All trials were separated by seven days and at ambient room temperature . During the 2 h recovery period after the initial ~60 min running at 75% V·O2PEAK, participants were provided with 1.38 ± 0.51 L of either deep ocean mineral water (DOM) or a taste-matched placebo (PLA), both mixed with 6% sucrose. DOM increased high-intensity running capacity by ~25% compared to PLA. There were no differences between DOM and PLA for blood lactate concentration, blood glucose concentration, or urine osmolality. The minerals and trace elements within DOM, either individually or synergistically, appear to have augmented high-intensity running capacity in healthy, recreationally active male soccer players after short-term recovery from an initial bout of prolonged, high-intensity running in thermoneutral environmental conditions.This study examined whether deep ocean mineral (DOM) supplementation improved high-intensity intermittent running capacity after short-term recovery from an initial bout of prolonged high-intensity running in thermoneutral environmental conditions. Nine healthy recreational male soccer players  completed a graded incremental test to ascertain peak oxygen uptake (V After 24 h, V·O2MAX increased by ~6% compared to baseline with DOM, whilst it was ~13% lower with placebo. The authors opined that specific elements in DOM, such as boron, magnesium and rubidium, were likely to have contributed to the observed ergogenic effects. Interestingly, using a similar protocol, Stasiule et al. [Hydrothermal systems in the deep ocean have been proposed to be among the most credible settings for the biological origins of life ,2. Indeee et al.  reportede et al. .Given the importance of optimising recovery for individuals who train/compete multiple times per day with limited time between bouts of exercise ,11, and ·O2, heart rate (HR), ratings of perceived exertion (RPE), and respiratory exchange ratio (RER) were 46 ± 4 mL kg min−1, 197 ± 4 beats min−1, 19.3 ± 0.7, and 1.07 ± 0.05, respectively.At the end of the graded increment test, mean relative V·O2PEAK  or the subsequent intermittent running until volitional exhaustion . There was no interaction  or main effect of treatment  for HR during the bout of running at 75% V·O2PEAK. However, there was a main effect for time  or main effect of treatment  for RPEL during the bout of running at 75% V·O2PEAK, although there was a main effect for time  or main effect of treatment  for RPEO during the running at 75% V·O2PEAK; however, there was a main effect for time  or main effects of treatment  for HR, RPEL and RPEO, respectively, during the 20 min running at 75% V·O2PEAK post-recovery; however, there were main effects of time for all variables . In contrast, DOM increased exercise capacity by 56%  compared to PLA . ·O2PEAK and at volitional exhaustion. There was no interaction  or main effect of treatment  for HR; however, there was a main effect for time  with HR greater at the end of exercise (174 ± 7 beats min−1) compared to after 1 min  and 2 min . Furthermore, there were no interactions  or main effects of treatment  for RPEL and RPEO, respectively. As with HR, there were main effects of time for RPEL  and RPEO , whereby RPEL (9.4 ± 0.7) and RPEO (19.2 ± 1.2) were greater at volitional exhaustion compared to after 1 min  and 2 min , respectively.Due to the variability in exercise capacity between treatments and participants, HR and perceptual data were only analysed for the first four shuttles at 90% V2 = 0.2) or main effect of treatment  or time  for urine osmolality , whereby body mass was greater for DOM vs. PLA  at baseline only (2 = 0.8), whereby body mass was greater  at baseline (80.0 ± 10.9 kg) compared to after 60 min running at 75% V·O2PEAK (79.3 ± 10.9 kg) after 2 h recovery (79.4 ± 11.0 kg) and at the end of the exercise capacity test (79.3 ± 10.9 kg). There was no main effect for treatment .There was an interaction for body mass  or main effect of treatment  for blood lactate; however, there was a main effect for time  or main effect of treatment  or time  for blood glucose  excretion via sweat and urine [In the present study, body mass was greater at baseline compared to after 60 min running at 75% Vnd urine . As Mg ind urine , it seemnd urine . Moreovend urine .+; [+ is crucial, such as ATP production and calcium signalling [Boron (B) has been shown to boost Mg absorption, reduce levels of inflammatory biomarkers such as high-sensitivity C-reactive protein (hs-CRP), tumour necrosis factor α (TNF-α), and interleukin 6 (IL-6), as well as contribute to the synthesis and activity of crucial biomolecules such as nicotinamide adenine dinucleotide . Therefgnalling . Albeit gnalling ), which gnalling . Furthergnalling . Howevergnalling ), and thgnalling  reported−1 [+) [+ and considerable loss of K+ from active muscle during high-intensity exercise. Thus, it is likely that the resultant electrical changes induced by ionic shifts across the muscle surface membrane are principal contributors of fatigue [+ and facilitate a prolonged status of optimal electrical activity and, hence, contraction in the muscle, thus delaying fatigue. Furthermore, Rb has been found to be 49% and 34% greater in professional footballers than sedentary controls and long-distance runners, respectively [Rubidium (Rb), present in all human tissues and ranging from 8 to 30 mg·kg−1 , has bee−1 [+) . It is w fatigue . Therefoectively . Howeverectively ,25 and, ectively ,25.In support of our hypothesis, the present study found no differences in urine osmolality between treatments. This is in contrast to Keen et al.  who repo·O2MAX. Importantly the absolute values of blood lactate after 60 min running at 75% V·O2PEAK in the present study were similar to those observed in various other studies examining blood lactate responses in the second half of a soccer match , highli−1) after DOM but 117% for PLA (+8.23 mmol L−1) when compared to values at the end of 60 min running at 75% V·O2PEAK. Excluding P2, the mean increase in blood glucose between these time points was 18% and 22% for DOM and PLA, respectively. Furthermore, only part of the difference observed for P2 might be explained by the differences between treatments of −1.39 mmol L−1 at baseline and of +1.35 mmol L−1 after completion of 60 min running at 75% V·O2PEAK for PLA vs. DOM, respectively. The potential of DOM to facilitate greater glucose uptake is supported by Ha et al. [Arguably the most intriguing finding from the metabolic data relates to a specific participant who, after completion of this study, was diagnosed with type I diabetes. The participant confirmed there was a familial history of this, which was undisclosed prior to participation  behind the ergogenic effects of DOM to maximise its potential performance and health benefits in the wider community.After reading an information sheet about the proposed research, and subsequently providing written informed consent, nine healthy, nonelite male soccer players ; and mean nonsoccer-specific training time : 37 ± 44 min (range: 0 to 90)) volunteered to participate in the study, which had received university ethical approval (reference: S2BR2016). The experimental cohort was recruited via opportunistic and convenience sampling. Participants were eligible if they were nonelite soccer-playing males who were healthy, free from injury, and were between 18 and 30 years of age. ·O2PEAK) followed by two familiarisation trials. Using a repeated measures, crossover, and counterbalanced design, participants then completed two experimental trials. All trials were completed on a motorised treadmill , separated by 7 d, undertaken at ambient room temperature , and commenced at similar times of day  to minimise any potential circadian rhythmic effects on soccer-specific endurance [Participants completed five visits to the laboratory. Firstly, they completed a graded incremental test to ascertain peak oxygen uptake  24 h prior to each trial. Due to the extended washout period, participants were verbally screened for the use of beta-alanine and creatine monohydrate supplementation. No participant was excluded from the study due to prior ingestion of either.