B. monnieri extract (BME) is an abundant source of bioactive compounds, including saponins and flavonoids known to produce vasodilation. However, it is unclear which components are the more effective vasodilators. The aim of this research was to investigate the vasorelaxant effects and mechanisms of action of saponins and flavonoids on rat isolated mesenteric arteries using the organ bath technique. The vasorelaxant mechanisms, including endothelial nitric oxide synthase (eNOS) pathway and calcium flux were examined. Saponins (bacoside A and bacopaside I), and flavonoids (luteolin and apigenin) at 0.1–100 µM caused vasorelaxation in a concentration-dependent manner. Luteolin and apigenin produced vasorelaxation in endothelial intact vessels with more efficacy (Emax 99.4 ± 0.7 and 95.3 ± 2.6%) and potency (EC50 4.35 ± 1.31 and 8.93 ± 3.33 µM) than bacoside A and bacopaside I . Pretreatment of endothelial intact rings, with L-NAME (100 µM); an eNOS inhibitor, or removal of the endothelium reduced the relaxant effects of all compounds. In K+-depolarised vessels suspended in Ca2+-free solution, these active compounds inhibited CaCl2-induced contraction in endothelial denuded arterial rings. Moreover, the active compounds attenuated transient contractions induced by 10 µM phenylephrine in Ca2+-free medium containing EGTA (1 mM). Thus, relaxant effects occurred in both endothelial intact and denuded vessels which signify actions through both endothelium and vascular smooth muscle cells. In conclusion, the flavonoids have about twice the potency of saponins as vasodilators. However, in the BME, there is ~20 × the amount of vaso-reactive saponins and thus are more effective. Bacopa monnieri (L.) Wettst. or Brahmi, is an Ayurvedic medicine traditionally used as a memory enhancer. Along with memory improvement, it is known to promote mental health, as a neurotonic and cardiotonic agent. B. monnieri extract (BME) clearly has a cognitive enhancing potential and neuroprotective effects . Th. Th50 4.However, higher potency of aglycone flavonoids compared to saponin glycosides may be due to sugar moieties interfering with the molecule interacting with the binding sites responsible for the vasorelaxation as suggested by previously, i.e., lipophilic groups in the ring skeleton of flavonoids increased their vasorelaxant activity . This pr50 and reduced Emax which imitated the effect of endothelial denudation, suggesting the relaxation was mainly medicated by NO. This accords with observations made by Jin et al. that a cyclooxygenase (COX) inhibitor did not affect the relaxation induced by apigenin  HEPES, 10; KH aerated ,49,50,51B. monnieri active compounds (0.1–100 µM), including luteolin, apigenin, bacoside A or bacopaside I were added cumulatively.Following stabilization, endothelial intact rings of mesenteric arteries were pre-contracted with 10 µM PE. After the contraction had become constant, the B. monnieri active compounds  induced vasorelaxation, endothelial denuded arteries were used. The data of effect of active compounds were presented as %relaxation.Successful endothelial denudation was confirmed by the absence of relaxation upon addition of 10 µM ACh. For investigation of the role of endothelium in 0.1–100 µM B. monnieri active compounds  induced vasorelaxation were evaluated in endothelial intact ring pre-treated with NG-nitro-L-arginine methyl ester , an inhibitor of eNOS, for 30 min prior to 10 µM PE exposure.The role of the endothelial relaxing factor, NO, in 2+-free Krebs’ solution (containing (mM): ethylene glycol-bis (ß-aminoethyl ether)-N,N,N,N tetra acetic acid (EGTA), 0.01; NaCl, 122; KCl, 5; HEPES, 10; KH2PO4, 0.5; NaH2PO4, 0.5; MgCl2, 1 and glucose, 11 (pH 7.3)) for 30 min followed by replacing with Ca2+-free Krebs’ solution containing 80 mM K+ for 10 min which depolarizes the vascular smooth muscle cells, thus opening VOCCs. Various concentrations of CaCl2 were then added (0.01–10 mM) in a logarithmic progression. After obtaining the maximum response, the baths were washed out and replenished with Ca2+-free Krebs’ solution for 30 min. The Ca2+-free 80 mM K+ solution was then re-applied following pre-incubation for 10 min with either: 10 µM active compounds or 1 µM nicardipine (antagonist of VOCCs). Concentration-response curves to cumulative addition of CaCl2 were then repeated and compared with maximum contraction evoked by previous control CaCl2 challenges.Endothelial denuded mesenteric arteries were equilibrated in Ca2+ loading of the SR Ca2+ stores, endothelial denuded mesenteric arteries were exposed to 80 mM K+ solution for 5 min, and then washed out with Ca2+-free Krebs’ solution containing 1 mM EGTA for 10 min. The arterial rings were then challenged with 10 µM PE  which release Ca2+ from the SR thereby eliciting a transient contraction [+ solution for 5 min, and washed out with Ca2+-free Krebs’ solution containing 1 mM EGTA and 10 µM active compounds for 10 min. The arterial rings were again challenged with 10 µM PE. The PE-induced contractions were compared in the presence or absence of active compounds.To stimulate initial Catraction . The sam50 and Emax were compared using unpaired Student’s t test. Values are expressed as mean ± SEM. A p-value < 0.05 was considered significant. ‘n’ is the number of vascular rings used, each ring originating from a different animal.Statistical analyses used GraphPad Prism version 5.00 for Windows, . Data from each concentration-effect curve was analysed using non-repeated two-way ANOVA. Curve fitting in the figures was generated by the same software using non-linear regression. ECB. monnieri active components, including both saponins and flavonoids, produced vasodilatory effects on rat isolated mesenteric arteries partially via endothelial dependent release of vasodilators and also by direct effects on vascular smooth muscle cells via blockade of Ca2+ influx and its release from SR. This study for the first time reports the comparative vasodilatory effects of saponins and flavonoids found in B. monnieri extract. However, B. monnieri extract, flavonoids i.e., luteolin and apigenin would be more potent vasodilators but saponins have a greater effect because of their greater contents. Accordingly, the clinical benefits on enhanced blood flow and cognitive function may arise from a combination of flavonoids and particularly the saponins.This study demonstrated that


AbstractZeamaysssp.mays Linnaeus, 1753) and Z.perennis  Reeves & Mangelsdor, 1942, by producing a series of autopolyploid and allopolyploid maize hybrids. The intra-genomic and inter-genomic meiotic pairings in these polyploids were quantified and compared using dual-color genomic in-situ hybridization. The results demonstrated higher level of chromosome stability in allopolyploid maize during meiosis as compared to autopolyploid maize. In addition, the meiotic behavior of Z.perennis was relatively more stable as compared to the allopolyploid maize. Moreover, ten chromosomes of "A” subgenome in maize were homologous to twenty chromosomes of Z.perennis genome with a higher pairing frequency and little evolutionary differentiation. At the same time, little evolutionary differentiation has been shown by chromosomes of "A” subgenome in maize, while chromosomes of "B” subgenome, had a lower pairing frequency and higher evolutionary differentiation. Furthermore, 5IM + 5IIPP + 5IIIMPP and 5IIMM + 5IIPP + 5IVMMPP were observed in allotriploids and allotetraploids respectively, whereas homoeologous chromosomes were found between the "A” and "B” genome of maize and Z.perennis.This study was aimed to investigate the stability of chromosomes during meiosis in autopolyploid and allopolyploid maize, as well as to determine an association of chromosomes between maize ( Zea Linnaeus, 1753, belongs to the tribe Maydeae Candolle, 1882, and consists of two sections: section Luxuriante and section Zea  is an important economic crop and polyploid genetic model with many duplicated genes in all of its ten chromosomes  Reeves & Mangelsdor, 1942 (section Luxuriantes), 2n=40 is one of the perennial teosintes having an inferred octoploid origin  to give systematic understanding of chromosome relationship between maize and Zeaperennis; (ii) to observe the meiotic chromosome stability in autopolyploid and allopolyploid maize (iii) to reveal the origin and differentiation process of "A” and "B” subgenomes (iv) to validate the chromosome paring pattern by using general cytology and dual-color genomic in situ hybridization (GISH) in a number of autopolyploid and allopolyploid hybrids that were synthesized by the cross of maize and