Since this study was undertaken retrospectively, the requirement for informed consent to be obtained from the participants to enable them to participate in this study was waived. This study was conducted with a priori approval from Ehime University Hospital’s Research Ethics Board in accordance with the 1975 Declaration of Helsinki , and it fulfilled all local ethical and legal requirements. All of the procedures were performed in accordance with the Good Clinical Practice guidelines.th and 75th percentiles. The assumption of normality was analyzed using the Shapiro-Wilk test or the Kolmogorov-Smirnov-Lilliefors test, because the continuous variables were non-normally distributed. The Mann-Whitney U test was used to analyze the between-group differences in the baseline characteristics, which included age, the BMI, the blood pressure, and the biochemical variables derived from the routine blood tests. The categorical variables, including gender, the subjects’ baseline lifestyle habits, and the onset of fatty liver disease, are expressed as numbers and percentages, and they were analyzed using the chi-squared test. Univariate Cox proportional hazards regression analyses that used forward likelihood ratio tests were performed to evaluate the importance of each variable in relation to the incidence of fatty liver disease onset. The multivariate Cox proportional hazards regression analyses, which calculated the adjusted hazard ratios of the incidence of fatty liver disease associated with cigarette smoking, were adjusted with the following variables that are associated with metabolic disease: age, gender, the BMI, the systolic blood pressure, the total cholesterol, triacylglycerol, fasting plasma glucose, uric acid, and creatinine levels, exercise and snacking habits, and the sleep duration (Model 1). Alcohol consumption was additionally included as an adjustment to Model 1 to analyze the data irrespective of the amount of alcohol consumed. Model 2 included all of the factors that were found to be significant at p < 0.05 in the univariate analyses. The Cochran-Armitage test was used to assess the trends in the incidence rates of fatty liver disease based on numbers of cigarettes smoked. The Kaplan-Meier method was used to plot the fatty liver disease onset curves, and they were compared using log-rank tests. All of the reported p values are two-tailed, and a value of p < 0.05 was considered statistically significant.The statistical analyses were performed using JMP software for Windows, version 12 . The continuous variables are presented as the medians and the interquartile ranges that comprise the 25The mean observation period was 3.96 (standard deviation 2.5) years and the median observation period was 3.61 (range 0.44–10.13) years. Data were available from all of the subjects for all of the variables examined, except for the one subject’s BMI (0.023%). During the observation period, 443 subjects met the criteria for fatty liver disease onset. Tables A comparison of the nondrinker and low alcohol consumption groups with respect to the baseline lifestyle characteristics is shown in the Tables The variables found to be associated with metabolic disease were included in the multivariate analyses. Model 1 included all of the parameters investigated, namely, age, gender, the BMI, the systolic blood pressure, the total cholesterol, triacylglycerol, fasting plasma glucose, uric acid, and creatinine levels, the exercise and snacking habits, and the sleep duration, while Model 2 included the parameters that were significant in the univariate analyses, namely, age, being male, the BMI, the systolic blood pressure, the total cholesterol, triacylglycerol, fasting plasma glucose, uric acid, and creatinine levels, and snacking. A significant association was found between cigarette smoking and the onset of fatty liver disease in the nondrinker group .p = 0.001) (An investigation into the relationship between the number of cigarettes smoked and fatty liver disease onset in the nondrinker group showed that the incidence of fatty liver disease increased significantly as the number of cigarettes smoked increased (= 0.001) .The current smokers had a significantly higher risk of fatty liver disease onset based on the univariate analyses . In addiModel 1 included all of the parameters, namely, age, gender, the BMI, the systolic blood pressure, the total cholesterol, triacylglycerol, fasting plasma glucose, uric acid, and creatinine levels, exercise and snacking habits, and the sleep duration, while Model 2 included those parameters that were significant in the univariate analyses, namely, age, being male, the BMI, the systolic blood pressure, the total cholesterol, triacylglycerol, fasting plasma glucose, uric acid, and creatinine levels, snacking, and the consumption of low levels of alcohol. A significant association was found between smoking and fatty liver disease onset in Model 2 .p < 0.001). In the smoker and nonsmoker groups, the 1-year fatty liver disease onset rates were 3% and 1.4%, respectively, the 3-year fatty liver disease onset rates were 12.1% and 6.2%, respectively, the 5-year fatty liver disease onset rates were 22.9% and 11.6%, respectively, and the 10-year fatty liver disease onset rates were 48.4% and 20.1%, respectively Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Figp < 0.001).Kaplan-Meier curves of the event-free durations according to the subjects’ smoking habits. In the smoker and nonsmoker groups, the 1-year fatty liver disease onset rates were 3% and 1.4%, respectively, the 3-year fatty liver disease onset rates were 12.1% and 6.2%, respectively, the 5-year fatty liver disease onset rates were 22.9% and 11.6%, respectively, and the 10-year fatty liver disease onset rates were 48.4% and 20.1%, respectively. The cumulative onset rate of fatty liver disease was significantly higher in the smokers compared with that in the nonsmokers ((EPS)Click here for additional data file.S1 Questionnaire(PDF)Click here for additional data file.S2 Questionnaire(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes and TM7. At genus level, many of the dominant rhizosphere genera  displayed different patterns of temporal changes in the rhizosphere as opposed to the bulk soil, showing rhizosphere has more impact on soil microorganisms. Besides, we found that significant growth-related dynamic changes in bacterial community structure were mainly associated with phylum Bacteroidetes, Proteobacteria and Actinobacteria , indicating that different growth stages affected the bacterial community composition in maize soil. Furthermore, some unique genera in especial Plant-Growth Promoting Rhizobacteria (PGPR) such as Nonomuraea, Thiobacillus and Bradyrhizobium etc., which were beneficial for the plant growth appeared to be more abundant in the rhizosphere than bulk soil, indicating that the selectivity of root to rhizosphere microbial is an important mechanism leading to the differences in the bacteria community structure between rhizosphere and bulk soil.In this study, we designed a microcosm experiment to explore the composition of the bacterial community in the rhizosphere of maize and bulk soil by sequencing the V3-V4 region of the 16S rRNA gene on the Illumina system. 