−1 [−1, participants were excluded from the trial. Two participants required small amounts of water in addition to the 500 mL bolus, but all participants were found to be adequately hydrated before each trial. A heart rate (HR) monitor  was subsequently attached. After 5 min seated rest, HR was recorded, and a finger prick capillary blood sample was taken and subsequently analysed for blood lactate (BLa) and blood glucose (BGl) concentrations .On the first visit to the laboratory, the participant’s age, stature (cm), and body mass (kg) were recorded, with only body mass being recorded on subsequent visits. One hour prior to exercise, participants were asked to ingest 500 mL of tap water to ensure an adequate and consistent level of hydration status . Hydrati−1 . If part·O2PEAK was achieved using an incremental exercise test on a motorised treadmill . After completing an initial 5 min warm up at 8 km h−1, participants commenced running at 9 km h−1 for 3 min. Incremental increases of 1 km h−1 were applied every three minutes until volitional exhaustion. At the end of each stage, and upon completion of exercise, HR was telemetrically recorded, and ratings of perceived exertion  and the respiratory exchange ratio (RER) over the last 60 s of rest and the last 30 s of exercise.Assessment of Vn  were co−1. Immediately after completing the warm up, participants then ran for ~60 min at a running velocity corresponding to 75% V·O2PEAK , until volitional exhaustion. At the end of each 1 min interval at 90% V·O2PEAK, HR, RPEO, and RPEL were recorded. This protocol was selected because it replicated the activity patterns and metabolic stress of speed endurance training sessions and was therefore relevant to individuals who competed in invasion sports such as soccer [After collection of initial baseline measures as previously described , participants commenced a 5 min warm up on the treadmill at 8 km hO; 6–20; ) and locL; 0–10; ) were cos soccer . Upon coParticipants subsequently recovered for 2 h in a semisupine position. During this period, they were provided with two litres of tap water to ingest ad libitum. The mean amount consumed during familiarisation sessions (1.38 ± 0.51 L) was subsequently used to calculate the volume of fluid provided during experimental trials. Additionally, after 30 min recovery, participants were given a standardised cereal bar  that contained 192 kcal (850 kJ) and constituted 7.2 g of fat (1.0 g saturated), 27.1 g carbohydrates (11.9 g sugar), 3.4 g protein, and 2.4 g of fibre. −1. Subsequently, participants then ran for 20 min at a running velocity corresponding 75% V·O2PEAK. For our primary performance outcome measure, the same pattern of intermittent running until volitional exhaustion  was then completed a final time with HR, RPEO, and RPEL collected at the end of each interval at 90% V·O2PEAK. Upon completion of exercise, participants completed 2 min active recovery, after which body mass was measured a final time, and further urine, BLa, and BGl samples were collected and analysed as previously indicated. Participants were then free to leave the laboratory.After the two-hour recovery period, further urine, BLa, and BGl samples were collected and analysed as previously indicated. Baseline HR was then recorded prior to commencing a 5 min warm up at 8 km hExperimental trials were identical to familiarisation trials with one exception. During recovery, participants were provided with 1.38 ± 0.51 L of either deep ocean mineral water  or a taste-matched placebo . Treatme2 (Pη2). Otherwise, for normally distributed data, the ES (d) was calculated using the difference in means divided by the pooled standard deviation (SD) of the compared values [Data were analysed using IBM SPSS . For all data, normality (via Shapiro–Wilk’s test) and homogeneity of variance/sphericity (via Mauchly’s test) were checked prior to choosing the appropriate statistical tests. With the exception of the exercise capacity data (Wilcoxon Signed Ranks Test), a number of two-way  repeated measure ANOVAs were subsequently conducted. If sphericity was violated, degrees of freedom were corrected using Greenhouse-Geisser values, and Bonferroni adjusted pairwise comparisons were applied. Additionally, Tukeys’ HSD post hoc analysis was undertaken for interactions by calculating the difference required between means for a minimum level of P = 0.05 . Data wed values  with 95%d values ,45. For d values . Unless


Incompatibility between mitochondrial genome and nuclear genome leads to non-functional male reproductive organs and results in cytoplasmic male sterility (CMS). CMS has been used to produce FGossypium hirsutum sterile male lines CMS-2074A and CMS-2074S, as well as their restorer and maintainer lines. First, we noticed the mitogenome organization and sequences were conserved in these lines. Second, we discovered the mitogenomes of 2074A and 2074S underwent large-scale substitutions and rearrangements. Actually, there were five and six unique chimeric open reading frames (ORFs) in 2074A and 2074S, respectively, which were derived from the recombination between unique repetitive sequences and nearby functional genes. Third, we found out four chimeric ORFs that were differentially transcribed in sterile line (2074A) and fertile-restored line.Here we compared the mitochondrial genomes (mitogenomes) of These four novel and recombinant ORFs are potential candidates that confer CMS character in 2074A. In addition, our observations suggest that CMS in cotton is associated with the accelerated rates of rearrangement, and that novel expression products are derived from recombinant ORFs.The online version of this article (10.1186/s12864-018-5122-y) contains supplementary material, which is available to authorized users. Cytoplasmic male sterility (CMS), a phenomenon that the male reproductive structures fail to develop, is an important agronomical trait in higher plants. The CMS character is frequently used in crop breeding and commercial seed production to increase the yield of the crops such as rice, maize, rapeseed, and cotton –5. CMS i1 and restorer lines’ in the context of their nuclear genomes can provide insights into cytonuclear-related phenotypes, such as cytoplasmic male sterile [Gossypium harknessii and G. hirsutum to determine candidate CMS factors. We also analyzed the expression patterns of uncharacterized ORFs, some of which are candidate genes for CMS. The results give some interesting clues about mitochondrial evolution and CMS generation, as well as generate a background for future studies on CMS molecular diversity and phenotypic variability in cotton.Next-generation sequencing technology (NGS) has been applied to plant chloroplast (cp) genomes, with over 1200 species sequenced –25. Howe sterile , 38. In Gossypium harknessii Brandegee CMS-D2–2 cytoplasm, was from its original sterile line DES-HAMS277. Line 2074S, an upland cotton cytoplasmic male sterile line, was from G. hirsutum L. CMS-AD1. These two lines were genetically stable cotton sterile lines derived from 20 to 30 generations of backcross. Line 2074B, a cultivar of upland cotton ‘Sumian No. 20’, was the maintainer of these two cytoplasmic male sterile lines. The restorer E5903 is a nuclear restoring line with normal nuclear and normal male-fertile Gossypium harknessii Brandegee. 2074A, 2074S and E5903 cotton materials used in this study are developed in our own lab [Line 2074A, an upland cotton cytoplasmic male sterile line with  own lab . We bree own lab .The mitogenome Fosmid library was constructed according to the manufacturer’s protocols (MaxxPlaxTMLambda Packing Extract)/. All these three mtDNAs Fosimd libraries have been constructed and screened with probes from sequences of conservative genes and scaffolds. From those libraries, 1000 clones were randomly selected and screened with 28 probes designed from sequences of mitochondrial genes. At last, 22, 26 and 21 positive clones were obtained from 2074A, E5903 and 2074S, from which 23 clones  were selected to cover larger repeats and sequenced the double-ends by shotgun strategy, with insert size of about 36.2 kb- 38.4 kb. Sequenced fragments were aligned using Blastn to determine the exactness of assembly .https://github.com/JGI-Bioinformatics/Kmernator) to remove potential undesirable artifacts, including adapters or low-quality or N bases or short sequences. The filtered reads Q30 > 85%. These filtered reads were a mixture of reads derived from chloroplast, mitochondrial and nuclear genomes; firstly, we removed the chloroplast and nuclear contaminant contigs through Blastn against nt/nr database  and assembled primarily using SOAPdenovo . The Illum 2074B , G. barbrbadense , G. raimarboreum , were usGossypium species, the Illumina reads covered all parts of the genomes consistently, achieving an average sequencing depth of 214.3× in 2074A mitogenome , 28.8× in 2074S mitogenome, 27.3× in E5903 mitogenome.Combined with the scaffolds’ information and one whole-genome backbone with positive clones, three procedures were adopted to finish the physics gaps. Firstly, we screened the library of the whole mitogenome according to the splicing sequence and the functional genes, constructed