Zeaperennis.From previous research, it is evident that crosses could be made between maize and  species . Differeraploids . The comraploids . Maize aectively . Besidesectively . Furthers of Zea . HoweverGISH Genomic in situ HybridizationRCC Relative chaotic coefficientPMCs Pollen mother cellsUSDA), Zeaperennis  was obtained from International Maize and Wheat Improvement Center (CIMMYT). The plant material was raised at experimental farm of Sichuan Agricultural University, Jinghong, China. Three crosses were made by hand pollination that were (1) between diploid maize inbred wf9 and Z.perennis, (2) between tetraploid maize Twf9 and Z.perennis and (3) between diploid maize inbred line wf9 and tetraploid maize Twf9. In next year, the pre-germinated hybrid seeds were planted in soil filled plastic pots  and placed in experimental station of Sichuan Agricultural University for initial identification. The seedlings at 5-leaf stage were transplanted into larger plastic pots  for further root tips collections.Plant materials are shown in Table The roots collected from parents and interspecific hybrids were immediately fixed in a saturated solution of α-bromonaphthalene for three hours, subsequently, transferred in Carnoy’s solution I  for 24 hours and, finally submerged in 70% ethanol solution after which these were preserved at 4 °C. Pre-mature anthers of hybrids and parents were collected and treated with Carnoy’s solution for a minimum of 12 hours and then preserved in 70% ethanol solution at 4 °C.in situ hybridization. Total genomic DNA from young leaves of maize and Z.perennis was extracted according to modified 2 × CTAB methods  and 1% pectinase  for 2.5–5.0 hours at 37 °C. Root tips and anthers were again thoroughly cleaned with distilled water in order to wash enzyme solution and finally, squashed onto glass slides in a drop of Carnoy’s solution I and dried with ethanol flame. The preparations showing well-spread and clean mitotic and meiotic chromosomes were selected by phase-contrast light microscopy  and stored at -20 °C for  methods .Z.perennis were labeled with DIG-Nick Translation and BIOTIN-Nick Translation Mix , respectively according to manufacturer’s protocol. The selected slides were preheated in an air blowing oven at 60 °C for one hour and then transferred into 0.1ug/ml RNase  in 2 × SSC solutions in a thermostat water bath at 37 °C for one hour. Afterwards, slides were washed twice in 2 × SSC for 5 minutes each at room temperature, followed by chromosome denaturation in 70 percent deionized formamide (FAD) solution at 70 °C for 2.5 minutes, then immediately dehydrated in an ice-cold 70 percent, 95 percent and 100 ethyl alcohol series and finally air dried at room temperature. The hybridization mixture comprised 150 µl 50% FAD, 60 µl 10% dextran sulfate (DS), 30 µl 2 × SSC, 15 µl 0.5% sodium dodecyl sulfate (SDS), 30 μg salmon sperm DNA (SSDNA) and 18 µl labeled probes for six slides. Hybridization mixture was denatured in a thermostat at 85 °C for 10 minutes, followed by quick cooling in ice for 10 minutes. A total 46 μl hybridization mixture was loaded on each slide and hybridization was accomplished in an incubator at 37 °C for 20–24 hours. After hybridization slides were immersed in 20% FAD, 2 × SSC, 0.1 × SSC, respectively for 15 minutes each, at 42 °C. After that, the slides were washed in 0.1% Triton X-100 once and in 1 × PBS thrice for 5 minutes each and then air dried, at room temperature. All further steps were performed in dark, 50 µl antibody diluent, which contained anti-digoxigenin-fluorescein  and streptavidin-Cy-3 fluorescein  was applied onto air dried slides and immunodetection was done at 37 °C for one hour in an incubator. Consequently, slides were washed in 1 x PBS thrice for 5 minutes each at room temperature and air dried finally. The chromosome counterstaining was performed by 4, 6-diamidino-2-phenylindole (DAPI) solution containing 86% 1 × PBS and 14% DAPI 10ug/ml (Solarbio), and slides were observed with fluorescence microscope  coupled with pre-fixed filter sets named as U-MNAU2 , MWIBA3  and U-MWIG3 . The images were captured with Media Cybernetics CCD 700 (Charge Coupled Device) and Image Pro Plus 6.0 . Captured images were processed by Adobe Photoshop 5.1.The genomic DNA of maize and Z.perennis (9475) to produce polyploid hybrids, and those synthetics are shown in Fig. GISH) had 10 maize and 20 Z.perennis chromosomes, while allotetraploid maize MP40 (2n=4x=20) was consisted of 20 maize and 20 Z.perennis chromosomes , and an average pairing configuration was of 0.18I+10.46II+0.13III+4.62IV  and five (40.30%) with the range of 3–18 and 1–8, respectively , as shown in Table Diploid maize genome exhibited regular meiosis and the most frequently observed meiotic configuration was 10II . Nearly half of analyzed PMCs did not show univalents and the average (range) number was 0.71 (0–3). The most frequent number of bivalents was four (24.00%) with an average (range) number of 3.31 (0–9) that suggests some of the paired chromosomes in maize genome didn’t share complete homology. MM30 had abundant trivalent, the most repeated number was eight (24.00%) and an average (range) number was 7.17 (2–10). On the contrary with wf9, most of PMCs in MM30 did not contain quadrivalents and an average (range) number was 0.28 (0–3), as shown in Tables RCC was 4.87, higher than that of wf9  with the average of 0.71I+3.31II+7.19III+0.28IV, while 10III (11.72%) was also found in some Cs Table 1. Nearly9.67% witPMCs of MP30 frequently showed lagging chromosomes at meiotic anaphase I . Dual-color genomic in situ hybridization was carried out to study cryptic chromosome pairing in synthesized allopolyploid , and most common number was five (18.30%) with an average number (range) of 3.79 (0–8). There were also some univalents from Z.perennis (IP) with most repeated number of zero (57.75%) and an average number (range) was 0.77 (0–7). Most autosyndetic bivalents in MP30 were from Z.perennis (IIPP) with the most common number of five (26.76%) and an average number (range) of 4.34 (1–7). The other autosyndetic bivalents were from maize genome (IIMM) with an average number of 0.25 (0–1), whereas allosyndetic bivalents (IIMP) were rarely seen in MP30, as their average numbers (range) was 0.85 (0–4). Most of the trivalents were allosyndetic trivalents (IIIMPP), which were composed of one chromosome from maize and two chromosomes from Z.perennis, and most the frequent number was five (36.62%) with an average number of 4.55 (0–7) as shown in Table Z.perennis. Moreover, quadrivalents and autosyndetic trivalents were rarely seen in MP30  of 3.23 (0–6), Fig. Z.perennis were discrepant homologous  was lower than in MP30 (9.00) and autosyndetic bivalents of maize genome in MP30 were much lower than MM30. The Z.perennis chromosomes in MP30 had lower RCC than that of maize in MM30 and MP30. Thus, overall RCC of MP30 was lower than that of MM30.For more detailed analysis of chromosomes in MP30, the configuration of IIIses Fig. . The mosous Fig. . Additionis Fig. . ComparaPMCs were found commonly at meiotic anaphase I. More than half of PMCs did not show univalents and trivalents with average number (ranges) for univalents and trivalents being 0.26 (0–2) and 0.14 (0–2), respectively. Moreover, commonest number of bivalents was zero (25.00%) with an average number (range) of 3.61 (0–10). MM40 had abundant quadrivalents and the most frequent number was nine (24.17%) with an average (range) number of 8.03 (4–10), as shown in Tables RCC was 4.83, higher than wf9 (Table The most frequent meiotic configuration of MM40 was 10IV (21.67%) with the average of 0.26I+3.61II+0.14III+8.03IV  number of maize and Z.perennis genome’s univalents were 0.81 (0–8) and 0.36 (0–7), respectively. The autosyndetic bivalents from maize and Z.perennis were frequently appeared and the most prevalent number was five  for both, while average number (ranges) for maize and Z.perennis were 4.30 (2–7) and 4.71 (1–9), respectively. Most PMCs did not possess allosyndetic bivalents and the average number (range) was 0.96 (0–4). The trivalents existed in several PMCs; in addition, maize autosyndetic trivalents were not found. Most PMCs did not contain autosyndetic quadrivalents and most allosyndetic quadrivalents (IVMMPP) consisted of two chromosomes from Z.perennis and two chromosome from maize, with most prevalent number of five (24.64%) and an average number was 4.29 (1–7) as shown in Tables Z.perennis genome.The most common meiotic configurations of allotetraploid maize MP40) were 8II+6IV (15.94%) and 12II+4IV (15.94%), and the lagging chromosomes found at meiotic