978–1239 OTUs (cut off level of 3%) were found in rhizosphere and bulk soil samples. Rhizosphere shared features with the bulk soil, such as predominance of  Soil microorganisms are not only an important part of soil, but also the main driver of soil nutrient cycling . Soil miacillus, Pseudomonas, Enterobacter, Acinetobacter, Burkholderia, Arthrobacter and Paenibacillus are the common PGPRs in the rhizosphere [Plants exert selective pressure on the structural and functional diversity of microbial populations through the root exudation, and in relation to soil properties, plant species, growth stage, and many other stress factors . Rhizospzosphere .Nelson  suggesteIt has been estimated that the microorganisms which can be cultured in laboratory conditions occupied only a small part of these populations (<1%). With the continuous advances of high-throughput pyrosequencing technologies, the research on the microbial flora in the soil has become more and more thoroughly, thus provides a possibility for comprehensive analysis of the taxonomy, phylogenetic, and functional diversity of microbial communities , 11–12.Since maize is one of the most important economic crops and the microbe structure in rhizosphere plays a crucial role in plant growth, it is imperative to give a better understanding of their individual roles. Thus, in this work we have carried out a microcosm experiment, with maize as an ideal plant for investigation. The aim was to assess and add some new insight into the influence of growth stage on composition of the bacterial community in the rhizosphere and bulk soil. It was also of interest to identify dominant and sensitive soil bacteria and in the hope of contributing to provide relevant data for further research.-1 organic carbon content, a pH of 6.23, an ion exchange capacity of 18.0 cmol·kg-1, 19.4% clay, 75.8% silt, and 4.8% sand. Manure and soil were mixed with an electric mixing machine at a ratio of 1:25 (w/dw), and the soil was filled into polystyrene tubes  that were planted with maize (Zea mays L.). The plants were kept in green house with a day/night cycle of 16:8 h. Water loss was replenished daily to keep soil moisture varied between 8.5 and 15.2% (w/dw).The study was permitted and approved by the Ministry of Environmental Protection, China. The land accessed was not privately owned or protected. No protected species were sampled. Manure was collected from pigs at the experimental farm of Jiangsu Province Tomorrow Agriculture and Animal Husbandry Science and Technology Co., Ltd. The soil used for microcosm were paddy soil from the experimental farm of Nanjing Institute of Environment Science, MEP with 49.8 g·kgSoil samples were collected after maize had grown for 14d, 35d, and 63d. The maize roots were shaken vigorously to remove loosely adhered soil which were defined as bulk soil (BK), while the soil that tightly adhered to the roots was regarded as rhizosphere (RH), soil samples were named BK-1, BK-2, BK-3, and RH-1, RH-2, RH-3, respectively. All samples transported on ice to be stored at 4°C before analysis in the lab. The total DNA extraction from rhizosphere and bulk soil samples was performed employing a PowerSoil DNA Isolation Kit  according to the protocol. The concentration and quality of the extracted total DNA were examined by UV spectrophotometry  and 1% agarose gel electrophoresis.5′–ACTCCTACGGGAGGCAGCAG-3′ and V4 reverse primer was 5′- TACNVGGGTATCTAATCC-3′. The PCR procedures were as follows: 94°C for 3min as an initial denaturation, 30 cycles of 94°C for 30s, 62°C for 30s, 70°C for 45s, and 72°C for 7 min as a final extension. A total volume of reaction mixture was 50μL which consisted of 1×Pfx Amplification Buffer , 0.4 mM dNTP, 2 mM MgSO4, 0.4 μM each primers, 1 μL of template DNA and 2 U of Platinum® Pfx DNA Polymerase . The extracted DNA was subject to high-throughput pyrosequencing using TruSeq SBS Kit V3 on Illumina Hiseq 2000 . The Illumina pyrosequencing strategy was “Index 101 PE” , which generates nearly equal amount of clean reads for each sample. For quality control, the raw reads containing three or more “N” or contaminated by adapter (>15 bp overlap) were removed.For gene libraries construction, the bacterial 16S rRNA gene was amplified by PCR with a set of primers targeting the hypervariable V3–V4 region (about 460 bp). The V3 forward primer was After 454 pyrosequencing, the clean high-quality sequence were grouped into operational taxonomic units (OTUs) using the Quantitative Insights into Microbial Ecology (QIIME) at 97% sequence identity . RepreseAfter sequencing the V3-V4 region, a total of 218905 raw reads were generated from soil samples. As shown in For detail, 978–1239 OTUs (cut off level of 3%) were found in rhizosphere and bulk soil samples. The OTUs first increased and then decreased in all soil samples as maize growth progressed, and were consistently higher in rhizosphere than those in bulk soil.The Shannon index yielded values ranging from 5.330 to 6.228 for rhizosphere and from 5.240 to 5.769 for bulk soil, suggesting a relatively higher diversity of bacterial sequences in the rhizosphere. The values of ACE for samples from rhizosphere were ranged from 1285.612 to 1479.729, whereas those for samples from bulk soil were 1337.056 to 1335.411. Overall, both the index of Chao, ACE, Shannon and Simpson revealed similar trends, with higher values observed for rhizosphere samples, when compared with samples from bulk soil, indicating that the higher abundance of bacterial community in rhizosphere, however, the difference among them was not significant.Rarefaction curve was the common used method to compare species diversity in the ecosystem. The rarefaction curves of the two soil groups were also drawn in this study . ResultsAcidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes and TM7, accounted for more than 90% of bacterial sequences in both rhizosphere and bulk soil.The average relative abundances are presented in Proteobacteria (19.3%~40.2%), Bacteroidetes (19.1%~30.8%), Firmicutes (5.4%~19.4%), Actinobacteria (6.3%~11.7%) and Acidobacteria (6.5%~15.0%). Bulk soil was mostly comprised by Proteobacteria (15.8%~38.3%), Bacteroidetes (27.2%~32.2%), Firmicutes (8.1%~16.2%), Acidobacteria (6.3%~14.1%) and Actinobacteria (5.4%~11.9%). WS3, Chlorobi, Cyanobacteria, Elusimicrobia, Nitrospirae, Planctomycetes and Tenericutes were present in most soil samples, but at relatively low abundance (bundance .Many sequences could not be classified but were abundant in rhizosphere and bulk soil (1.22%~18.95%), demonstrating that soil remains a challenging reservoir of biodiversity. Moreover, the ratio of sequences in rhizosphere and bulk soil was 63d>35d>14d, indicating