genome physical map and then sequenced the positive clones . SecondlG. raimondii genome. The syntenic regions of mitogenomes between different cultivars were detected using Nucmer of the MUMmer package (v3.06) [https://www.R-project.org/).Intersubspecific polymorphisms were firstly identified based on the MUMmer package (v3.06) . The res (v3.06)  with 50  (v3.06)  and were (v3.06) . The ann (v3.06)  and R PrWe used YASS to analyze the genome complexity that was defined as the complete sequence information of a genome with only one copy of each duplicate > 500 bp). We set parameters as follows: E-value < 1e-30 (with the score “+ 1” for one match and “-3” for one substitution); the rate of substitutions and insertions/deletions < 5% [0 bp. We https://www.ncbi.nlm.nih.gov/orffinder/) and EMBOSS (6.3.1: getorf) [− 5. ORFs containing at least 30 bp of an identified mitochondrial gene were characterized as chimeric, excluding any ORFs that overlap the genomic position of an identified gene. Transmembrane domains in each candidate ORF were predicted using TMHMM Server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).Based on the previous reports showing that CMS genes are chimeric , 51, 52, getorf) . All ORF1 (2074A × E5903) were sequenced on an Illumina HiSeq2000 at BGI (Beijing Genomics Institute). Ribosomal RNAs were removed from the extracted mitochondrial RNA using Ribo-Zero  and the mitochondrial RNA libraries were prepared using Illumina’s TruSeq RNAseq Sample Prep kit. Libraries were sequenced on one lane with 4 Gb clean reads/samples of an average length of 90 nt for paired-end. RNA sequence data quality was checked using FastQC to remove the adapters, low-quality, containing N bases and short sequences with reads Q30 > 85%. The reads were mapped to the assembled mitogenome of CMS line 2074A using bowtie2 [p-value of less than 0.05.The extracted mitochondrial RNA from the flower buds (3–5 mm size) in CMS line 2074A, its maintainer 2074B and fertile material F bowtie2  with the bowtie2 . The bow bowtie2 . DiffereG. harknessii (CMS-D2–2) into tetraploid G. hirsutum; while, 2074S was a result of alloplasmic G. hirsutum with G. hirsutum L. CMS-AD1-derived cytoplasm [Cotton is the first species that the mitogenome is sequenced among the large numbers of malvales. We performed de novo sequencing of three mitogenomes lines: a) CMS-2074A, b) CMS-2074S, and c) E5903 (a restorer line). Lines 2074A and E5903 were derived from integrating the cytoplasm of diploid species ytoplasm . The mitytoplasm , 57. ComThe main cycle (MC) molecules of two male sterile lines (2074A and 2074S) are 47 kb larger than that of the maintainer line (2074B). The reason is that they contain three large repeats, for examples, one is 10 kb larger than that of 2074B, which are similar as observed in the sterile line Ks3 that contains four repeats larger than 20 kb , and therpl2 in cotton lacks an intron [nad 1, nad 2, nad 3, nad 4, nad 4 L, nad 5, nad 6, nad 7 and nad 9), two subunits of complex II (sdh 3 and sdh 4), one subunit of complex III (cob), three subunits of complex IV , and five subunits of complex V ; four genes involved in biogenesis of cytochrome c ; 10 genes encode for ribosomal proteins , while the numbers of the ribosomal proteins are usually variable among different species [mttB) is involved in independent membrane targeting and translocation system, and one maturase gene (matR) is mapped within the 4th intron of nad1. 9 protein-encoding genes  contain a total of 23 group II introns, among of which three are trans-spliced . As previously noted, the cotton mitogenomes presented here lack rps1, rps2, rps11, rps13, rps19 and sdh2, and only partial sequences of these genes were detected in cotton mitogenomes.Cotton mitogenomes typically contain 36 genes Table ; Fig. 1 n intron , 43, 58)G. hirsutum maintainer line, those of 2074A, 2074S and E5903 contain duplicated trnM (CAU) and ccmFC in large repeats. In addition, congruent with prior results, rps3 is located at a repeat’s boundary and varied in structure among these four Gossypium mitogenomes. In cotton, rps3 contains a central domain (pfam00013) which has been lost in the incomplete duplicates (pseudogene rps3–2) of 2074A, 2074S, and 2074B.As reported previously, several mitochondrial genes exist in repeat regions and in multiple copies /E5903 and 2074B, might represent suitable candidates.A total of 47 SNPs exists within 21 protein-coding genes in four mitogenomes analyzed, and only 11 SNPs were synonymous mutations , and AUU was modified into AUG in one gene (mttB). There were five cases where gene editing generated stop codon, thereinto, four cases were the conversion of TAG into CGA in rps10, ccmC, atp9 and ccmFC genes; however, TAG was converted to CAA in atp6 gene. of Evolutionary rates analysis (ka/ks or ω) revealed that the ratios of ka to ks of nine genes  were greater than 1, which implied a positive selection. In addition, that of two genes (rpl5 and cox3) were less than 1, which implied a purifying selection. By contrast, the non-coding regions appeared to be rapidly diverged  were similar in two CMS lines, but were absent in the maintainer and restorer lines , ranging from 2824 to 147,683 bp, which possessed at least 98% identity ; 2) Special ORFs in unique regions of sterile lines, which always have short-sequences homology to chloroplast or mitochondrial sequences of other plants; such as Aorf2, Aorf18, Sorf15, Sorf16 and Sorf2 that were found in unique sequences of two sterile lines. Mostly, they are similar to chloroplast or mitochondrial sequences of other plants, or have no homology sequences in NCBI-NR database. In the third group, the ORFs are comprised of homologous sequences of 2074B and unique sequences such as Aorf14, Sorf13 and Sorf14.We categorized the specific ORFs into three basic groups: I) ORFs near the functional genes, which is transcribed in the same direction with adjacent positioned genes either up or down stream, and could be co-transcript relevant to CMS (1 (2074A × E5903). Among all the three lines, the expression of mitochondrial genes was highest in F1 and lowest in 2074B . The expression levels of shd3 and rpl10 genes were higher in 207A than in 2074B . Taking the sequences of 28 predicted ORFs in 2074A as a pool; we used Blastn to match all three-transcript data . As a result, 10 ORFs were expressed at high levels (10 fold) as compared to the similar sequence (with 1–3 gap) in 2074B; five ORFs were expressed at high levels as compared to the similar sequence in F1; the five ORFs were not expressed in 2074B  have same expression trend both in 2074B/2074A and F1/2074A, therefore, these four ORFs might be co-transcribed with functional genes and relevant to CMS.To further verify whether these ORFs were functionally associated with CMS, we profiled the expression of mitochondrial genes and ORF’s based on RNA-seq data of flower buds (3–5 mm in size) in CMS 2074A, maintainer 2074B, and the fertility-restored FSorf25, Sorf4, Sorf4–2, Sorf29, Sorf8 and Sorf27) and five ORFs in CMS2074A  were close to functional genes within 565 bp, and six  of them are the products of rearrangements by large repeats. Additionally, these ORFs have transmembrane domain  and their functional genes  might have higher expression in CMS-2074A compared to F1. Aorf4 (561 bp) is found at the downstream 565 bp of atp8. Besides, the first 45 bp of Aorf4 are derived from rps3, while other partial sequences are identical to sdh3 (the 5′-end of orfH79 has 84 bp homology to cox1) and have same expression trends with atp8 in 2074B/2074A (− 0.3) and F1-A/2074A (2.6~ 2.9). Aorf4–2 (561 bp) is found in the downstream 444 bp of rpl2 and have same expression trends with rpl2 in 2074B/2074A (− 0.2~ 0.5) and F1-A/2074A (2.9~ 3.2). Aorf28 (867 bp), located at the downstream 241 bp of cox1 and the upstream 311 bp of cox3 (331 bp in 2074S), shows 66% identity with Arabidopsis mitogenome and is close to AR1. In addition, the expression trend of Aorf28, cox1 and cox3 were same. Aorf9 (357 bp), located at the downstream 19 bp of rrn26, keeps same expression trends with rrn26 in 2074B/2074A (− 0.7) and F1-A/2074A (2.4); as well, Aorf9 also has 76 bp identity with nad7 and 89% identity with Ricinus mitogenome. These four ORFs show the characters of CMS-associated genes and are similar to other ORFs, such as T-urf13 of maize [orf355/orf77 [orf224 of rape [orf256 of wheat [orf125 of radish [orfs, are needed to confirm the real CMS gene of upland cotton.Furthermore, we analyzed 16 reported CMS-associated ORFs. We found that these ORFs 78–488 bp) are near to co-transcribed genes and form a bicistronic complex with many functional genes. In this