anaphase I Fig. . However0 were 8IMMPP were not similar. The most frequent configuration of IVMMPP was of "ring type” with an average number (range) of 0.72 (0–6), while another form of "rod type” also found  and common number (range) of autosyndetic quadrivalent in MM40 was nine (4–10), that indicated some paired chromosomes in "A” subgenome or in "B” subgenome have been differentiated. Furthermore, the Z.perennis belongs to another section of genus Zea  revealed that there were not only "frying pan type”, but "rod type” also existed in allotriploid with a maximum number of five (Table MMPP) was not only the "rod type” but "ring type” was also found (Table MPP and IVMMPP, the degree of chromosome pairing was variable e.g. relatively tight chromosome pairing between maize and Z.perennis, maize and maize, Z.perennis and Z.perennis were observed, while the loose chromosome pairing between maize and Z.perennis, maize and maize, Z.perennis and Z.perennis was also seen (Figure Z.perennis and their hybrids is built (Fig. Detailed examination of allosyndetic trivalents (IIIve Table . These rve Table . In addind Table . In IIIMn Figure . These rilt Fig. .Z.perennis cytoplasm are represented by light green and light pink circles, respectively. The blue and green strips represent maize chromosomes, while pink, orange, brown and dark red strips represent Z.perennis chromosomes. The centromeres in middle of all chromosomes are labeled red; moreover, both of them have red centromere in the middle. Black parentheses represent paired homologous chromosomes and red parentheses represent expected chromosomes combinations.The maize and Z.perennis genome were proposed: First, the genome composition of "AABB” hybrids was an ancestral cross between two closely related "AA” and "BB” genomes that was followed by evolutionary fractionation; Second, Zea species were originated through chromosome duplication, followed by homoeologous genomes "A” and "B” differentiation (Z.perennis appropriately. Thus, we put forward a third possible evolutionary mechanism: Firstly, duplication event occurred in two closely related species with "AA” and "BB” genome, as a consequence, autopolyploid of "AAAA” and "BBBB” genome were formed. Secondly, evolutionary fractionation took place in two autopolyploids that turned both genomes into "AAA’A’” and "BBB’B’”. thirdly, crossing between those two autopolyploids followed by probable limited compatible coevolution in "A” and "B” subgenomes led to the formation of "AmAmBmBm” genome with barely deviation of maize intra-subgenomes; Lastly, second duplication event of hybrids "AABB’” followed by differential degree of evolutionary fractionation in "A” and "B” subgenomes, led to creation of Z.perennis with genome of "ApApAp’Ap’Bp1Bp1Bp2Bp2” (Fig. Z.perennis than "B” genome and also suffers less genes losses, as well as, has higher expression for genes located in this subgenome, while "B” genome has a faster differentiation. So it is concluded that "B” subgenome was responsible for species isolation, domestication, and as well as further speciation in genus Zea (In previous studies, two possible evolutionary mechanisms for maize and ntiation . Howeverp2” Fig. . Moreoveenus Zea .


The phosphatidyl-inositol 3 kinase (PI3K) δ inhibitor, idelalisib (IDE), is a potent inhibitor of the B-cell receptor pathway and a novel and highly effective agent for the treatment of chronic lymphocytic leukemia (CLL). We evaluated the activities of IDE in comparison to bendamusine (BEN), a commonly used alkylating agent, in primary CLL cells ex vivo. In contrast to BEN, IDE was cytotoxic to cells from extensively-treated patients, including those with a deletion (del)17p. Cross-resistance was not observed between BEN and IDE, confirming their different modes of cytotoxicity. Marked synergy was seen between BEN and IDE, even in cases that were resistant to BEN or IDE individually, and those with deletion (del) 17p. CD40L/interleukin 4 (IL4) co-treatment mimicking the CLL microenvironment increased resistance to IDE, but synergy was retained. PI3Kδ-deficient murine splenic B cells were more resistant to IDE and showed reduced synergy with BEN, thus confirming the importance of functional PI3Kδ protein. Although IDE was observed to induce γH2AX, IDE did not enhance activation of the DNA damage response nor DNA repair activity. Interestingly, IDE decreased global RNA synthesis and was antagonistic with 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB), an inhibitor of transcription. These findings add to the increasingly complex cellular effects of IDE, and B cell receptor (BCR) inhibitors in general, in CLL. Most patients with chronic lymphocytic leukemia (CLL) will require therapy at some point during their disease course, and typically receive chemoimmunotherapy as initial treatment . StandarHowever, the algorithm for therapy has recently shifted, with the advent of novel targeted therapies ,5. TheseIn the present study we have evaluated the cytotoxicity of IDE in primary CLL cells ex vivo, demonstrating a lack of cross-resistance between this agent and chemotherapy, significant cross-resistance between IDE and IBR and a decrease in IDE activity when the microenvironment was simulated with CD40L/IL4. There was synergy between IDE and FLU, CLB or BEN, particularly in the presence of CD40L/IL4. Interestingly, synergy between BEN and IDE appeared unrelated to enhanced DNA damage but rather via transcriptional modulation. Our findings suggest a novel biological role for IDE in anti-CLL therapy. 50) varied significantly (32-fold) for IDE , while the variation was less (16-fold) for IBR . Cells from four patients with del 17p were sensitive to treatment with these agents . There was a significant correlation between the IC50 values of IDE and IBR (p < 0.0001), demonstrating cross-resistance and similar mechanisms of action, consistent with their function as inhibitors of the BCR pathway. In contrast, there was no correlation between the IC50 values of IDE and BEN (p = 0.39), IDE and CLB (p = 0.085), or IDE and FLU . Moreover, in contrast to IBR and IDE, cells from the patients with a del 17p were resistant to BEN and the other chemotherapies  co-treatment of CLL patient cells has previously been shown to be a highly-effective and consistent method to simulate the CLL microenvironment ,22. CLL esistant A. All sar others A. Further others  B,C, a lr others A. 50 (50 > 80 µM), while the response to BEN was decreased slightly  and the synergy shifted to lower (IDE)  were isolated from age-matched healthy donors (HD) and BEN/IDE synergy was tested with and without CD40L/IL4 stimulation. As expected, since the composition of PBMCs in HD is mainly T cells, all PBMC samples were resistant to single agent IDE A, while 50 A. Synerg50 B. At cli50 A. Upon ser (IDE) A–C. To der (IDE) D.50, 20.1 µM) compared to wild-type (WT) counterparts (IC50 of 1.7 µM), however, the residual response suggests additional PI3Kδ-independent molecular targets of IDE , which lack BCR signaling A. In eacAlterations in chromatin architecture is well-documented to modulate transcription and gene expression . TherefoTo determine if the synergistic cytotoxicity of IDE/BEN was related to alterations in transcription by IDE, we examined combinations of IDE, BEN and DRB. When cytotoxicity was assessed after 18 h drug treatment, IDE was antagonistic with DRB, indicating that these agents likely affect common molecular targets, particularly with short-term treatment C. SimilaWe have demonstrated that inhibition of PI3Kδ by IDE is cytotoxic to primary CLL cells, with the sensitivity of cells differing to that of the standard chemotherapeutic agents, BEN, CLB and FLU. Indeed, some patients who were previously treated with chemoimmunotherapy showed sensitivity to IDE, consistent with clinical experience ,13. MorePrevious studies have demonstrated synergy between IDE and BEN , IBR andxL and Bfl-1 expression over Mcl-1 in CLL, and these are also likely reduced by inhibition of the BCR pathway [A number of mechanisms may account for this synergy. First, there might be an interaction between the BCR and DNA repair pathways, although we did not observe an effect of BEN on p-AKT levels or of IDE on p-ATM production. Second, synergy may be related to increased BEN-induced DNA damage by IDE. However, this also appeared unlikely, as the combination of BEN and IDE did not produce a synergistic increase in γH2AX formation, to parallel the changes seen in cytotoxicity. Third, the synergy may be related to alterations in transcription by IDE. We observed transcriptional suppression with IDE and IBR treatments