that bacterial diversity was modified with root activity at different sampling times.Chitinophaga, Flavisolibacter, Nitrospira, Pseudomonas and Streptomyces which were predominantly found in the rhizosphere and bulk soil, belong to Bacteroidetes, Proteobacteria and Actinobacteria, respectively.A thorough investigation at the genus level showed an enrichment trend of predominant bacterial groups in soil samples. The relative abundance of any genus was <7% in each sample, implying high bacterial diversity in the two sample groups. As shown in Tables Streptomyces, Burkholderia, Caulobacter, Devosia, Glycomyces and Ralstonia was higher in rhizosphere than bulk soil, while DA101, Dyadobacter, Niastella, Pseudomonas and Sorangium demonstrated the reverse.The relative abundance of Vrhodoplanes, Streptomyces, Nocardia, Actinomadura, Amycolatopsis, Dokdonella and Niastella significantly increased in 35d and/or 63d in bulk soil, rising by 119.01%-3549.86%. In the rhizosphere, Nocardia, Pseudoxanthomonas, SMB53, Sphingobacterium, Turicibacter, Actinomadura, Amycolatopsis and Dokdonella was elevated by 308.97%-3747.85%, compared to the last sampling time.The OTU abundance varied from sampling times. For example, Niastella and Flavisolibacter in rhizosphere was decreased in 35d and then increased in 63d, while difference occurred in bulk soil, with a continued increase from 14d to 63d. The results suggest that bacterial community composition was altered markedly to a greater extent in the rhizosphere than bulk soil, indicating that rhizosphere bacteria may respond first to environmental changes ahead of bulk soil.The abundance of Nonomuraea), nitrogen fixing bacteria , phosphate solubilizing bacteria (Thiobacillus and Erwinia), and pathogenic bacteria (Rickettsia); while, 5 unique genera detected in bulk soil were divided into: phosphate solubilizing bacteria (Gemmatimonas), nitrogen fixing bacteria (Azospirillum and Actinomycetospora), pathogenic bacteria (Aquicella and Conexibacter). In rhizosphere, most unique genera bacteria belongs to PGPR, and the main function were phosphorus solubilizing, nitrogen fixation, and degrade cellulose, while in bulk soil belongs to PGPR and pathogenic bacteria.In this study, some unique genera in rhizosphere and bulk soil were found, respectively . Among tSoil is a very complex ecological system, and the soil microorganism plays an important role in the soil. High-throughput pyrosequencing is considered to be a good method to study soil microbial diversity, which can be used to carry out high quality sequencing of DNA specific sections of soil microbial communities. In this study, the diversity, composition, and relative abundance of bacterial community in rhizosphere and bulk soil were represented at different sampling times during the maize growth process, via Illumina high-throughput sequencing. The results provided insights into the effects of alterations in maize root exudates on microbial community.After sequencing the V3-V4 region of 16S rRNA, 215903 effective sequences were obtained in total with 35983 sequences per sample on average, then clustered into 978~1167 OTUs at 97% similarity level. By comparing with the database, we found that the composition of microbial community in rhizosphere was similar but higher than bulk soil on the phylum level. However, with the growth period of maize, the relative abundance of each phylum varying in soil samples, which may be related to soil structure, nutrients, root exudates and other factors.Proteobacteria is the predominant phylum in rhizosphere may due to their rapid growth rates and, this nutrient-rich environment was suit for the phylum Proteobacteria, or certain classes within this phylum [Acidobacteria were similar regardless of crop variety (grass or wheat) and land management practice [Bacteroidetes in intestinal accounted for more than half of total bacteria, thus can be widely detected in manure and manure-amended soil. Actinobacteria are widely founded in soil and water ecosystem and play a critical role in the decomposition and humus formation process [Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes, Firmicutes and Plantcomycetes, although recognizing that their relative abundances vary with the study site.s phylum . This gepractice . The num process . Overall process , 18. TheChitinophaga, Nitrospira, Flavobacterium, etc.) displayed different patterns of temporal changes in the rhizosphere as opposed to the bulk soil . Members of Burkholderia were enriched in the rhizosphere, possibly due to their versatile abilities to utilize root metabolites, degrade aromatic compounds [Pseudomonas has rapid growth and is therefore good colonizers in soil. Pseudomonas can use various substrates as nutrients and survive under different stressing conditions. Also its ability to produce various compounds, such as antibiotics, polysaccharides and siderophores are crucial to its success. Li et al [Flavobacterium may be copiotrophic and fast-growth bacteria, able to exploit a transient niches for nutrition and others at the early growth stage of maize. The decline of Flavobacterium at later stages may be explained by the reduction of quantity and quality of organic carbon, or interspecies competition for resources.There were several key microbial groups that displayed different temporal dynamics in rhizosphere from that in the bulk soil. In fact, many of the dominant rhizosphere genera (l Tables  and 3, sompounds  and prodLi et al  reported29 live cells in soil [There were more than 2.6×10 in soil , but maj in soil .It is essential to give insight to the microbial biodiversity since the bacterial diversity and soil function are correlated. Most of the bacteria in the rhizosphere are phosphate solubilizing, nitrogen, fixing and plant disease suppressing which helps to improve the crop yield and protection. In rhizosphere, most unique genera bacteria belongs to PGPR, and the main function were phosphorus solubilizing, nitrogen fixation, and degrade cellulose, while in bulk soil belongs to PGPR and pathogenic bacteria.These changes in the bacterial species may have a close relationship with the root exudates of maize. For example, Nitrogen fixing bacteria are very sensitive to sugar and amino acids in root exudates, thus leading to a large number of bacteria with nitrogen fixing function in the rhizosphere . BesidesAt this present study, we have analyzed the maize bacterial community in the rhizosphere and bulk soil at different growth stages. Overall, the abundance and diversity of microbial community in rhizosphere were higher than bulk soil, and related closely with maize growth stages. Besides, some growth-promoting microorganisms beneficial for the plant growth appeared to have been more abundant in the rhizosphere than bulk soil, indicating that selectivity of root to rhizosphere microbial is an important mechanism leading to the differences in the bacteria community structure between rhizosphere and bulk soil.