context, six ORFs in CMS2074S  have been lost and 38 nonsynonymous mutations occurred in 21 protein-coding genes, though they are functionally irrelevant. Out of 28 ORFs in CMS 2074A, four ORFs  are close to the functional genes and show similar characters to CMS-associated genes in other plants. These four ORFs may be the potential candidates conferring CMS in cotton.The two almost identical male sterile lines, 2074A and 2074S, share high identity with the restore line E5903 but are different from their maintainer line 2074B, especially in non-coding regions. The cotton mtDNAs are 621,884–668,584 bp in length, and harbor 36 known protein-coding genes, three rRNAs  as well as 18 different tRNAs. The rates of the coding genes (including duplicated genes) accounting for the total genomes’ length are almost similar, but the repeat sequences show a few differences. In addition, five genes Additional file 2:Table S2A. The verification about breaking point of scaffolds between 2074A and 2074B. (DOCX 15 kb)Additional file 3:Table S2B. The verification about breaking point of large repeats between 2074A and 2074B. (DOCX 15 kb)Additional file 4:Table S3. The chloroplast-derived sequences (> 70 bp) found in four mitogenomes. (DOCX 19 kb)Additional file 5:Table S4. List of multi-copy genes in cotton mtDNA. (DOCX 16 kb)Additional file 6:Table S5. Nucleotide differences relative to the 2074B mitogenome. (DOCX 14 kb)Additional file 7:Figure S1. The end sequencing positive clones in E5903 and 2074A. (DOCX 63 kb)Additional file 8:Figure S2. The syntenic regions in four mitochondrial genomes. (DOCX 80 kb)Additional file 9:Figure S3. Differential expression of CMS candidate ORFs in 2074A, 2074B and F1-A. Log2 transformations of the expression fold changes (2074B/2074A and F1-A/2074A) are represented by bars. Y axis denotes the levels of transformed expression fold changes. (DOCX 19 kb)


We found that vorinostat (SAHA) and MC1568 inhibit tube formation by rat glioma C6 cells. Moreover, at sublethal doses for GBM cells, SAHA, trichostatin A (TSA), entinostat (MS275), and MC1568 significantly decrease tube formation by U87MG and by patient-derived human GBM cancer stem cells (CSCs). The reduced migration and invasion of HDACis-treated U87 cells, at least in part, may account for the inhibition of tube formation. In conclusion, our results indicate that HDACis are promising candidates for blocking vascular mimicry in GBM.Glioblastoma (GBM), a high-grade glioma (WHO grade IV), is the most aggressive form of brain cancer. Available treatment options for GBM involve a combination of surgery, radiation and chemotherapy but result in a poor survival outcome. GBM is a high-vascularized tumor and antiangiogenic drugs are widely used in GBM therapy as adjuvants to control abnormal vasculature. Vasculogenic mimicry occurs in GBM as an alternative vascularization mechanism, providing a means whereby GBM can escape anti-angiogenic therapies. Here, using an in vitro tube formation assay on Matrigel O6-methylguanine-DNA methyltransferase (MGMT) levels, to inactivation of mismatch repair enzymes MLH1 and MSH29 and over-expression of multidrug resistance proteins [Glioblastoma (GBM) is the most common and lethal among nervous system tumors, classified as grade IV glioma by the World Health Organization (WHO) ,2,3,4. Tproteins . Moreoveproteins ,12,13,14proteins ,16,17,18proteins ,20. Vascproteins  and highproteins . Howeverproteins  such as proteins , vasculaproteins ,25, intuproteins ,27 and vproteins . An impoproteins ,29,30,31Indeed, the ability of aggressive and genetically dysregulated tumor cells to form fluid-conducing network  is well ® (ECM), under serum-free conditions, they form clear-cut tubes developing from elongated cell bodies that connect to each other and form polygonal networks similar to those formed by HUVECs . The experiments were carried out at 50 nM and the effects were evaluated after 3h in Boyden chamber assay and after 24h in xCELLigence® Real-Time Cell Analysis  . It is noteworthy that all inhibitors are able to completely inhibit the chemotactic response to FBS, reducing migration rate to the basal level of random migration (migration of untreated cells in the absence of chemo-attractant) for TSA and below for MS275 and MC1568 B. Also xas 100%) C.Moreover, we tested the capability of MS275, the most effective compound in the migration assays, to impair U87MG cells ability to invade ECM. In Boyden chamber invasion assays, after 20 h, MS275 decreased cell invasion toward FBS to 44%, as compared to the control  . It is oIn CSCs (GBM1 and GBM2), isolated from two different human GBMs, MC1568, SAHA and MS275, reduce cell ability to form vascular tube, after 24 h of treatment, with a higher efficacy in GBM2 cells A,B. Agai® , a reliable in vitro assay useful to evaluate, in quantitative manner, the cell ability to form tube-like structures. Even if in vivo models could give more information on the angiogenesis processes, this assay has been widely used to study both normal and alternative angiogenesis, including vasculogenic mimicry [Here we show that in vitro tube-like structure formation by GBM cell lines and GBM CSCs can be impaired by HDACis. For this purpose, we performed tube formation assay on Matrigel mimicry ,49,50 si mimicry . In fact mimicry ,51. The  mimicry ,32. The ® assays. MS275 was the most effective HDACis in decreasing cell migration and, thus, was tested in invasion Boyden chamber assay. We found that the tested HDACis significantly inhibit FBS-dependent directional cell migration and invasion, suggesting that they affect important components underlying vasculogenic mimicry process.Cellular migration and invasion are central hubs in the tube formation process. In particular, cellular invasion depends on migration through the dense extracellular matrix barrier and requires, in addition to adhesion and migration, the proteolysis of ECM components . For theWe found that all the HDACis tested are able to impair tube formation in human GBM U87MG cell line and in human GBM CSCs. In rat glioma C6 cells, at the same concentrations used in U87MG, only SAHA and MC1568 interfere with vessels formation, whereas TSA and MS275 do not display activity to prevent tube-like structures. However, it is possible that in C6 cells TSA and MS275 need higher concentrations to be effective. These findings may suggest that these HDACis could discriminate among different species (human vs. rat) or among tumors harboring different genetic mutations. Furthermore, HDACis affect the ability of CSCs, derived from two differently classified human tumors , to formThe finding that all the HDACis, with different specificity for HDAC classes , are actFurther studies are underway to confirm the effect on a larger number of cell lines and CSCs and to identify the molecular mechanisms of HDACis in reducing vasculogenic mimicry in GBM cells. In particular, we are investigating the molecular effects of these compounds in inhibiting cell motility and their interference on the epithelial-mesenchymal transition during tube formation by GBM cells.2, 95% air atmosphere.Human glioblastoma U87MG cell line was cultured in DMEM  containing 10% fetal bovine serum (FBS) (Gibco-Life Technologies). Rat glioma C6 cell line was cultured in MEM/F12 (1:1) (Gibco-Life Technologies) 10% FBS as previously described . The cel® , experimental conditions that allow cells to retain CSCs features, as reported [4 cells/mouse, in 6–8-week-old NOD/SCID mice ; in compliance with guidelines approved by Ethical Committee for animal use in cancer research at IRCCS Ospedale Policlinico San Martino (ethics code 17/12 approved 14 September 2012) .Two tumor specimens were obtained from Neurosurgery Dept. of IRCCS Ospedale Policlinico San Martino , after Institutional Ethical Committee approval of the informed consent provided to patients and the ex vivo human sample study . Both samples were diagnosed as primary WHO grade IV GBM and individual characteristics are reported in reported . This cureported , we demoTSA , Tubacin (Sigma Aldrich), MS275 , MC1568  and SAHA  HDACis were dissolved in dimethylsulfoxide (DMSO)  at appropriate concentration and stored at –20 °C until further use. In the different experimental setting, cells were exposed either to HDACi or to DMSO.4 cells/well and 0.5 × 104 cells/well respectively; 24 h later, the cells were treated with different HDACis at specified concentration; the cellular proliferation was measured at 24 h by Thiazolyl Blue tetrazolium bromide (MTT) assay as previously described [In 24-well plates, U87MG and C6 cells were plated at 1 × 10escribed . BrieflyThe cell viability was calculated as follows:®  