in CLL cells in vitro, which was most marked when RNA synthesis was initially increased by CD40/IL4. Similarly, a recent study in mantle cell lymphoma also showed that IDE can inhibit protein synthesis which correlated with a reduction in cell size and growth . While a pathway . Finally pathway . Our comprehensive analysis confirmed that IDE alone produces a measurable increase in γH2AX in CLL cells, suggesting the induction of DNA double strand breaks ,25. ATM In summary, we have demonstrated marked synergy between IDE and BEN in CLL cells, both when quiescent and under conditions to simulate the microenvironment. These beneficial outcomes have been seen clinically, but were associated with increased immunosuppression and associated infections at the commonly-used drug doses . Based o2 in a humidified atmosphere.Patients were selected from the Manitoba CLL Clinic to ensure variation in prior treatments and prognostic markers. B cell isolation from CLL patients was performed as previously described ,39. InfoBEN, CLB, FLU, and DRB were purchased from Millipore Sigma and IDE and IBR were purchased from Selleckchem . All agents were reconstituted to 100 mM in dimethyl sulfoxise (DMSO) , aliquoted and stored at −80 °C, except DRB which was reconstituted to 50 mM and stored at −20 °C. Aliquots were then diluted in SFM prior to use. DMSO alone treatment was used in all experiments as a negative control and, unless otherwise stated, for normalization.For synergy and dose-response determinations, CLL cells were treated in 96-well plates with 6 increasing concentrations of drug for ~18 or ~72 h. Concentration ranges were selected and optimized to ensure that the exponential phase of the dose-response curve was obtained for the majority of the samples. Ranges were 0–160 µM for BEN, 0–80 µM for IDE and CLB, 0–20 µM for IBR and 0–10 µM for FLU. Synergy experiments with DRB were performed using 3 or 4 increasing doses of drug for ~18 h with the concentration ranges 0–40 µM for BEN and IDE, and 0–20 µM for DRB. The concentration of DMSO was constant between samples. After 18 or 72 h incubation, cell death was determined by flow cytometry by annexin-V-FITC and 7AAD  . Cells w4CL, 10 mM KHCO3, and 0.1 mM Na EDTA pH 7.2–7.4) for 2 mins at room temperature. 15 mL Media was then added, samples were centrifuged and resuspended in 1× PBS  with 2% FBS . B cells were isolated using the EasySep™ Mouse Pan-B Cell Isolation Kit  as per the manufacturer’s instructions. Isolated splenic B cells were then stimulated for 24 h with CD40L (1µg/mL) and IL4 (5ng/mL) in RPMI with 1% Penicillin/Streptomycin. 2 × 106 cells/mL were then treated with drug, incubated in 100 μL for ~72h at 37 °C and 5% CO2 in a humidified atmosphere, and cell death was then measured. Experiments were performed twice with similar results. All mice experiments were approved by University of Manitoba animal care committee  under title “Phosphoinositide-dependent signalling pathways controlling B lymphocyte activation” on 1 April 2019 for period from 30 May 2019 to 29 May 2020.C57BL/6 mice (8–10 weeks old) were purchased from and maintained in a pathogen-free facility at the GMC, University of Manitoba, according to the Canadian Council on Animal Care guidelines. Mice were either WT or expressed a mutated inactive form of p110δ (p110δD910A) . SpleensDSBs were measured for immunoreactivity to anti-γH2AX antibody after cells were treated for ~18 h with drug. Cellular irradiation  using a RS-2000 Rad Source  served as a damage-positive control. Thirty min post-irradiation, samples were washed in PBS (MilliporeSigma), resuspended in 70% ethanol (MilliporeSigma), and stored at −20 °C for at least 1 h (up to 4 days). Cells were then washed 3 times with cell staining buffer  and 1.75 µL of Alexa 647conjugated anti-H2AXγ antibody or Alexa647-Mouse-IgG1 Isotype control (Biolegend) was added to 50 µL of cell staining buffer at room temperature in the dark for 30 min. Samples were washed and analyzed via a NovoCyte flow cytometer.Following 24 h treatment with IDE, adherent non-B lineage cells  were treated followed by immunostaining with 1/1000 diluted Alexa 647-conjugated anti-H2AXγ antibody in 3% bovine serum albumin (BSA)-PBS . Images The alkaline comet assay was performed as previously described , with thFor synergy and dose-response determination, CLL cells were treated in 96-well plates with 6 increasing concentrations of IDE from 0–80 μM. The concentration of DMSO was constant between samples. Plates were then irradiated with 0, 1, 2, 5, 8, 10 or 20 Gy and cell death and γH2AX were measured after 72 h. In IR recovery experiments, CLL patient cells were stimulated with 50 ng/mL of CD40L and IL4, or with PBS as a control. DMSO or 10 μM of IDE was then added to the cells, the plates were incubated for ~18 h, and the cells were analyzed for γH2AX and comet assay. Simultaneously with the IDE incubation starting at the beginning of drug treatment, at 3 h, at 0.5 h, or immediately prior to γH2AX or comet assay analysis, cells were irradiated with 10 Gy and allowed to recover.Primary CLL cells were treated with 10 µM IDE and/or 40 µM BEN for ~18 h with or without CD40L/IL4 treatment and pelleted. Protein extracts were prepared and underwent western analysis as per . Blots w® RNA Alexa Fluor® 488 HCS Assay (Life Technologies) was performed as per the manufacturers’ instructions with the following modifications (in consultation with manufacturer). One h prior to the end of drug treatment, 100 μL of 2 mM 5-ethynyl uridine (EU) was added directly to the 100 μL of cells and incubated under normal culture conditions. The cells were then stained for 30 mins at 4 °C with the eBioscience  Fixable Viability Dye eFluor™ 780 prior to fixation with 3.7% formaldehyde (MilliporeSigma) and permeabilization with 0.5% Triton® X-100 (MilliporeSigma). After incubation with Click-iT® reaction cocktail and washing with Click-iT® reaction rinse buffer, cells were resuspended in 100 μl of PBS (MilliporeSigma) and analyzed by flow cytometry using a NovoCyte flow cytometer. Cells stained without the viability dye, the Alexa Fluor® azide, or both were run as controls. Click-iT® median fluorescence intensity (MFI) was measured in the viable cells. AV/7AAD was run in parallel as a control for viability. All wash steps were performed in PBS followed by microplate centrifugation at 250 g and removal of the supernatant.Primary CLL cells were treated with either CD40L/IL4 or PBS as a control and incubated for 18 h. 10 μM IDE, 5 μM IBR, 30 μM DRB, or 25 μM BEN were also added either immediately following CD40L/IL4 treatment or after 12 h (for the 6 h drug treatment time point) to cells for a final volume of 100 μL in a 96- well plate. Following drug treatment, the Click-iTIGHV) mutational status of the patients was determined from RNA, as previously described [The immunoglobulin heavy chain variable region (escribed . Fluorescence in situ hybridization (FISH) analysis was carried out on fresh or stored cells, as previously described . p < 5 × 10−2; ** p < 10−3, *** p < 10−4). Synergy was also assessed according to the method of Chou and Talalay, where the CI is defined as: CI = (d1x/D1x) + (d2x/D2x); where d1x and d2x are doses of drug 1 and drug 2, respectively, required to produce a given reduction in cell viability (x) when given in combination, and D1x and D2x are doses of drug 1 and drug 2, respectively, required to produce the same effect in single-agent treatments [p-value < 0.05 was considered significant.Graphical representation and statistical analysis were performed using MS Excel and GraphPad Prism. Drug synergy was assessed using Combenefit software  and Grapeatments . CI < 1,eatments . A p-val50 of the drugs and the average CI values, the Pearson correlation coefficient (r) was calculated and the p-value was determined using a two-tailed T test with a 95% CI. To test the significance of the Click-iT® (Life Technologies) data, a paired two-tailed t-test was performed with a 95% confidence interval . To determine the correlation between the ICIDE is a potent inhibitor of the δ isoform of PI3K, a key enzyme in the BCR pathway. IDE has significant clinical efficacy in CLL, but its use is limited by autoimmune colitis, hepatitis and pneumonitis. In the present study, we show significant synergy between IDE and BEN in CLL cells in vitro, both when incubated alone or with CD40/IL4, and this also affects normal B cells but not T cells. These findings emphasize the interaction between the BCR and DNA repair pathways, and the importance for initial dose-escalation studies when combining BCR inhibitors with chemotherapy.