Eptesicus that cannot be distinguished from recent North American Eptesicus fuscus. The remaining specimen, a horizontal ramus with m3, is from a smaller vespertilionid bat that cannot confidently be assigned to a genus. Although many vespertilionid genera can be excluded through comparisons, and many extinct named taxa cannot be compared due to nonequivalence of preserved skeletal elements, the second taxon shows morphological similarities to small-bodied taxa with three lower premolar alveoli, three distinct m3 talonid cusps, and m3 postcristid showing the myotodont condition. It resembles especially Nycticeius humeralis and small species of Eptesicus. Eptesicus cf. E. fuscus potentially inhabited eastern North America continuously since the late Hemphillian land mammal age, when other evidence from the Gray Fossil Site indicates the presence in the southern Appalachian Mountains of a warm, subtropical, oak-hickory-conifer forest having autochthonous North American as well as allochthonous biogeographical ties to eastern Asia and tropical-subtropical Middle America.Thousands of vertebrate fossils have been recovered from the Gray Fossil Site, Tennessee, dating to the Miocene-Pliocene boundary. Among these are but eight specimens of bats representing two different taxa referable to the family Vespertilionidae. Comparison of the fossils with Neogene and Quaternary bats reveals that seven of the eight specimens pertain to a species of  Sinomenium, having subtropical-tropical Asian affinities), and subject to occasional drought and fires and browsing by large mammalian herbivores  is a small but significant late Miocene-early Pliocene vertebrate fossil locality occurring within the Valley and Ridge Zone of the Appalachian Mountains physiographic province. In this region the Valley and Ridge Zone exhibits Paleozoic rocks distorted by numerous broad folds and synclinoria that have also been cut by thrust faulting. The Paleozoic rocks include limestone, dolomite, and marble, providing soluble rocks of the Valley and Ridge karst region, one of the most extensive and important karstic regions in the United States . Prior trbivores . Non-mameloderma . Preserveloderma . Remainse 5.5 °C . GFS is e 5.5 °C . Certaine 5.5 °C . AlthougThe first eight fossils of bats recovered from sedimentary deposits at the Gray Fossil Site by the staff and affiliates of the Gray Fossil Site and East Tennessee State University Museum of Natural History as of 2016 were graciously loaned to the author for study. The fossils were identified by comparison with casts of Neogene North American fossil bats and with skeletal material of recent bats in the Oklahoma Museum of Natural History. Occlusal terminology of the teeth and humeral terminology follow Material: East Tennessee State University Museum of Natural History (ETMNH) specimen number 9714, right M1; ETMNH 19286, left edentulous dentary fragment with partial alveoli for p4-m3; ETMNH 19287, left partial dentary with m2 and condyloid and angular processes; ETMNH 19288, right m2 possibly associated with 19287; ETMNH 9654, partial right petrosal; ETMNH 14022, left distal humerus; ETMNH 9755, left proximal radius.Measurements (in mm): ETMNH 9714, M1 anteroposterior length (apl), 1.8; transverse width, 2.05. ETMNH 19286 depth of dentary beneath posterior alveolus of m1, 1.7. ETMNH 19288, right m2 apl, 1.8; trigonid width, 1.1; talonid width, 1.2. ETMNH 19287 left m2 apl, 1.9; trigonid width, 1.1; talonid width, 1.2. ETMNH 14022 distal humerus midshaft diameter, 1.5; greatest transverse distal width, 3.5; extension of trochlea beyond spinous process, 0.3; proximo-distal diameter of trochlea (in anterior view), 1.8; anteroposterior diameter of trochlea , 2.2; anteroposterior diameter of lateral ridge of capitulum , 1.9; transverse width of medial epicondyle from medial edge (lip) of trochlea, 0.6; width of distal articular surface (trochlea to capitular tail), 2.9. ETMNH 9755 proximal radius greatest width, 2.5 mm.The right upper molar ETMNH 9714 appears to be M1, because its length and width are more nearly equal to one another than in M2s, which are anteroposteriorly shorter and transversely wider –1C. In oE. fuscus. The condyloid and angular processes of the dentary are similar in size and shape to those in E. fuscus, in which some intraspecific variation occurs among the comparison specimens.The m2s in ETMNH 19287 –1H and 1E. fuscus. The edentulous fragment includes the alveoli for a two-rooted p4. Mesial and slightly lingual to the p4 anterior alveolus is the bottom of another premolar alveolus ).ETMNH 9654 is a damaged right petrosal missing most of the pars cochlearis and parts of the crista parotica and lateral semicircular canal –1L. The The distal humerus ETMNH 14022 represents a moderately large vespertilionid –2E. The E. fuscus.The proximal radius ETMNH 9755 is typical of that of vespertilionids –2J. On tCorynorhinus, Myotis, Nycticeius, Parastrellus, Perimyotis, and Plionycteris in being larger and more robust and in details of cusp and crest shapes. It differs from the M1s of Antrozous and Lasiurus mainly in that the postprotocrista does not connect to the base of the metacone, and from Simonycteris in having the lingual cingulum absent at the base of the protocone. It is essentially identical to the M1 in modern specimens of Eptesicus fuscus.In morphological features of the M1, the GFS specimen differs from E. fuscus. In the edentulous partial dentary ETMNH 19286, no portion of a large canine alveolus is apparent mesially due to breakage, so it is not possible to determine whether more than one small lower premolar was present between the lower canine and the large p4 in this specimen. However, because the preserved alveolar configuration of the ramus fragment and its size and are consistent with those in dentaries of E. fuscus, the specimen is referred to E. cf. E. fuscus along with the teeth and tooth-bearing dentary in the GFS sample.The crown morphology and size of the m2s, one of which is still retained within a partial dentary with the ascending ramus, closely match the same elements in Plecotus in lacking a completely laminated posterior semicircular canal, in having a much larger stapedial fossa, and in possessing an anterior petrosal process. It differs from Corynorhinus in having a less completely laminated posterior semicircular canal, in possessing an anterior petrosal process, and in having a lower fenestra cochleae relative to its width. Compared to Barbastella the fossil has a broader stapedial fossa, a narrow U-shaped notch immediately anterior to the anterior petrosal process instead of a broad connection of the process to the bone near the anterior ampulla, and thin rather than thick bone making up the prefacial commissure bridging the facial canal. It differs from Antrozous in lacking a sharp flange along the length of the anterior semicircular canal  and a curved rather than nearly straight opening of the fenestra cochleae. It differs from Lasiurus in having a narrow U-shaped notch anterior to the anterior petrosal process, a broader stapedial fossa, and a larger opening of the vestibular aqueduct that extends higher along the common crus. The unbroken portion of the pars cochlearis lacks evidence of the broad flange extending medially toward the basioccipital in Nyctalus, Vespertilio, and Lasiurus , and bears a rather stout tubercle ventral to the fenestra cochleae that is absent in Nyctalus, Vespertilio, and Myotis. The GFS petrosal further differs from that of Myotis in having a less completely laminated posterior semicircular canal, a small tubercle ventral to the fenestra cochleae, and a larger, flared opening of the vestibular aqueduct extending higher along the common crus. It differs from the petrosal of Lasionycteris in having an open (unlaminated) posterior semicircular canal. It differs from that of Perimyotis in having a less completely laminated posterior semicircular canal and a small tubercle ventral to the fenestra cochleae. It differs from the