with ECM proteins concentration of 9.3 mg/mL. The matrigel was allowed to polymerize at 37 °C for 30 min. The U87MG and C6 cells were dissociated by trypsinization, washed in PBS  and re-suspended in serum-free medium at 2 × 105 cells/mL and 1.7 × 105 cells/mL respectively. To evaluate the effect of HDACis on tube formation, the cell suspensions were divided into control groups (DMSO 0.1%) and several treatment groups to receive the different HDACis at final concentration of 10 nM or 50 nM. Subsequently, 0.1 mL/well cell suspensions were plated onto the surface of Matrigel® and incubated at 37 °C for 24 h or 16 h in U87MG or C6 experiments respectively. In order to perform tube formation assay on Human GBM CSC μ-Slide Angiogenesis  were coated with Matrigel® (BD Biosciences) and allowed to polymerize at 37 °C for 30 min. 104 cells were subsequently seeded on Matrigel® and incubated at 37 °C for 24 h, in Endogro medium  to induce endothelial transdifferentiation [® in 96-well plates at 2 × 104 cells/well, and were incubated at 37 °C for 24 h as previously described [For U87MG and C6, tube formation assay was performed in 96-well tissue culture plates coated using 50 µL/well of Matrigelntiation . Followiescribed  as tube ® for Tube Assay as previously described; tubes were fixed with 4% PFA solution for 20’ at room temperature. The fixed tubes were treated with PBS 10% FBS blocking solution for 1 h at room temperature and incubated with monoclonal I antibodies anti VE-cadherin  (1:250) or anti-CD31  for 3 h at room temperature. The samples were then incubated with II Ab anti mouse IgG HRP conjugated  (1:5000 in PBS) for 1 h at room temperature. Antibody positivity was revealed by peroxidase substrate DAB substrate Kit . Cells were observed by a light inverted microscope at 10× magnification. Negative controls were incubated with anti-mouse IgG only.U87MG and HUVEC cells were seeded on Matrigel6 U87 MG cells were treated with 50 nM of indicated compound for 24 h. Cells were harvested and washed twice with cold 1× PBS and lysed in Triton extraction buffer (TEB: PBS containing 0.5% Triton X-100 (v/v), 2 mM PMSF, 0.02% (w/v) NaN3) for 10 min on ice, with gentle stirring. After a brief centrifugation at 2000 rpm at 4 °C, the supernatant was removed and the pellet was washed in half the volume of TEB and centrifuged as before. The pellet was suspended in 0.2 M HCl and acid extraction was left to proceed overnight at 4 °C on a rolling table. Next, the samples were centrifuged at 2000 rpm for 10 min at 4 °C, the supernatant was removed and protein concentration was determined using the Bradford assay. Immunoblotting was performed using acetylated histone H3 (H3K9K14) and H4  antibodies.1 × 105 cells/sample were added to the upper compartment, while the chemoattractant, consisting of 10% FBS diluted in DMEM/0.1% BSA, was added to the lower compartment. After incubation at 37 °C for 3 h, the cells, able to migrate toward the lower compartment, were fixed, stained with hematoxylin and counted under microscope along fields of the filter diameter. Number of migrated cells is expressed as a percentage of the cells migrated in the absence of chemoattractant (random migration), taken as 100%. As a control, we analyzed the FBS-dependent migration of cells in the presence of DMSO, the HDACis molecules solvent. Experiments were performed in triplicate.U87MG directional cell migration has been assessed by Boyden chamber assay; 8 µm pore size filters  coated with 50 µg/mL collagen type IV (Sigma-Aldrich) were employed. The cells were detached by mild trypsinization, washed in PBS, re-suspended in DMEM/0.1% bovine serum albumin (BSA) (Sigma-Aldrich) and pre-treated with the indicated HDAC inhibitors at final concentrations of 50 nM for 1 h at 37 °C. 104) were starved in a serum-free media and treated with several HDAC inhibitors such as MS275, MC1568 and TSA at a final concentration of 50 nM. Treated and control (DMSO) cells were added in triplicates to the upper chambers and leaved at room temperature for at least 30 min to let cells settle. RTCA DP instrument was placed in a humidified incubator maintained at 37 °C with 5% CO2 for 24 h. The xCELLigence software was set to collect impendence data (reported as percentage of cell index) at least once every 15 min for 24 h. For quantification, the cell index at 24 h, for each HDACis treatment, was averaged from at least three independent measurements.To monitor cell migration in real time, we used the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16 . The CIM-plate 16 is a 16-well system in which each well is composed of upper and lower chambers separated by an 8-μm microporous membrane. These specially designed “trans-wells” measure capacitance on the underside of the dividing membrane . Migration was measured as the relative impedance change (cell index) across microelectronic sensors integrated into the bottom side of the membrane. FBS (10% in DMEM) was used as a chemoattractant. U87 MG cells (3 × 10® (Corning®) was used. Cell suspension was prepared and pre-treated as described for Boyden chamber migration assay. 0.5 × 105 cells/sample were added to the upper compartment, whereas the chemoattractant, consisting of 10% FBS diluted in DMEM/0.1% BSA, was added to the lower compartment. After incubation at 37 °C for 20 h, the migrated cells were fixed, stained with hematoxylin and counted under microscope along fields of the filter diameter. Number of migrated cells is expressed as a percentage of the invaded cells in the absence of chemoattractant (random invasion), taken as 100%. As a control, we analyzed the FBS-dependent invasion of cells in the presence of DMSO, the HDACis molecules solvent.U87MG cell invasion have been assessed by Boyden chamber assay; 8 µm pore size filters were coated with 50 µg/mL collagen type IV, dried and then a second coating with 50 µg/mL Matrigelp-value < 0.05 was considered as statistically significant.Data were expressed as mean ± SD. T test were performed and Altered HDACs expression and/or function, modifying the normal epigenetic arrangement, play an important role in tumor initiation and progression. Unlike genetic mutations, epigenetic alterations are reversible and can be targeted by specific drugs. Here, we provide substantial in vitro evidences that HDACis inhibit human GBM cells ability to form vascular tubes, the cellular ability underlying vascular mimicry process in vivo ,44,45. T


The supramolecular recognition behavior of each polymer was evaluated via potentiometric measurements against selected anions in aqueous media. Interestingly, this proof of concept study shows that these systems were selective against only fluoride (F−) or both, fluoride and acetate (OAc−), by tailoring the relative content of Zn-Salphen monomer, thus making them a promising starting point for modular design of chemical sensors through straightforward synthetic approaches.In this work, we present the synthesis of a novel Zn-Salphen complex containing an allyl group, which was used as building block in the further preparation of a new family of functional terpolymers. These polymers were obtained through radical co-polymerization with methyl metacrylate (MMA) and  Among these characteristics, their advantageous easy tuning properties through chemical functionalization of the aryl groups from the ligand part has facilitated their use in a wide range of applications: e.g., chemosensors for anions  2 and poly[4%-stat-(nBuA)-stat-(MMA)] 3 were synthesized by radical co-polymerization of nBuA and MMA varying the relative amount of Zn-Salphen 1 present therein (2% wt. for 2 and 4% wt. for 3), preserving the nominal weight ratios of nBuA:MMA = 8:2. The synthesis was performed under inert atmosphere without solvent, at 80 °C for 20 min in presence of AIBN as initiator, affording the terpolymers 2 and 3 in good yields (98% and 97% respectively). In the 1H NMR spectra recorded in CDCl3 (nBuA:MMA = 8:2) .Zn-Salphen 1-containing terpolymers 2 and 3, namely poly), C29H23N2O2Zn requires 495.10510. For 1H NMR, 13C{1H}, and HSQC spectra see To a solution of ketimine I  in 10 mL of MeOH we added a solution of 3-allyl-2-hydroxybenzaldehyde II  and Zn(OAc)2 atmosphere, a mixture of the monomer Zn-Salphen complex 1, nBuA, and MMA in different ratios was homogenized at room temperature and N2 was additionally bubbled through the solution for 5 min. Then, 10 mg of azobisisobutyronitrile (AIBN) were added. The reaction mixture was stirred for 20 min at 80 °C, and the resulting polymer was dissolved in CHCl3 and solvent was evaporated under a high vacuum to eliminate volatile nBuA and MMA monomers.Under an N2%-stat-(nBuA)-stat-(MMA)], 2. Mass