Lipoprotein fractionation analysis revealed the predominant distribution of ApoL1 in large HDL fraction, and the significant increase of ApoL1 in large LDL fraction in high ApoL1 samples with insulin resistance. In T2DM, ApoL1 was higher in T2DM with metabolic syndrome, however ApoL1 was lower with β cell dysfunction. Insulin significantly promotes ApoL1 synthesis and secretion in HepG2 cells. In conclusion, circulating ApoL1 may be associated with abnormal HDL metabolism in insulin resistant status. This may suggest a regulation of insulin signal on the ApoL1 level, leading to offer a novel insight to the ApoL1 biology.Circulating ApolipoproteinL1 (ApoL1) is a component of pre-β-high-density lipoprotein (HDL), however little is known about the relationship of ApoL1 with cardiometabolic factors. Considering previous studies reporting the correlation of ApoL1 to triglyceride, we have hypothesized that ApoL1 associates with insulin-related metabolism. The current study examined their associations in 126 non-diabetic subjects and 36 patients with type 2 diabetes (T2DM). Non-diabetic subjects demonstrated triglyceride (standardized coefficients [s.c.] = 0.204,  APOL1 gene on chromosome 22 comprises putative sterol response elements in the promoter region2, through which the transcription of genes involved in lipid metabolism is presumed to be activated. Circulating ApoL1 levels correlate with the plasma triglyceride (TG) levels, and increases in subjects with a deficiency of cholesteryl ester transfer protein (CETP) that exchanges cholesterol and TG between the TG-rich lipoprotein and HDL3. Recent research reported that ApoL1-containing HDL subsets that comprise ApoA-I and ApoE are huge pre-β-HDL particles4. The evidence indicates that the HDL particles carrying ApoL1 are the characteristic subspecies in terms of HDL metabolism or bioactivity.Apolipoprotein L1 (ApoL1) was originally recognized as a component of human high-density lipoprotein (HDL) particles. ApoL1 possesses amphipathic helices, which confer a high level of lipophilicity; therefore, ApoL1 is entirely bound to HDL during the circulation of normolipidemic populationTrypanosoma brucei that causes sleeping sickness6. Additionally, the association between plasma ApoL1 and the clinical characteristics including lipid-related factors in normolipidemic, dyslipidemic, or diabetic subjects, has also been explored. Duchateau et al. reported the independent positive correlation of ApoL1 with total cholesterol  and TG in normolipidemia, and with TG in dyslipidemia. Moreover, it was observed that ApoL1 was elevated in the non-insulin dependent diabetic patients with hypertriglyceridemia3. Albert et al. reported that TG and hyperglycemia are the strongest predictors of plasma ApoL1 in dyslipidemic coronary artery disease subjects with low HDL cholesterol (HDL-C)7. Intriguingly, proteomic analysis demonstrated the positive correlation of the in vitro antioxidative capacity and ApoL1 peptide count on purified HDL determined by mass spectrometry8.In innate immunity, HDL ApoL1 is the trypanolytic factor in human serum, which forms anion channels on the lysosomal membrane of These data suggest that ApoL1 may be associated with lipid and glucose metabolisms or may exert an antioxidative effect as a distinct HDL subspecies in certain dyslipidemic subjects. We postulate that serum ApoL1 levels may elevate and exert certain biological activity in subjects with insulin resistance that frequently presents dyslipidemia marked by hypertriglyceridemia; however, the association between ApoL1 and insulin resistance has not been reported. The present study aimed to examine this association in the nondiabetic volunteers and patients with type 2 diabetes (T2DM) characterized by insulin resistance.2, and the mean systolic and diastolic blood pressure (BP) values were 113.8 ± 12.0 and 73.0 ± 11.0 mmHg, respectively. The percentage values of the habitual drinker and present smoker were 28.6% and 16.7%, respectively. The median value of serum ApoL1 was 24.0 (20.0–29.0) µg/mL. All characteristics in the serum and urine samples were within normal limits. Univariable linear regression analysis revealed the significant correlation of log ApoL1 with sex , log WC , log BMI , systolic BP , diastolic BP , low-density lipoprotein (LDL)-C , log HDL-C , log TG , log γ-glutamyltransferase (γGTP) , uric acid , fasting blood sugar (FBS) , log insulin , and log adiponectin   were enrolled, and their median age was 34.0 (26.3–41.0) years. Their median waist circumference (WC) and body mass index (BMI) were 79.0 (70.0–87.0) cm and 21.9 (19.5–24.9) kg/mp < 0.05), log TG , and log HDL-C  as the independent determinant of serum ApoL1 levels . The male percentage in the pre-Mets/Mets group was 81.8%, which was significantly higher than that of the non-obese group. In the non-obese group, the median WC and the mean BMI were 74.0 (68.0–80.8) cm and 20.7 ± 2.2 kg/m2 and all other characteristics were within normal limit. In the abdominally obese group defined as having central obesity without metabolic abnormality, the median WC and the mean BMI were 90.0 (87.0–96.3) cm and 25.5 ± 3.4 kg/m2, which were significantly higher than that of the non-obese group; however, all other characteristics revealed similar levels with the non-obese group. In the pre-Mets/Mets group, metabolic characteristics significantly increased compared to the non-obese group. Between the abdominally obese group and the pre-Mets/Mets group, characteristics except for the BP and fasting insulin revealed not significant difference, which was presumably due to lesser participants in these two groups or the large deviation. The median ApoL1 was 30.0 (24.8–35.8) µg/ml in the pre-Mets/Mets group, which was significantly higher than that in the non-obese  or abdominally obese group . No significant difference was observed in ApoL1 between the non-obese and abdominal obese groups /Mets, and compared the ApoL1 levels between these groups. No significant difference was observed in ApoL1, BMI, HDL-C, and TG between pre-Mets and Mets; hence, we combined both groups as pre-Mets/Mets. All data are listed in Table ups Fig. . Insulinp < 0.05), BMI (p < 0.01), TG (p < 0.05), and insulin level (p < 0.05) were higher, whereas HDL-C (p < 0.05) and adiponectin (p < 0.01) levels were lower  and high ApoL1 subjects (N = 5). Serum ApoL1 levels were 24.2 ± 3.3 and 42.6 ± 9.9 µg/mL in low and high ApoL1 subjects, respectively. In high ApoL1 group, the WC  mg/dL and the mean HbA1c was 9.7 ± 2.5%, thus indicating poor glycemic control. The median urine albumin-to-creatinine ratio was 29.1 (7.2–284.3) mg/gCr, in which overt albuminuria is 25.0%, microalbuminuria is 22.2%. ApoL1 revealed positive correlation with WC  and log TG , and negative correlation with log adiponectin  in overall patients with T2DM .We examined ApoL1 expression in 36 patients with T2DM characterized by insulin resistance (Table 10. We incubated HepG2 cells in the absence of insulin or in the presence of 100 nM insulin. Incubation with 100 nM insulin for 6 h results in the significant upregulation of ApoL1 mRNA (p < 0.05)    Fig. . ApoL1 p05) Fig. . In addi05) Fig. . ApoL1 p11. Specifically, serum ApoL1 in Mets was significantly higher than in non-obesity or abdominal obesity without insulin resistance. The abnormal lipid metabolism in Mets is characterized by elevated VLDL, hypertriglyceridemia, high levels of LDL-C, increased CETP activity, and low levels of HDL-C, in which insulin resistance and increased circulating free fatty acid (FFA) resulting from adipose tissue hyperplasia and hypertrophy are considered as fundamental pathogenesis15. This study also found a significant correlation between serum ApoL1 and HOMA-IR in all non-diabetic volunteers. These findings suggest that serum ApoL1 may not increase in obesity alone, but it may be elevated in non-diabetic Mets marked by insulin resistance-mediated dyslipidemia.This observational study revealed several novel findings regarding insulin resistance-mediated ApoL1 biology in Mets and T2DM, including the elevation of serum ApoL1 in Mets. In nondiabetic volunteers, high BMI, hypertriglyceridemia, and low HDL-C were identified as independent characteristics that determined serum ApoL1 levels and are typical features associated with Mets. To the best of our knowledge, this is the first study to report a correlation between ApoL1 and HDL-C or BMI3 is consistent with the results of this study because insulin resistance is the common basal pathophysiology of Mets and T2DM. However, patients with T2DM unexpectedly demonstrated no significant increase in serum ApoL1 compared with the non-obese volunteers in this study. In contrast, those with T2DM exhibited a significant increase in WC, BMI, TG, and adiponectin compared with non-obese volunteers, thus suggesting a difference