petrosal of Nycticeius in having a less completely laminated posterior semicircular canal and a long anterior petrosal process. It differs from that of Pipistrellus in having a tubercle ventral to the fenestra cochleae and a less completely laminated posterior semicircular canal. In morphology the petrosal is a close match with petrosals of E. fuscus, and the size also matches that of E. fuscus. Without comprehensive samples of modern bat petrosals for comparisons, and without assessments of petrosal variation within and among genera and species, it is difficult to judge individual variability in this element and provide a precise identification of ETMNH 9654. Because of its close similarity to the petrosal of modern E. fuscus, and in light of the relative abundance of other craniodental fossils referred to Eptesicus herein, this petrosal is tentatively referred to the same taxon.Petrosals of very few extinct late Paleogene and Neogene North American bats have been described in detail; they belong to families other than Vespertilionidae . By comparison with petrosals of several families of recent bats available for comparison, the GFS petrosal is representative of the structure in certain vespertilionids. Although exhaustive comparisons were not made throughout the diverse and globally-distributed family, the GFS petrosal resembles that of many vespertilionids in having a flattened anterior petrosal process separated anteriorly from the prefacial commissure by a narrow U-shaped notch, in having the anterior ampulla visible externally as a prominent swelling, and in having the fenestra cochleae much wider than high. Some obvious differences serve to differentiate the Gray Fossil Site petrosal from petrosals of several widespread genera of vespertilionids. The fossil differs from the petrosal of Miomyotis and Suaptenos based solely on nearly complete humeri from the early Miocene Thomas Farm locality in Florida. Another Thomas Farm bat, Karstala, is also represented by fossils of its humerus , have aEptesicus from other European vespertilionids in having the distal humerus with a spinous process that does not extend beyond the trochlea, a transition between the trochlea and medial ridge of the capitulum on the joint surface that is concave proximally, and the proximal tip of the trochlea reaches the contour of the epiphysis in anterior view. In the GFS humerus the first two of these characters are met, but the proximal tip of the trochlea does not reach the outline of the epiphysis. The bone is virtually identical to the humerus of modern Eptesicus fuscus in details of shape, except that the notch between the spinous process and distal edge of the trochlea is deeper and more distinct. However, this feature can be individually variable: one of four specimens of modern E. fuscus examined had a shallower notch whereas the other three had no notch in this area. Alternatively, if additional fossils are eventually found that consistently bear the distal notch, they might help determine whether the Hemphillian GFS bat is a distinct species of Eptesicus.Material: ETMNH 19285, right dentary horizontal ramus with m3 and alveoli for all of the other lower teeth.Measurements (in mm): The m3 measures 1.2 mm in anteroposterior length, 0.7 mm in trigonid width, and 0.6 mm in talonid width. Dentary depth below the anterior alveolus of m1 is 1.25 mm. Alveolar length from c1-m3 is 5.0 mm.E. fuscus. It indicates an animal near the size of the extant eastern North American species Lasiurus borealis. The alveolar formula indicates 3i, 1c, 2 or 3 p, 3m. The canine alveolus is relatively small. The three lower premolar alveoli are subequal in size, with the first and third equal and the middle one slightly smaller. For the lower premolars these three alveoli ostensibly do not allow a determination of the presence of a single-rooted p3 and double-rooted p4 or of three single-rooted lower premolars p2, p3, p4. However, in bats with three single-rooted premolars, the p3 root is typically smallest, that of p2 larger, and that of p4 largest, usually much larger than that of the adjacent p3, whereas in bats with a two-rooted p4 the roots are nearly equal in size. The m3 is relatively unreduced with low trigonid cusps and a narrower but relatively wide, basined talonid that retains three distinct and well-developed cusps, hypoconid, entoconid, and hypoconulid. The m3 postcristid exhibits clear myotodonty.Description: The ramus is from a small vesper bat, much smaller than Antrozous, Barbastella, Bauerus, Eptesicus, Histiotus, Ia, some Lasiurus, Nycticeius, Otonycteris, Perimyotis, Parastrellus, Rhogeessa, Scotomanes, and Vespertilio, whereas the 1 + 1 + 1 count occurs in Corynorhinus and Idionycteris. Among northern hemisphere Neogene bats of North America and Eurasia, ETMNH 19285 differs from Ancenycteris, Hanakia, Eptenonnus, Quinetia, and Submyotodon, and from the extant species  of Lasionycteris, Myotis, some Lasiurus, Plecotus, Euderma, Scotoecus, and Scotozous in having three lower premolar alveoli. ETMNH 19285 differs from Barbastella, Nyctalus, Scotoecus, and Scotozous in showing myotodonty rather than nyctalodonty of the molar postcristid. It differs from Otonycteris and Scotomanes in having m3 with a well-developed rather than greatly reduced m3 talonid. ETMNH 19285 differs from Karstala, Samonycteris, Idionycteris, Histiotus, Ia, Antrozous, Bauerus, Otonycteris, Nyctalus, Scotoecus, Scotomanes, and Scotozous in its much smaller size, and from Parastrellus in its much larger size . Compared to Corynorhinus and Idionycteris, premolar alveoli in the GFS fossil are more nearly equal to one another in size rather than having the p4 alveolus larger than those of p2 and p3. Moreover, the m3 trigonid cusps–especially the protoconid–are distinctly shorter than in Corynorhinus, and the m3 hypoconulid is better developed. Compared to Lasiurus ( subgenus Lasiurus), Lasiurus (Dasypterus), Rhogeessa, and Scotomanes, the GFS fossil differs in having a stronger talonid on m3 with better developed hypoconulid and hypoconid. It has a deeper, more robust horizontal ramus and more robust teeth than Perimyotis subflavus. Comparisons cannot be made with the Neogene vespertilionids Paleptesicus , Nycticeinops, Vespertilio, Miostrellus, and Pipistrellus. Because these genera cannot be distinguished from one another based on the morphological features preserved in ETMNH 19285, it is not possible to assign the GFS specimen to a genus.Discussion and Comparisons: Among recent vespertilionid genera with three root sockets between the lower canine and the first lower molar, there are two possible premolar configurations, a single-rooted p3 and double-rooted p4 (1 + 2), or single-rooted p2, p3, and p4 (1 + 1 + 1). The premolar alveolar count of 1 + 2 is known in nly; see ), PlionyEptesicus of several species have been described from the Miocene of Europe  occurred in the late Miocene  of the Redington fauna, Arizona  at Beck Ranch, Texas (Eptesicus sp. in the late Pliocene (late Blancan) at Inglis 1A, Florida (Eptesicus fuscus in the Pliocene (Blancan) in San Bernardino County, California  as far north as the Arctic Circle, and southward through many parts of Eurasia and Africa. In the Neotropical region it extends southward through Central America and South America, south to central Argentina, as well as in northern and southern Africa, and southeastern Asia . The genus has considerable recent diversity and contains about 25 extant species of small to large body size for the family  cannot be distinguished by morphological characteristics of skeletal elements. At one time E. serotinus, but later authors reversed this designation  having eastern Asian affinities (Pristinailurus and Arctomeles, related to the red panda and Eurasian badger (E. fuscus should also be looked for in eastern Asia.In any case, the few GFS fossils cannot address these questions unless better more complete specimens are found. A Miocene biogeographic connection for tropical forest between eastern