ratio :nBuA:MMA = 2:78.4:19.6 . Transparent yellow highly viscous liquid (98%). −1, GPC): 20887, 71029, 175060, 207234. Td (TGA): To = 345 °C, Tf = 390 °C, Tp = 378.3 °C, loss 92%, residue 10%. Tg (DSC): –28.5 °C. For 1H NMR spectrum see For poly[4%-stat-(nBuA)-stat-(MMA)], 3. Mass ratio :nBuA:MMA = 4:76.8:19.2 . Transparent yellow highly viscous liquid (97%). −1, GPC): 48103, 81197, 126498, 139350. Td (TGA): To = 338 °C, Tf = 390 °C, Tp = 373 °C, loss 85%, residue at Tf 17%. Tg (DSC): -51.6 °C. For 1H NMR spectrum see For poly[nBuA)-stat-(MMA)], 4. Mass ratio nBuA:MMA = 8:2. Transparent colorless highly viscous liquid (99%). −1, GPC): 21194, 84378, 224387, 266924. Td (TGA): To = 318 °C, Tf = 393 °C, Tp = 378 °C, loss 95%, residue at Tf 9%. Tg (DSC): -28.6 °C. For 1H NMR spectrum see For blank copolymer poly[(2) was carried out following the doctor blade’s technique, using a solution of 20% m/v of the corresponding polymer 2–4 containing (6 × 10−3)% m/v of tetrabutylammonium hexafluorophosphate as an inert lipophilic counterion in CHCl3. Then, the deposited films were dried for 24 h under vacuum, thoroughly washed with de-ionized water, and dried again prior to further evaluation to guarantee that the solvent and remaining traces of monomers nBuA/MMA were completely removed. The thickness of the testing films was controlled through this process to a final value of 50 μm in the solid state using variable deposition speed, blade height, and sample volume depositing as needed. These films were mechanically stable for at least four weeks before testing, whereas the deposition technique allowed for easily prepared homogeneous surfaces.In a typical experiment, testing film deposition over ITO  in a stepwise mode (10−12 mol/L to 10−4 mol/L in the cell) and following the variations on the recorded values. The dilution was corrected for all the stepwise concentration experiments. K+ was used as a counterion for all the anion stock solutions used. The stock solutions (1 mol/L) used in stepwise additions were prepared by diluting the required mass of the salt’s potassium fluoride, potassium acetate, potassium thiocyanate, and monopotassium gluconate, in 100 mL of the aforementioned KNO3 background solution, and the pH was adjusted to neutral.Supramolecular recognition assessments were carried out using the zero-current potentiometry technique, where the selectivity and response of the polymers vs. different anions was followed by measuring the electromotive force (EMF) against anion concentration. All the EMF measurements were taken in a cell containing 50 mL of a KNOg is decreased by the bulky Zn-Salphen component. This situation paves the way for more simplified strategies in chemical sensor design, departing from one single functional monomer that only depends upon the composition, thereof.In conclusion, the present work shows that a straightforward modulation of the supramolecular recognition behavior of a functional polymer could be achieved by the molecular design of a polymerizable Zn-Salphen monomer and its co-polymerization at different ratios with an acrylic backbone. The origin of such tailored recognition behavior is mainly an interplay between attractive host-guest interactions and either repulsive or attractive guest-polymer backbone interactions, depending on the nature of the guest, together with an increased polymer free volume when T


Olea europaea protects endothelial cells against oxidative stress generated by reactive oxygen species (ROS). Methods: OLE total polyphenols (TPs) were characterized by the Folin–Ciocalteu method. Endothelial cells were grown in conventional cultures  and on a biomaterial scaffold  fabricated via electrospinning. Cell viability and ROS measurement after H2O2 insults were performed. Results: OLE TP content was 23.29 mg GAE/g, and oleuropein was the principal compound. The dose-dependent viability curve highlighted the absence of significant cytotoxic effects at OLE concentrations below 250 µg/mL TPs. By using OLE preconditioning at 100 µg/mL, cell viability decrease was observed, being in 3D lower than in the 2D model. OLE was protective against ROS in both models. Conclusions: OLE represents a high-value antioxidant source obtained by biowaste that is interesting for biomedical products. Using a 3D scaffold could be the best predictive model to mimic the physiological conditions of vascular tissue reaction.Olive leaf extract (OLE) can be obtained as biowaste and is extensively used a food supplement and an over-the-counter drug for its beneficial effects. New studies have investigated OLE concerning the role of oxidative stress in the pathogenesis of vascular disease. This in vitro study aims to evaluate if OLE extracted from the Tuscan  Olea europaea is one of the most ancient trees of the Mediterranean region. Olive leaves, an agricultural waste by-product obtained during the harvesting or pruning process of olive fruit, contain considerable bio-phenols as the other parts of olive tree  powder (70:30 % mol) was purchased from Piezotech Arkema , Methyl ethyl ketone (MEK) was bought from Merck . Medium 199 (M199), fetal bovine serum (FBS), penicillin-streptomycin solution, L-glutamine, HEPES buffer were supplied by Hospira S.r.l. . HPLC-grade acetonitrile and methanol were purchased from Labscan . Acetic acidof analytical grade (assay >99.5%) was purchased from Fluka . Water was purified by using a Milli-Q System . Other reagents unmarked were of analytical grade. Methanol (MeOH) was purchased from Carlo Erba , H2DCFDA) was supplied by Invitrogen. EGM-2 medium bullet kit was bought from Lonza , alamarBlue® was obtained from Thermo Fisher Scientific .4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolium]-1,3-benzenedisulfonate (WST-1 assay), was purchased from Roche Applied Science , 5-(and-6)-chloromethyl-2’,7’-dichloro-di-hydro-fluorescein diacetate, acetyl ester , Italy. Leave collection was carried out manually and at different months between February 2018 and March 2019. The leaves were stored at 25 °C after harvesting. For each lot, 20 g of leaves were placed in liquid nitrogen and crushed manually. A final weight ratio of 0.9 g OLE/g  was obtained and used for future experiments. Olive leaves were obtained from To measure TP content, 1 mg of OLE powder was dissolved in water and therefore analyzed with Folin–Ciocalteau’s method , as per A high-performance liquid chromatography analysis (HPLC) was carried out to identify and quantify the major phenolic compounds of the OLE using a slightly modified method developed in our previous study .The retention times and UV absorbance spectra of phenolic compounds present in OLE were compared with those of the commercial standard and quantified at 278 nm, using p-hydroxyphenyl acetic acid as the internal standard, according to the previously reported method. Sample concentrations were determined by linear regression. For each calibration curve, the correlation coefficients were >0.99. 2O/AcOH (97.5:2.5 v/v) (A) and MeOH/ACN (1:1 v/v) (B), programmed as follows: a gradient from 5% (B) to 30% (B) in 45 min; 30% (B) for 5 min and then from 30% (B) to 5% (B) in 5 min. The flow rate was 1 mL/min, and the injected volume was 50.0 μL.HPLC analysis was performed using an HPLC instrument  equipped with a System Gold Solvent Delivery module (Pumps) 125 and a System Gold UV/Vis Detector 166, set to 280 nm, and using a Phenomenex Gemini reverse-phase C18 column . The mobile phase was a mixture of H2 using growth culture medium consisting of M199 (PAN-Biotech), supplemented with 10% FBS, penicillin-streptomycin solution, l-glutamine, HEPES buffer. After 24 h, the growth medium was replaced to remove excess red blood cells.Tissue samples were collected and treated anonymously and in conformity with the Declaration of Helsinki. Human umbilical vein endothelial cells (HUVECs) were isolated from the umbilical vein endothelium of healthy human donor cords, following the procedure described by Jaffe et al. . Isolatew/v and stirred at 300 rpm for 12 h at room temperature to allow complete dissolution. The polymer solution was loaded into a 10 mL glass syringe, fitted with a blunt tip stainless steel needle (21G × 3/4”), and placed into a syringe pump . The ground terminal of high voltage supply  was connected to the metal needle, whereas the positive terminal was connected to the collector; 35 kV potential was applied. A cylindrical collector , was placed at a distance of 15 cm from the tip of the needle. The polymer solution was injected from the needle in the presence of the electric field at a constant flow rate of 0.016 mL/min and collector velocity of 500 rpm, room temperature, and 46% relative humidity. The