in ApoL1 regulation between Mets and T2DM. Considering that insulin secretory capacity in T2DM varies, we examined the presence of Mets in T2DM. T2DM without Mets had significantly lower c-peptide and ApoL1 levels, despite high FBS levels similar to T2DM with Mets, thereby indicating that absolute insulin deficiency due to β cell dysfunction is associated with low ApoL1 levels in T2DM without Mets. ApoL1 protein levels in the circulation are primarily determined by liver secretion16. We postulated that insulin, not hyperglycemia, promoted ApoL1 secretion in the Mets liver. We finally showed that insulin regulated ApoL1 synthesis and secretion in HepG2 cells. Hence, we conclude that ApoL1 synthesis and secretion in the liver attribute, in part, to insulin signaling in Mets or T2DM.The ApoL1 increase in T2DMet al. reported that patients with HAT presented with an altered lipid and lipoprotein profile, including hypertriglyceridemia, high LDL-C, low HDL-C, and reduced ApoA-117. Lamour et al. found hypertriglyceridemia and lowered plasma levels of phospholipid and sphingolipid as metabolic markers in HAT18. Vervet monkeys infected with Trypanosoma brucei presented with hypoglycemia, hypertriglyceridemia, and low HDL-C19. Further, Trypanosoma infection-induced TNF has been shown to inhibit lipoprotein lipase activity in a rodent model20; therefore, increased lipolysis from adipose tissue may result in lipid abnormalities. These findings indicate some similarities in the pathophysiology of dyslipidemia between Mets and HAT. In addition, circulating ApoL1 expression was reported to be increased by Trypanosoma brucei infection21. The interaction of the altered lipid profile and increased ApoL1 expression remains unclear; however, some metabolic factors or pro-inflammatory cytokines induced by trypanosoma infection may alter lipid and lipoprotein metabolism in HAT.Alteration of the lipid and lipoprotein profile induced by human African trypanosomiasis (HAT), wherein ApoL1-containing particles function as lytic factors, has been reported. Huet et al. reported that ApoL1 existed in the large HDL and VLDL fractions under nondenatured conditions, and that ApoL1-containing lipoproteins exhibited pre-β mobility1. Weckerle et al. also found ApoL1 in the LDL and large HDL fractions by FPLC separation, in which the ApoL1 protein was highly enriched in the large HDL particle size. They also found that these ApoL1-carrying HDL subspecies expressed ApoA-I and ApoE and migrated in the β/pre-β position4. Interestingly, plasma levels of pre-β HDL have been reported to increase in parallel with TG and BMI in Mets24. Lipoprotein fractionation analysis in this study indicated the predominant expression of ApoL1 in the large HDL fraction, independent of ApoL1 levels. However, ApoL1 in the LDL fraction, which corresponds with the larger ApoL1-containing complex, presented a significant increase (1.7-fold) in the high ApoL1 subjects, thus suggesting that two different size ApoL1-enriched complexes may be differentially metabolized in the abnormal HDL metabolic cycle of Mets.A second major finding was the distinct expression and distribution pattern of ApoL1 in subjects with increased ApoL1 levels resulting from insulin resistance. Circulating ApoL1 was reported to show a bimodal distribution among lipoprotein fractions. Duchateau 1. Compared with low ApoL1 subjects, high ApoL1 subjects exhibited a significant increase in total ApoL1 and 39 kDa ApoL1, thus indicating that circulating ApoL1 in Mets may be transcriptionally increased and proteolytically activated. The alteration in HDL apoprotein expression levels or distribution contributes to HDL metabolism or bioactivity in pathological conditions, including Mets29. These data suggest the possibility that the change in distribution or proteolytic activation of ApoL1 may be associated with ApoL1-containing HDL activity or metabolism in Mets.Moreover, we detected serum ApoL1 at two different sizes of 42 and 39 kDa by western blotting, which are regarded as a precursor and a signal peptide-deleted activated form, respectively7. These data support that ApoL1 is not a strong proatherogenic molecule, although serum ApoL1 levels have been shown to be associated with known proatherogenic markers such as hypertriglyceridemia or low HDL-C.The function of circulating APOL1 is another important point to consider. Therefore, we explored the association of ApoL1 levels with arterial stiffness and oxidative stress in T2DM. ApoL1 did not correlate with arterial stiffness, as evaluated by pulse wave velocity in this study, which may be consistent with the previous observation that ApoL1 levels did not correlate with the progression of coronary artery disease30. U-8OHdG/Cr in T2DM without Mets was significantly higher than in T2DM with Mets, and log U-8OHdG/Cr was inversely correlated with serum ApoL1 levels. One of the possible explanations for this result is the antioxidative activity of ApoL1-enriched HDL. Little is known about the anti-oxidative capacity of ApoL1. Davidson et al. reported a positive correlation of ApoL1 peptide count on HDL by mass spectrometry and the anti-LDL oxidative capacity of HDL. In that study, the correlational network analysis found a strong relationship between ApoL1, paraoxonase1, paraoxonase2, and ApoF, thus suggesting potent anti-oxidative activity of these apoproteins8. Another possibility is that the oxidative stress-mediated β cell dysfunction may result in reduced insulin secretion31 and ApoL1 production in T2DM without Mets. A third possible explanation is the association between urinary 8OHdG and ApoL1 in kidney cells. ApoL1 protein is expressed in glomerular podocytes or proximal tubular cells, and the expression levels have been shown to decrease in diseased kidney. Conversely, urinary 8OHdG excretion increases in human and rodent kidney diseases34. ApoL1 in kidney cells is assumed to be derived from both endogenous synthesis and extracellular resource, and to serve a functional role36. ApoL1 localized in kidney cells may affect urinary 8OHdG excretion. Our data suggest several hypotheses on the ApoL1 effect; however, further investigation is required to clarify the causal association and mechanism between ApoL1 and oxidative stress.Additionally, we measured urinary 8OHdG to evaluate oxidative DNA damage throughout the body37. Proteomic analysis revealed variant-related quantitative changes in apoproteins on ApoL1-containing HDL subspecies4; however, it remains unclear if these variants alter lipid metabolism, HDL function, or cardiovascular disease susceptibility40. Furthermore, the gene variants have not been examined in the Japanese population. Therefore, our study may provide insight into variant-independent ApoL1 biology.This study had certain limitations. First, this study was conducted with our institute volunteers, in which the job types were slightly biased; therefore, the baseline data may differ from the general population. However, the pre-Mets/Mets ratio in our subjects  did not differ from recent data of the Japan National Health and Nutrition Survey. Thus, the correlation between ApoL1 and clinical factors in this study is similar to that in the Japanese population with insulin resistance. Another limitation is that we did not examine gene mutations related to kidney disease in black Americans. The gene variant is known to determine trypanolytic activity or prognosis of kidney diseasesin vitro examination demonstrated insulin resistance-mediated ApoL1 synthesis and secretion in the liver and alteration of ApoL1 distribution between lipoprotein fractions in Mets. These data may provide new insights into ApoL1 biology in insulin resistance-mediated abnormal lipid metabolism.In summary, the current observational study and This study enrolled 126 non-diabetic volunteers from age 20 to age 60. These non-diabetic volunteers who consisted of doctor, nurse, clinical laboratory technician, medical representative and medical student were recruited from our institute with their consent. We measured BP, height, body weight and WC, and collected serum and urine after an overnight fasting. According to the criteria of International Diabetes Federation, Mets was defined as having more than any two of the four factors including raised TG (2) = 194 X Age−0.287 X Creatinine (mg/dL)−1.094 . BMI was calculated as weight in kilograms divided by height in meters squared. HOMA-IR, insulin resistance index, was calculated from FBS and insulin as follows; HOMA-IR = fasting insulin (μU/mL) X FBS (mg/dL)/405.Venous blood and urine were collected after an overnight fasting. Serum was then isolated by centrifugation  and stored at −80 °C until analysis. Serum levels of Total-C, LDL-C, HDL-C, TG, γGTP, albumin, uric acid, creatinine, blood sugar, insulin, HbA1c and adiponectin, and urine levels of albumin, creatinine and 8OHdG were measured. 