Asia and western North America via Beringia is already well established . The fosfinities , as welln badger , respectEptesicus in the late Neogene of eastern North America, the other being the Florida Pliocene record. Recent bats of the genus Eptesicus occur on six continents and are tolerant of a wide range of habitats and environmental conditions from lowlands to highlands and rain forest to desert (Eptesicus fuscus itself is widespread in the extant biota of northwestern South America, Central America, many Caribbean Islands, and North America throughout the United States and much of southern Canada. It is a habitat generalist and utilizes a variety of habitats across this broad range (E. cf. E. fuscus and the second unidentified vespertilionid cannot provide new information relevant to paleoenvironmental interpretations of the GFS; nevertheless, they are consistent with earlier interpretations of subtropical oak-hickory forest for the area in the late Hemphillian. Stable isotopes recovered from GFS browsing mammals suggest that in the late Miocene-early Pliocene there was minimal seasonal variation in temperature and precipitation at GFS (This report includes the first records of bats from the Gray Fossil Site, and the second record of o desert . For exaad range ; foraginad range . As a ren at GFS . Togethen at GFS  and a prn at GFS , these p


Madurella mycetomatis. Two months prior to presentation to the MRC, a discharging sinus in the left lateral side of the chest wall was noted by the patient. The discharge was purulent and contained black grains. Just before presentation, air leak from the sinus was noted and she developed general weakness and deterioration.We report on a 60-year-old housewife who presented to the Mycetoma Research Centre (MRC) at the University of Khartoum on the 7 December 2016 with a 30-year history of chest wall eumycetoma due to The patient was noncompliant with ketoconazole therapy for eumycetoma and has had 3 previous surgical excisions of her chest wall lesion; the first one was in 1991, the second one was in 1995, and the third excision was done in 2002. However, she ceased treatment and follow-up in 2002.The patient has had diabetes mellitus for 14 years and hypertension for 1 year. She has various diabetic microvascular complications, diabetic nephropathy, and diabetic septic foot, leading to left above-knee amputation in 2015, and she later developed right gangrenous middle and fourth toes. Her drug history includes insulin 10/5 units, furosemide 40 mg, aspirin 81 mg, atorvastatin 20 mg, and amlodipine 10 mg per day. She has had multiple hospital admissions and blood transfusions due to chronic kidney disease secondary to diabetic nephropathy.She is a housewife with a low socioeconomic status. A family history of mycetoma was noted; her son has bilateral lower limb eumycetoma.Clinically she looked unwell and pale. She was haemodynamically stable, with a pulse rate of 72 beats per minute, respiratory rate of 20 breaths per minute, and blood pressure of 130/70 mmHg. Her head and neck examinations were unremarkable. A respiratory examination showed no signs of respiratory distress, and the trachea was central, but the movement of the left side of the chest was reduced. A 2 cm in diameter sinus discharging an exudative material with black grains was noted in the midaxillary line of the left chest. The discharge was noted to increase with cough . There w+ of 4.6 mmol/l. A liver function test showed an elevated alkaline phosphatase (ALP) of 228 IU/l; otherwise, other enzymes were normal, with aspartate aminotransferase (AST) 6 IU/l and alanine aminotransferase (ALT) 4 IU/l. Total protein was normal at 6.9 g/dl, but a low serum albumin (1.8 g/dl) was noted. C-reactive protein was 11 mg/l, and a viral screening was negative. A urinalysis was positive for pus cells of 8–12/hpf, albumin 1+, and sugar 2+. After the stimulation of whole blood with culture filtrate of M. mycetomatis, cytokines interleukin (IL)-10 and interferon gamma (IFN-γ) levels were measured. The levels of these cytokines were higher in the patient compared with control subjects. This indicates a mixed type of immune response in which both Th1 cytokines and Th2 cytokines are secreted at the same time.Investigations showed a haemoglobin level of 5.0 g/dl with normal leucocyte count and platelets. Her random blood glucose level was elevated at 324 mg/dl. There was evidence of chronic kidney disease, with urea 94 mg/dl and creatinine 3.2 mg/dl. She was hyponatremic with a serum sodium of 129 mmol/l and a normal KShe underwent imaging investigations, including chest X-ray, computed tomography (CT), and MRI scan examinations. The chest X-ray revealed soft tissue in the left lateral part of the chest wall, a left lower lung mass, reduced lung volume, and left- sided pneumothorax .A CT scan showed a left lower chest wall thickening with a wall defect, left side hydropneumothorax, and lower lobe collapse and consolidation of the lingual .An MRI examination revealed marked volume loss in the left hemithorax, pneumothorax, soft tissue lesion in the left lingular region, left lower lobe atelectasis, and a small amount of pleural effusion .M. mycetomatis. A molecular diagnosis of causative organism was done by a grains PCR examination, and it confirmed the diagnosis of M. mycetomatis.A grains culture was done on Sabouraud agar, and it revealed evidence of Escherichia coli, which is sensitive to imipenem and amikacin. Imipenem 500 mg twice a day was prescribed for 5 days. An intercostal chest drain was inserted to drain the intrathoracic fluid collection. Treatment for eumycetoma was commenced with itraconazole 400 mg per day. Her condition showed a gradual improvement, with a plan to stay on antifungal treatment. Given her poor general condition, malnutrition, and chronic kidney disease, surgical exploration was deferred. She was seen in April of 2017 at the clinic. Her condition is stable, and the secondary infection is reduced, but the fistula is still open.She was admitted to the hospital and received 5 units of blood to correct the anaemia, as well as 18 units of albumin. A swab culture from the sinus discharge revealed Mycetoma is one of the most destructive neglected tropical diseases, characterised by the formation of a chronic inflammatory granuloma, caused either by fungal (eumycetoma) or bacterial (actinomycetoma) microorganisms. If left untreated, it will induce massive deformities, destructions, and disabilities. It has enormous damaging effects on patients, family, and community in endemic regions ,2,3.In mycetoma, the foot and hand are the most affected sites. The chest wall is a rare site, accounting for 0.1% of 6,792 mycetoma patients seen at the MRC in the period 1991–2014 . Only 2 The triad of subcutaneous mass, sinuses, and discharge-containing grain is pathognomonic of the disease ,9. It isA broncho-pleuro-cutaneous fistula is potentially fatal; however, in the reported patient, the process was slowly progressive, with the formation of a large intrathoracic granuloma with thick capsule leading to lung volume reduction and encapsulated pneumothorax. The formation of fistula in this patient is likely to be due to the long-standing progressive disease, diabetes, and its complications and due to her age. Surgical repair of the fistula is the treatment of choice in such patients. However, the large intrathoracic granuloma, her poor general condition, and her chronic kidney disease hindered the surgical intervention .A search of the medical literature failed to reveal any report on such presentation, and hence it is worth reporting this patient presentation in order to share the experience.Written informed consent was obtained from the patient.Chest wall eumycetoma is an unusual condition.The reported intrathoracic pathology is rare.Chest CT scan and MRI are essential to confirm the diagnosis.Management of such presentation is challenging.Health education and early presentation are crucial to avoid such presentation.


DGK genes in response to beneficial elements and other metal and metalloid ions, including silver (Ag), aluminum (Al), arsenic (As), cadmium (Cd), chromium (Cr), mercury (Hg), and sodium (Na). In all plant genomes explored, we were able to find DGK representatives, though in different numbers. The phylogenetic analysis revealed that these enzymes fall into three major clusters, whose distribution depends on the composition of structural domains. The catalytic domain conserves the consensus sequence GXGXXG/A where ATP binds. The expression profiling data demonstrated that DGK genes are rapidly but transiently regulated in response to certain concentrations and time exposures of beneficial elements and other ions in different plant tissues analyzed, suggesting that DGKs may mediate signals triggered by these elements. Though this evidence is conclusive, further signaling cascades that such elements may stimulate during hormesis, involving the phosphoinositide signaling pathway and DGK genes and enzymes, remain to be elucidated.Diacylglycerol kinases (DGKs) are pivotal signaling enzymes that phosphorylate diacylglycerol (DAG) to yield phosphatidic acid (PA). The biosynthesis of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a crucial signaling process in eukaryotic cells. Next to PLD, the PLC/DGK pathway is the second most important generator of PA in response to biotic and abiotic stresses. In eukaryotic cells, DGK, DAG, and PA are implicated in vital processes such as growth, development, and responses to environmental cues. A plethora of DGK isoforms have been identified so far, making this a rather large family of enzymes in plants. Herein we performed a comprehensive phylogenetic analysis of DGK isoforms in model and crop plants in order to gain insight into the evolution of higher plant DGKs. Furthermore, we explored the expression profiling data available in public data bases concerning the regulation of plant  N-acylethanolamines, and free fatty acids . After phosphatidylinositol 4,5-bisphosphate hydrolysis, IP3 diffuses into the cytosol and acts as a second messenger implicated in calcium (Ca2+) mobilization from intracellular compartments such as the vacuole, while DAG remains in the plasma membrane and may activate other physiological processes. DAG can then be phosphorylated by DGK to yield PA, which in turn can be further metabolized to PPI. Additionally, enzymes that dephosphorylate PA include lipid phosphate phosphatases and PA hydrolases (lipins). Moreover, PA can be further phosphorylated to diacylglycerol pyrophosphate by PA kinase, or metabolized to lyso-PA through PLA2 activity  as a product of the hydrolysis of structural phospholipids, such as phosphatidylcholine and phosphatidylethanolamine by the action of different isoforms of phospholipase D PLD; ; or (2) activity . Most ofactivity .In eukaryotic cells, PA levels are typically low, corresponding to 0.67% of total phospholipids  and PA fChlamydomonas moewusii, hyperosmotic stress triggers the activation of both PA biosynthetic pathways  and sodium (Na), as well as other metal and metalloid ions, including silver (Ag), arsenic (As), cadmium (Cd), chromium (Cr), and mercury (Hg). Beneficial elements are not required by all plants, but may be essential for certain taxa depending on environmental conditions, element concentration and plant species. When applied at low concentrations, they may elicit positive responses in growth, yield, and responses to environmental stresses  required for kinase activity . DGK actstresses . Indeed,stresses . HerewitDGK gene homologs in plant species, searches were performed based on the reported sequences of AtDGK (1 (2 and the UniProtKB server3. According to 4 (To identify of AtDGK  using thAtDGK (1  with a Bing to 4 . This meing to 4 . For thaDGK gene probes were retrieved from the Genevestigator software package5 (AtDGK), rice (OsDGK), tomato (SlDGK), soybean (GmDGK), wheat (TaDGK) and barley (HvDGK) DGK genes in response to Ag, Al, As, Cd, Cr, Hg, and Na from Genevestigator as well.Tissue-specific expression patterns of available plant package5 . FurtherIn order to reconstruct the DGK phylogeny from plants, a protein database search in the NCBI and UniProtKB was carried out. The queries were DGK sequences homolog to Arabidopsis DGKs using the BLAST tool. Subsequently, we added to our analysis all homolog DGK sequences previously analyzed phylogenetically and referred to in articles. From Arabidopsis, we took into consideration AtDGK1, AtDGK2, AtDGK3, AtDGK4, AtDGK5a, AtDGK5b, AtDGK6, and AtDGK7 , 2005a; Table 1), we aligned the sequences through Clustal-omega1.2.1 software  sequence and demonstrated that the ATP-binding site is conserved in all DGK catalytic domains in plants. Conversely, the consensus sequence in ScDGK1 has been replaced by the sequence H58LKSHE63 in the catalytic CTP domain. This catalytic domain exhibits a much simpler and less varied amino-terminal regulatory domain than its ATP-dependent counterpart . Our results confirm that plant DGKs fall into three phylogenetic clusters, which is in full agreement with other studies reported by Figure 2.In order to test whether our phylogenetic analysis was consistent with those previously reported, we constructed an additional rooted tree . Importantly, we were able to retrieve crucial expression data of DGK genes when Arabidopsis, barley, soybean, tomato, rice, or wheat plants were exposed to Ag, Al, As, Cd, Cr, Hg, or Na.GenevestigatorDGK genes were overexpressed during cell culture, seedling, and inflorescent stages. At cell culture, induced expression of AtDGK2, AtDGK4, and AtDGK5b was observed in sperm cell; HvDGK8 in protoplast; OsDGK1 and OsDGK8 in the whole cell culture and primary cell. In seedlings the gene HvDGK3c was found to be induced in roots. During inflorescence, the genes AtDGK4 and GmDGK1 were overexpressed in raceme. In general, all genes analyzed were expressed in all tissues, though at different levels. This is consistent with the expression data reported by AtDGK2, since such gene was found to be highly expressed in young tissues and flowers. In a study performed in reproductive organs of Arabidopsis, the published microarray data revealed that AtDGK1, AtDGK3, and AtDGK5 are primarily expressed in pistils, stamens, and petals, while AtDGK4 is highly expressed only in stamens. Instead, AtDGK5 is slightly higher expressed in stamens and petals than in pistils  were found to be highly expressed in stems and most of them in the flower as a whole .We explored the gene expression profiling data deposited in the Genevestigator platformDGK genes available in Genevestigator.Beneficial elements are not essential in most plant species. Instead, they may elicit hormetic responses by stimulatory effects when applied at low concentrations. Although up to now still largely unexplained, the plant growth enhancing effects of beneficial elements is gaining