fiber meshes were kept in an oven at 60 °C overnight to remove solvent traces. The morphology of the scaffolds was evaluated by scanning electron microscopy (SEM) using an FEI FEG-Quanta 450 instrument . The samples were sputtered with gold  before analysis. P(VDF-TrFE) was dissolved in MEK at a concentration of 20% n = 3).HUVECs between passage P2–P4 were treated for 2 and 24 h with different polyphenol concentrations of OLE  in growth medium with 5% FBS . The absorbance was directly correlated to the number of metabolically active cells, and viability was expressed as a percentage of viable cells. Cell viability was determined by colorimetric assay with WST-1, based on the cleavage of tetrazolium salt by mitochondrial dehydrogenases, present in viable cells. Briefly, at the end of the treatment, HUVECs were incubated with tetrazolium salt at 10 μL/well for 3 h at 37 °C, in 5% CO2O2 for 1 h. At the end of each treatment, cells were analyzed for viability and ROS production.The protective effect of OLE was evaluated pre-treating HUVECs with different OLE polyphenol concentrations for 2 and 24 h and then with 100 μM of H2DCFDA, a cell-permeable indicator for these compounds. Briefly, during the last 30 min of treatment with H2O2, the HUVECs were co-incubated with CM-H2DCFDA at 10 μM/well dissolved in PBS, in the dark at room temperature. ROS production was detected by measuring the increase in fluorescence over time by microplate reader (Thermo Scientific Fluoroskan Ascent Microplate Fluorometer), using excitation at 488 nm and emission at 510 nm.Intracellular ROS production was evaluated by ROS fluorescent probe CM-Hw/v) gelatin aqueous solution for 30 min and seeded with HUVECs at a density of 1 × 105 cells/sample. Cell/scaffold constructs were cultured in EGM-2 medium bullet kit  for 1 week in a standard incubator  by replacing the medium every 48 h. The P(VDF-TrFE) scaffolds were cut into 0.8 × 0.8 mm squares and sterilized in absolute ethanol overnight. The scaffolds were coated with a sterile-filtered 2% :Cell/scaffold construct (control);Cell/scaffold construct + OLE (100 µg/mL GAE) for 24 h;2O2 (100 µM) for 1 h;Cell/scaffold construct + H2O2 (100 µM for 1 h).Cell/scaffold construct + OLE (100 µg/mL GAE for 24 h) + HThe in vitro effect of OLE on metabolic activity and ROS production was tested on HUVEC/scaffold constructs following H® assay, according to the manufacturer’s instructions and expressed as the percentage of reduced alamarBlue® (%ABred). Data were acquired after 3 days of culture without treatments to evaluate cytocompatibility and post-treatment to evaluate the antioxidant activity of OLE. The absorbance of supernatants was measured with a spectrophotometer  under a double-wavelength reading (570 and 600 nm). Finally, %ABred was calculated by correlating the absorbance values and the molar extinction coefficients of the dye at the selected wavelengths, following the protocol provided by the manufacturer. Cell metabolic activity was assessed using the alamarBluew/v) neutral buffered formalin for 10 min at 4 °C. SEM was used on cell/scaffold constructs to image cell attachment, for morphology, and for spreading onto the scaffolds. The biological samples were dehydrated with 70% ethanol for 30 min and finally dried in oven at 37 °C for two h prior to sputter coating. For examination, the samples were mounted on aluminum stubs, sputter-coated with gold, and observed under SEM. At the endpoint, the cell/scaffold constructs were washed twice with 1× PBS and then fixed in 1% (n = 3).Intracellular ROS production was evaluated by ROS fluorescent probe CM-H2DCFDA. Briefly, after cell treatment, the cells/scaffold constructs were washed twice with 1× PBS and then stained with Hoechst 33342 (5 µM for 10 min) for nuclei visualization. The constructs were then washed twice with 1 × PBS and incubated with 10 μM CM-H2DCFDA in PBS for 30 min in the dark at room temperature. The cells/scaffold constructs were then washed three times with PBS and visualized under a fluorescent microscope . ImageJ software (Version 1.52r) was used to measure the integrated optical density (OD) of the stained cells, which was normalized for green + blue integrated OD  in 1x PBS for 10 min RT, to detect cell nuclei (fluorescent in blue) and washed in 1× PBS. Specimens were observed by an inverted fluorescence microscope equipped with a digital camera .p-value < 0.05 were considered statistically significant.Data are presented as mean ± standard deviation (SD). Three independent replications were evaluated for each treatment with OLE. The in vitro experiments were performed in three separate series of three independent replicates for each well. The difference among groups of values was evaluated by a one-way ANOVA, and a post hoc analysis was performed by Tukey’s or Bonferroni’s multiple comparisons test when appropriated. Differences with a Olea europaea after prunings, namely 18 g OLE/20 g leaves, and characterized. It resulted highly rich in polyphenols (23.29 mg GAE/g), among which oleuropein accounted for a concentration of 14.69 ± 0.92 mg/g of OLE corresponding to 1.47% (w/w%), and luteolin 7-O-glucoside for a lower concentration of 3.60 ± 0.25 mg/g of OLE, corresponding to 0.36% (w/w%).OLE was obtained from the leaves of the Tuscan 2O2 insult. A number of medical applications can potentially benefit from OLE, including cardiovascular disease and wound healing, especially for wounds compromised by ROS stress. Local administration of OLE may thus represent a valid and strategy in biomedicine, which is also compliant with a sustainable use of bio-resources.OLE administration to endothelial cells (HUVECs) showed a protective effect against ROS production, which was also confirmed in a 3D culture model. This model was set up under the tissue engineering approach by using electrospun P(VDF-TrFE) scaffolds. OLE showed good cytocompatibility and antioxidant activity, which revealed effectiveness in controlling the oxidative stress on HUVECs upon exposure to an H


Escherichia coli (E. coli). The device features three components: paper for its ability to extract protein molecules nonspecifically from cells, DNA superstructures for their ability to immobilize RNA-cleaving DNAzymes (RCDs) but undergo target-induced RNA cleavage on paper, and isothermal rolling circle amplification (RCA) for its ability to amplify each cleavage event into repetitive sequence units that can be detected by naked eye. This device can achieve detection of E. coli K12 with a detection limit of as low as 103 CFU·mL−1 in a total turnaround time of 35 min. Furthermore, this device allowed the sensitive detection of E. coli in complex sample matrices such as juice and milk. Given that more specific RCDs can be evolved for diverse bacteria, the integrated PAD holds great potential for rapid, sensitive and highly selective detection of pathogenic bacteria in resource-limited settings.Rapid detection of pathogenic bacteria is extremely important for public health and safety. Here, we describe for the first time an integrated origami paper-based analytical device (PAD) incorporating cell lysis, molecular recognition, amplification and visual detection of  Escherichia coli, Salmonella, Helicobacter pylori, Campylobacter and Vibrio cholerae [Escherichia coli (E. coli) [E. coli is a widely distributed Gram-negative bacterium that mainly lives in the intestines of humans and warm-blooded animals for facultative anaerobic activities. However, it can also cause serious symptoms such as sepsis and hemolytic uremic syndrome [E. coli pathogens are allowed in drinking water. Since E. coli can cause pathogenicity at low doses (most likely less than 100 organisms) [E. coli is of great significance in the fields of medical diagnostics, food safety, and environmental monitoring.In recent years, infectious diseases caused by food- and water-borne bacterial pathogens have a high incidence throughout the world ,2,3,4. Tcholerae ,6,7,8, aE. coli) . E. colisyndrome ,11, and ganisms) , the devTraditional culture-based methods for qualitative and quantitative detection of pathogens are powerful. However, the requirement for pre-enrichment in non-selective culture broth, selective enrichment and plating, biochemical analysis and serological confirmation makes these methods labour-intensive and time-consuming, as the entire process can take several days ,14. In rE. coli and Clostridium difficile [Recent advances in functional nucleic acids (FNAs) have enabled the use of them as molecular recognition elements for various bioanalytical and biomedical applications ,24. FNAsifficile ,26. Furtifficile ,28,29. HE. coli cells. To facilitate the attachment