increased attention in relation to enhancing crop production . In ordeAtDGK2 transcript levels are induced in response to Al, one of the beneficial elements referred to in the literature . When barley plants were exposed to ∼1 μM Hg the expression of HvDGK3a, HvDGK3b, and HvDGK3c was significantly induced in root tissues. Similarly, in Arabidopsis roots the genes AtDGK5b and AtDGK7 were highly transcribed in the presence of 10 μM Ag. In rice, plants exposed to 13.3 μM dibasic sodium arsenate (NaHAsO4) exhibited induced expression of OsDGK2 in leaves, whereas in tomato the application of 200 mM Na enhanced the expression of SlDGK1 also in leaves. Conversely, lower expression levels were detected in shoots, especially those of AtDGK5b and AtDGK7 in response to 200 μM Cd.We have previously observed that terature . Herein Plants have evolved effective response strategies to react against environmental stimuli and protect themselves from various stress factors. One of the most important signaling pathways that mediate these responses is represented by the PPI, in which one of the emerging components is PA . In the Catharanthus roseus, tobacco (Nicotiana tabacum), wheat, tomato, Arabidopsis, rice, and apple -binding domain (CBD). The two proteins lack the cysteine-rich domain (CRD) present in other eukaryotic DGKs, but are active in vitro. LeCBDGK (SlDGK1b in Table 1) is found both in associations with membranes and in soluble cell extracts. By contrast, LeDGK1 (SlDGK1a in Table 1) only associates with the membrane fraction, via a Ca2+/CaM-independent mechanism, which might represent a means of encoding specificity in cellular responses by alternative splicing  were cloned and their encoded enzymes were catalytically active in in vitro assays. AtDGK2 transcripts are found in the whole plant except in stems and are induced by exposure to cold (4°C), pointing to a role in cold signal transduction  followed by an accessory domain  at the C-terminus. Since DGKa is associated with DGKc, the former may contribute to the functionality of the catalytic domain. Whereas mammalian DGK enzymes can be classified into five groups according to sequence homology , plant DS. cerevisiae was initially thought to lack a DGK, though Dgk1p, a novel type of DGK, utilizes CTP rather than the common ATP was discovered  induced the expression of rice OsDGK2, but slightly downregulated that of OsDGK3a and OsDGK8. This fact, as well, points to a possible role of As in the PI signaling pathway.Arsenic (As) is a metalloid occurring in natural environments in some abundance (1.2–1.4 ppm) in the Earth’s crust and in small quantities in rocks, soils, water bodies, and air. Hence, plants have evolved in the presence of this element, and it has been postulated that As might induce hormetic effects on plant growth. Although the mechanism of action is still unknown, it has been suggested that the growth benefit triggered by As arises from As stimulation of Pi uptake . HoweverPanicum virgatum) under hydroponic conditions, since plants were capable of developing better in the nutrient solution containing 100–175 μM Cd at pH 4.1–5.9, showing enlargement of the root length and surface, enhanced absorption of essential nutrients and increased biomass  and carotenoids, as well as relative water contents in leaves of Lonicera japonica plants receiving 0.5–5.0 mg L-1 Cd in the nutrient solution, as compared to the control. In our analysis, Cd repressed the transcriptional activity of AtDGK1, AtDGK2, AtDGK3, AtDGK4b, AtDGK5b, and AtDGK7. In rice, Cd slightly enhanced the expression of OsDGK8. It has been previously demonstrated that Cd affects the content of PPI in mammalian cells  enhanced defense response and maintained photosynthetic activity in Eichhornia crassipes, implying that the antioxidant defense system enzymes attempted to ensure the redox homeostasis. In our analysis, Cr slightly induced the expression of most OsDGK isoforms. 2+-ATPase in mammals, which is connected with the regulation of phospholipid levels  accelerated catalase and peroxidase activities. In our survey, Hg application in barley resulted in a strong induction of all three HvDGK3 genes . Nevertheless, additional experimental evidence demonstrating the connection between Hg and the PI signaling pathway remains to be elucidated.Low levels of Hg salts induce stress-dependent increase in glucose uptake in mammalian cells . Further elegans , Anas plrhynchos , and pla2 during photosynthesis, since Na mediates the regeneration of PEP from pyruvate. Additionally, Na has been widely reported to trigger hormetic responses on most plant taxa, including C3 plants , can be fulfilled by Na+. Nevertheless, plant species significantly vary in their capacity to replace K+ with Na+ , including C4 or CAM species that use phosphoenolpyruvate (PEP) to fix CO3 plants . Importawith Na+ . Na is awith Na+ . In our bidopsis . These fDGK gene expression, the possible impact of dosages, chemical forms, time of application, and genotype responses remains to be further investigated. Additionally, understanding the roles of beneficial elements and other hormesis-inducing ions in different plant species under stress conditions may be a possible area of further study. Herewith we provide evidence for the first time that DGK genes encoding enzymes implicated in the synthesis of PA, an emerging second messenger, are responsive to Ag, Al, As, Cd, Cr, Hg, and Na. Importantly, we found most DGK genes ubiquitously expressed in plant tissues, and encoded proteins widely distributed in higher plants. PA, the protein product of DGK activity, has been proved to be a crucial second messenger. Its potential role as signaling mediator in response to beneficial elements and other ions could be of crucial significance for plants, providing the cell with spatial and transient information to perform better under limiting environment conditions, or simply to improve plant production and productivity. The discovery and description of the molecular bases of such relationships would be a noteworthy contribution to the field of agriculture.Beneficial elements are emerging as potential biostimulators for agricultural proposes, since they can induce desirable plant responses in a hormetic manner. In light of the growing need for sustainable agriculture, the general global challenges related to climate change and an increasing population demanding more food, beneficial elements open up new possibilities to attain food security. Though cumulative evidence points to beneficial elements like Al and Na having a role in the regulation of HE-S and FG-M developed and designed the experiments and the survey strategies. LT-T supervised the physiological and plant nutrition experiments. PP-R and JH-C supervised the bioinformatics analyses. HE-S and FG-M wrote the manuscript. LT-T, PP-R, and JH-C revised the manuscript.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.