and functionality of EC1 on paper device, we used our previously proposed 3D DNA strategy for expedient engineering of functional DNA-based surfaces [E. coli K12 with a detection limit of 103 CFU·mL−1. To investigate whether this paper sensor can specifically recognize E. coli, we selected two bacteria: (1) Bacillus subtilis, commonly found in soil, water sources and in association with plants, is the best-characterized member of the gram-positive bacteria [Providencia sp., one common gram-negative bacteria, can cause diseases such as diarrhea, extraintestinal infections and urinary tract infections [E. coli whereas other samples  showed negative results. This fully integrated oPAD holds great promise for future POC detection of pathogenic bacteria in resource-limited settings.In this study, we developed a disposable origami paper-based analytical device (denoted as oPAD) that fully integrates on-paper cell lysis for protein extraction, target-responsive RCDs for molecular recognition, rolling circle amplification (RCA) for signal amplification, origami technique for reagent diffusion and flow, and portable readout. For this work, we employed EC1, a reported RCD that can be activated by protein molecule secreted specifically by surfaces . The devbacteria ; (2) Profections . The gooAll DNA oligonucleotides were obtained from Takara Biotechnology Co.  and purified by standard 10% denaturing (8 M urea) polyacrylamide gel electrophoresis (dPAGE) or high-performance liquid chromatography (HPLC). T4 DNA ligase, dNTP, and T4 polynucleotide kinase (PNK) were purchased from Takara Biotechnology Co. . ϕ29 DNA polymerase was acquired from Thermo Scientific . Pullulan was obtained from Sangon Biotech . BCA Protein Assay Kit was acquired from Beyotime Biotechnology . Triton X-100 cell lysis buffer SS0890 was obtained from NOVON . Nitrocellulose paper (HF180), Whatman filter paper 1# and Whatman 3MM CHR chromatography paper were purchased from GE Healthcare . All other chemicals were purchased from Sangon Biotech  and used without further purification. Water was purified with a Greate Fun SUMMER-S-10 water purification system.The sequences of the DNA molecules were as follows:CDT1 (anti-EC1): GATATCATAT CACACAACTG TAAAGAAATC CATCCCCACA CAGTGTAGTG TTCCGGTCGC AGGTCTCGAC AACGCACATC (5′→3′)TP1: ATATGATATC GATGTGCGTT (5′→3′)DT1: ACGCACATCG ATATCATATC AC (5′→3′)CDT2: TAGCTAGGAA GAGTCCCAAC CCGCCCTACC CAAAATGTCT CGGAT (5′→3′)TP2: TTCCTAGCTA ATCCGAGACA (5′→3′)F-RS28: ACTCTTCCTA GCFrAQGGTTC GATCAAGA-invert dT (5′→3′)The fluorescent images of gels were obtained using a Typhoon 5 variable mode imager  and analyzed using Image Quant software . Paper well plates were printed using a Xerox ColorQube 8560N solid wax printer , then heated at 120 °C for 2 min to melt the printed wax.A circular DNA template (CDT1) containing anti-EC1 was first synthesized as previously described . 1 μL ofE. coli K12 grown on Luria Broth (LB) agar plate was taken and used to inoculate 50 mL of LB. After shaking at 37 °C until OD600 reached ~1, the bacterial culture was serially diluted in 10-fold intervals. 10 µL of each diluted solution were plated on the LB agar plates (3 repeats) and cultured at 37 °C for 18 h to determine the number of colony forming units per milliliter (CFU·mL−1) in the original bacteria cultures. Cells in the sample were then removed by centrifugation at 8000 g for 10 min at 4 °C. The crude intracellular mixture (CIM) from E. coli and B. subtilis cells (~107 CFU·mL−1) was prepared according to our previously reported method [A single colony of d method .w/v) pullulan, and then printed onto Panel C using the dispensing unit. For Panel D, 1 µL of 1 µM CDT2, 1 µL of 10 mM dNTPs, 1 µL of 100 µM hemin, 4 U of PNK and 5 U of ϕ29DP were first mixed with 20 µL of 10% (w/v) pullulan, and then printed. The bioactive paper was dried and stored at RT in a desiccant container. In order to assemble these four panels, a single sided adhesive film was used to enable bidirectional folding.Each device contains four panels, namely: an absorbent pad  for sample purification and washing; Whatman 3MM CHR chromatography paper  for cell lysis; Whatman Grade 1# filter paper  for DNAzyme recognition, and Nitrocellulose membrane HF180  for RCA. For dry reagent storage, 5 µL of cell Lysis Buffer was pipetted onto the Panel B. The Panel was dried in a vacuum oven at 50 °C for about 1 h. For Panel C, 5 μL of 3D EC1 was printed using a Biodot XYZ3060 automated dispensing unit . After immersion into the blocking buffer  for 10 min, the obtained bioactive paper was dried at RT. 5 µL of F-RS28 (100 nM) was then mixed with 5 µL of 10% (E. coli cells (10–107 CFU·mL−1) was applied to the Panel B and incubated for 3 min. After washed by 50 μL of pure water, protein levels were measured with the BCA Protein Assay Kit according to the manufacturer’s protocol. The colorimetric result was recorded within ~1 min by a digital camera and analyzed by ImageJ.5 μL of E. coli cells (10–107 CFU·mL−1) was applied to the Panel B. Following incubation for 3 min, 50 μL of pure water was then added. Panel B was then the folded onto Panel C and D to allow the flow of liquid. 50 μL of 1×RCA Buffer  was added to the back of Panel B to elute the protein. After the pullulan film was dissolved onto Panel D (~30 min), the flexible oPAD was folded to physically separate the Panels. 5 μL of 40 mM H2O2 and 5 μL of 20 mM TMB to initiate the colorimetric reaction on Panel D. The colorimetric result was recorded within ~1 min by a digital camera and analyzed by ImageJ.5 μL of E. coli. By folding Panel B onto Panel C and Panel D, the purified protein targets were then transferred to Panel C, where the printed fluorogenic RNA substrate  was released, and the following 3D EC1-mediated RNA cleavage can be performed. The fluid containing 5’ cleaved fragment will further migrate to Panel D, where the printed pullulan-encapsulated RCA reagents  was dissolved and the following RCA reaction was activated using the 5’ cleaved fragments as new primers. The detection of E. coli is therefore easily converted to the detection of RCA product, which was designed to contain a repetitive unit of PW17, a peroxidase-mimicking DNAzyme capable of producing a colorimetric signal by oxidizing the chromogenic substrate TMB in the presence of hemin and H2O2 [The working principle and key functionalities of the oPADs are illustrated in and H2O2 . E. coli cells; (2) protein purification. One wash to remove the potential inhibitors and surfactants on Panel B; (3) protein elution. One wash to elute the purified proteins from Panel B for the following DNAzyme-mediated cleavage on Panel C and RCA on Panel D; (4) signal readout. The chromogenic substrate and H2O2 were added to initiate the colorimetric reaction. The device can provide quantification of E. coli within 35 min, including 3 min of cell lysis, 30 min of cleavage reaction and RCA, and 1 min of signal read-out.The overall operating processes include four steps: (1) cell loading and lysis. The impregnated dry lysis buffer is rehydrated on Panel B and provides the chemical lysis of E. coli, resulting in a dramatic time-dependent increase in fluorescence. In contrast, no obvious fluorescence increase was observed upon the addition of B. subtilis. Taken together, these results indicated that 3D EC1 retained its high cleavage activity and specificity for E. coli.To create the bioactive paper surface, we used the micron-sized 3D DNA superstructure to immobilize EC1 for the molecular recognition. 3D EC1 containing concatemeric EC1 was first synthesized by RCA. Transmission electron microscopy  images in 6E. coli cells was pipetted onto Panel B containing dried lysis reagents. It took about 3 min for cell lysis  protein assay. As shown in 3 cells  of 103 CFU·mL−1. It was known that the colorimetric signal depended on the RCA product [B. subtilis. and Providencia sp.). These control bacteria were not able to induce a significant color change, indicating the high specificity of our device  into different samples, including milk and juice. It was observed that this device was able to produce a positive signal , is approximately 103–104 CFU·mL−1 in several hours [E. coli) require detection limit of as few as 1–100 CFU in food or water. Thus the challenge that must be addressed is how to make this paper device suitable for integration with sample enrichment for real-world application.In summary, we developed a fully disposable and integrated paper-based device by integrating paper-based protein extraction, paper-based FNAs sensor, paper-based isothermal DNA amplification and portable readout. The detection limit of our device was 10al hours . Thus, o