8OHdG was evaluated by ICR-001  and U-8OHdG/Cr was used as a marker of oxidative DNA damage. Estimated glomerular filtration rate of each participant was calculated from serum creatinine and age by using the 3-variable Japanese equation as follows; estimated glomerular filtration rate (ml/min/1.73 m41. The column was eluted with 50 mmol/L phosphate buffer (pH7.4) containing 150 mmol/L NaCl and 1 mmol/L EDTA at a flow rate of 0.5 mL/min. Eluted fractions (0.5 mL each) were subjected to lipid analysis and immunoassays. Lipoprotein fractions were defined by eluted fraction volume as follows; very low-density lipoprotein (VLDL) 7.5–9 mL, large LDL 10.5–12 mL, small LDL 12–13.5 mL, large HDL 16–17.5 mL, small HDL, and very small HDL 17.5–19.0 mL.Serum samples (0.3 mL) were fractionated by gel filtration choromatography on a Superose 6 column  in a liquid chromatography apparatus  using a procedure modified from a previous reportApoL1 concentration in serum and in lipoprotein fraction was measured by a human specific sandwich ELISA (KE00047) . Briefly, serum was diluted 1:10,000 and assayed in duplicate according to the manufacture’s instructions. As for the examination of lipoprotein fractions, samples were diluted 1:250.6 cells/well and cultivated for 24 h. The culture medium was replaced with a fresh one without fetal calf serum, and cells were additionally incubated for 12 h before insulin administration. Cells were incubated in the absence of insulin or in the presence of 100 nM insulin  RNA or whole cell extract was collected after 6 h or after 12 h incubation with insulin, respectively. Culture media for 24 h insulin incubation was collected and concentrated by concentration column  for the evaluation of ApoL1 secretion.HepG2 cells were cultivated in DMEM containing 10% fetal calf serum, 5%CO2 at 37 °C. For RNA isolation or protein extraction, HepG2 cells were seeded on 6-well plates at a density of 1 × 10One μL of serum, 15 μg of whole cell lysate, 16 μL of concentrated culture media of HepG2 or 10 μL of lipoprotein solution were heated to 70 °C for 15 minutes in SDS gel-loading buffer, and applied to SDS gel electrophoresis, thereafter proteins were transferred to nitrocellulose filters . The blots were incubated with rabbit anti-ApoL1 antibody  or rabbit anti-human GAPDH antibody , followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG . Signal intensity of ApoL1 was measured by Image J.The total RNA was extracted from cultured HepG2 cells by RNAiso Plus . Quantitative PCR was performed on BIO-RAD MJ Mini Personal Thermal Cycler  using SYBR Green PCR Master Mix (Applied Biosystems). The following PCR primers were used; human ApoL1: sense 5′- tgagattcaaaagccacactg-3′, antisense 5′- cgaggggcttactttgagga-3′ and human GAPDH: sense 5′-tttggctacagcaacagg-3′, antisense 5′-ggtctctctcttcctcttg-3′.This study was approved by the Ethics Committee of Tokushima University Hospital  and Hokkaido University (No.17–53–1), and was performed in compliance with the Helsinki Declaration. Written informed consent was obtained from all participants.t-test was used in parametric variables and Mann -Whitney’s U test was used in non-parametric variables for comparison between two groups. For the regression analysis, correlations were determined by univariate linear regression analysis, and multiple stepwise regression was used to confirm significant independent correlation. When data were non-normal distribution, log transformation was applied to correct skewness. For the assessment of potential differences in mean value, one-factor ANOVA was performed. Subsequently, as for the characteristic that presents significant difference between three groups, multiple comparison analysis was performed. Those that were normally distributed were subjected to multiple comparison by Tukey’s test, and those that were non-normal distribution were subjected to multiple comparison by Kruskal-Wallis test. Statistical analyses were performed with IBM SPSS statics. A p value The results are shown as mean ± standard deviation, as number (%) or as median (interquartile range) if skewed. A test for normality was performed using the Kolmogorov-Smirnov test. Student’s Supplementary Table S1, Supplementary Table S2, Supplementary Table S3 and Supplementary Figure S1


Current O3 pollution in the United States is estimated to decrease the yields of maize (Zea mays) up to 10%, however, many bioenergy feedstocks including switchgrass (Panicum virgatum) have not been studied for response to O3 stress. Using Free Air Concentration Enrichment (FACE) technology, we investigated the impacts of elevated O3 (~100 nmol mol−1) on leaf photosynthetic traits and capacity, chlorophyll fluorescence, the Ball–Woodrow–Berry (BWB) relationship, respiration, leaf structure, biomass and nutrient composition of switchgrass. Elevated O3 concentration reduced net CO2 assimilation rate (A), stomatal conductance (gs), and maximum CO2 saturated photosynthetic capacity (Vmax), but did not affect other functional and structural traits in switchgrass or the macro- (except potassium) and micronutrient content of leaves. These results suggest that switchgrass exhibits a greater O3 tolerance than maize, and provide important fundamental data for evaluating the yield stability of a bioenergy feedstock crop and for exploring O3 sensitivity among bioenergy feedstocks.Elevated tropospheric ozone concentration (O Intabolism ,78,79. Tficiency ,81,82, w cycling ,84,85. B DOY 225 , decreas on δ15N .3 tolerance, and suggest that C4 bioenergy crops including maize and switchgrass differ in O3 tolerance. However, the year of our experiment was extremely wet, and previous work in maize also showed that O3 sensitivity was greater in dry years [3 concentrations strongly vary over the land surface throughout the day and over the season, resulting in geographic variation in O3 pollution. Therefore, understanding variation in C4 bioenergy feedstock responses to elevated O3 could be used to better place specific feedstocks on a dynamic landscape.Although a successful bioenergy industry will require high productivity and yield stability of bioenergy feedstocks, how the bioenergy crops acclimate to a rapidly changing, more polluted environment should be considered seriously. Our results provide evidence that switchgrass exhibits Ory years , thus adwww.igb.illinois.edu/soyface/, 40°02’N, 88°14’W) in 2018. Six plots in octagonal shape of 20 m diameter were designed for this study: three at ambient O3 concentration (30–50 nmol mol−1) and three fumigated to elevated O3 concentration (~100 nmol mol−1). The weather conditions including daily maximum and minimum air temperature, averaged light intensity (9:00–18:00), precipitation, averaged daily relative humidity and O3 concentration (10:00–18:00) during growing season of 2018 were monitored by an on-site weather station at the FACE facility and shown in The study was conducted at the Free Air Concentration Enrichment (FACE) facility in Champaign, IL, USA  were transplanted in the central part of each plot on 24 May (DOY 144) in 2018. Elevated O3 plots were fumigated on 25 May (DOY 145) in 2018 and followed the protocol described in detail by Morgan et al. (2004) [3 fumigation was carried out using the O3-enriched air that was delivered to and released within the experimental plots with FACE technology. O3 was generated by an O3 generator  using pure oxygen and monitored by a chemiluminescence O3 sensor  that connected to the tube pumping air from the central point of plot. Fumigation was applied for 8 h per day from 10:00 to 18:00 when leaves were not too wet and when wind speed was not too low, with the target O3 concentration of 100 nL L−1 at the central point of elevated plots. O3 fumigation was stopped on 13 August (DOY 225) in 2018 once the second round of midday photosynthesis measurements were finished (3 concentrations within the elevated plots were within 20% of the target concentration 81.6% of the time.Seedlings of switchgrass (. (2004)  and Yend. (2004) ,54. Elevfinished e. In 2012 assimilation rates (A), stomatal conductance to water vapor (gs), intercellular CO2 concentration (Ci) and chlorophyll fluorescence  under illumination were measured with a portable photosynthesis system  following previously published protocols [2 concentration was 400 µmol mol−1, light intensity at the leaf surface was 1950 µmol m−2 s−1 and relative humidity was 60% for the DOY 206; leaf cuvette temperature was 31 °C, CO2 concentration was 420 µmol mol−1, light intensity at the leaf surface was 1750 µmol m−2 s−1 and relative humidity was 60% for DOY 225, 2018. The measurement was performed when photosynthesis had stabilized, typically 3–5 min after leaf enclosure. Considering the heterogeneity of physiology and structure within a given leaf [A/gs.In situ midday gas exchange and chlorophyll fluorescence measurements were made on fully expanded leaves between 11:00 and 14:00 on sunny days of 25 July (DOY 206) and 13 August (DOY 225) in 2018. The net COrotocols ,87,88. Bven leaf ,90, we mA to Ci using a LI-6400. Predawn on DOY 206, leaves were excised and recut immediately under water to prevent leaf water potential decrease, chloroplast inorganic phosphate concentration or maximum photosystem II efficiency decrease [2 concentration and saturating light levels to achieve a steady-state. The middle part of the leaf was then enclosed in cuvette and measurements were initiated at a CO2 concentration of 400 µmol mol−1, air temperature of 25 °C, light intensity of 1800 µmol m−2 s−1 and relative humidity of 60%. CO2 concentration within the cuvette was then changed sequentially as follows: 400, 300, 200, 100, 50, 400, 500, 600, 800, 1000, and 1200 µmol mol−1. The maximum carboxylation capacity of phosphoenolpyruvate (Vpmax) and CO2 saturated photosynthetic capacity (Vmax) were calculated according to Farquhar et al. (1980), von Caemmerer (2000) and Markelz et al. (2011) [Three sun-exposed leaves of different individuals within each plot were selected to measure the response of decrease . With th. (2011) ,93,94.gs is stomatal conductance to water vapor (mol (H2O) m−2 s−1); A is net CO2 assimilation rate (µmol (CO2) m−2 s−1); Hs and Cs are relative humidity (Pa (air) Pa (Saturated)−1) and CO2 concentration (Pa (CO2) Pa (air)−1) at the leaf surface, respectively; and a and b are the slope (mol (H2O) mol (CO2)−1) and intercept (mol (H2O) m−2 s−1) of the BWB relationship, respectively [The Ball–Woodrow–Berry (BWB) relationship was calculated as:ectively ,48.Hs was calculated as:Es is the partial pressure (Pa) of vapor at the leaf surface and Esat is the partial pressure (Pa) of vapor at saturation.Es was determined as:Tr is transpiration (mol (H2O) m−2 s−1) and P is air pressure (Pa). Ei is the partial pressure of vapor (Pa) at substomatal cavity and is assumed to be saturated:−1) and the gas constant of vapor and (set at 461 J kg−1 K−1) [Tl is leaf temperature (K).g−1 K−1) , respectCs was determined by the following equation:2O to that for CO2 and has dimensions of ((mol(H2O) m−2 s−1)/(mol(CO2) m−2 s−1)) and Ci is intercellular CO2 concentration (Pa (CO2) Pa (air)−1).A, gs, Tr, Ci, P and Tl were observed from a portable photosynthesis analyzer Licor-6400. The slope a and intercept b of the BWB relationship were estimated using linear regression with observed gs and calculated The values of 2 concentration was 400 µmol mo−1, relative humidity was 60% but light intensity at the leaf surface was 0 µmol m−2 s−1. Leaf dark respiration was measured after readings stabilized, typically 3–10 min after leaf enclosure. To examine the effects of elevated O3 on photosystem II (PS II) activity, dark-adapted chlorophyll fluorescence was measured. Following the respiration rates measurements, the leaf was further illuminated with a saturating irradiance (>7000 µmol m−2 s−1) to measure the minimum fluorescence yield (F0) and the maximum dark-adapted fluorescence yield (Fm). The spatially averaged maximum dark-adapted quantum yield of photosystem II (PSII), Fv/Fm was calculated as the ratio of (Fm − F0) to Fm.Leaf respiration rates and chlorophyll fluorescence under dark were also measured using the LI-6400. Immediately after each A/Ci curve was completed, the leaf was removed from the cuvette and kept in the cabinet under dark for at least 50 min. Environmental controls inside the cuvette were maintained to match the ambient conditions: leaf cuvette temperature was 27 °C, CO2 were excised with a razor blade and stored in 70% ethanol for further analysis in the laboratory. For each leaf sample, three hand-cut transverse sections were viewed under a microscope  and imaged using a digital camera . Using an image analysis software , the following leaf structural traits were measured followed previous published methods [−1), distance between secondary vein (mm), leaf thickness (µm), interveinal distance , conduit diameter (µm), conduit size (µm2), out bundle sheath size (µm2), inner bundle sheath size (µm2), vein size (µm2), colorless cell size (µm2), upper epidermis cell size (µm2), lower epidermis cell size (µm2), motor cell size (µm2), and sclerenchyma size (µm2).Immediately after each dark-adapted chlorophyll fluorescence measurement was completed, leaf samples of 16 cm methods ,96: bund2 [To measure stomatal density and guard cell length, clear nail polish impressions were collected from abaxial surface of the lamina using other leaf discs from the same leaf sample used for leaf anatomical measurement and viewed and imaged under microscope. The stomatal pore area index (SPI) was calculated as  × (guard cell length)2 .2 leaf disc from the same leaf sample used for leaf anatomical and stomatal measurement, minor vein density  was determined. After the epidermis was removed with a sharp razor blade, the remaining leaf samples were put in bleach  to clear mesophyll cells. The samples were then stained with toluidine blue  and imaged under microscope. The length minor vein per leaf area was measured with Image J manually.Using a ~2 cmAll plants were harvest on 23 August (DOY 235) in 2018. Three individuals of each plot were selected for the biomass, C and N content measurement. Leaf area was measured by an area meter  and the number of tillers of each plant was counted. Leaf and stem dry mass were determined after oven-drying for 1 week at 50 °C. Leaf dry mass per area (LMA) was calculated as dry mass/ area. Dried leaf and stem samples were then ground and weighted, and C and N content (%) was determined by a Costech 4010 elemental analyzer .TM, Thermo Scientific). Briefly, samples were submitted to a microwave acid sample digestion  . All samples were previously spiked with two internal standard solutions of gallium and rhodium for final concentrations of 10 and 2 μg L−1. After acid digestion, samples were diluted to 50 mL with Milli-Q water to obtain solutions containing 2.0% (v/v) of nitric acid, then filtered at 0.45 μm using a teflon filtration system. Quantification of each element was performed using external standard calibration curves. The quality of mineralization and analysis were checked using a certified reference material of Citrus leaves . Isotopic analysis of C and N was performed with a continuous flow isotope mass spectrometer  linked to a C/N/S analyser .Macro- and micronutrients were quantified as previously described  by induc3 were tested with one-way ANOVA followed by the Tukey’s post hoc test using SPSS 16.0 . The differences in slope and intercept of BMB relationship (observed stomatal conductance vs. 3 were tested with standardized major axis tests using SMATR v2.0 [p < 0.05.The differences in physiological and structural traits between ambient and elevated OATR v2.0 . All sta


There are some limitations of standard chemotherapy for acute leukemia. Vincristine and doxorubicin are commonly used for acute leukemia, but they may induce serious side effects such as cardiomyopathy and neurotoxicity. Furthermore, chemotherapy resistance occurs more and more frequently. Therefore, effective treatment strategies are needed. Histone deacetylase 6 inhibition is considered as a potential therapeutic strategy for acute leukemia, since it is observed that HDAC6 is overexpressed in acute leukemia and regulates tumor survival. Combination therapy for cancer is used to minimize adverse drug effects, reduce drug dosage, enhance efficacy, and prevent drug resistance. In order to improve efficacy of chemotherapy agents of acute leukemia, this study will investigate the effects of combination MPT0G211, a novel histone deacetylase 6 inhibitor, with doxorubicin or vincristine on human acute leukemia cells.MPT0G211 combined with doxorubicin induces DNA damage response on human acute myeloid leukemia cells. MPT0G211 can additionally increase Ku70 acetylation and release BAX to mitochondria. Ectopic expression of HDAC6 successively reversed the apoptosis triggered by the combined treatment. Moreover, co-treatment of MPT0G211 and vincristine may alter microtubule dynamics, triggering acute lymphoblastic leukemia cells arrest in mitotic phase followed by induction of the apoptotic pathway. Finally, MPT0G211 plus doxorubicin or vincristine can significantly improve the tumor growth delay in a tumor xenograft model.Collectively, our data highlighted that MPT0G211 in combination with chemotherapy drugs has significant anticancer activity, suggesting a novel strategy for the treatment of acute leukemia.The online version of this article (10.1186/s13148-018-0595-8) contains supplementary material, which is available to authorized users. Leukemia is a hematologic malignancy caused by the rapid proliferation of abnormal blood cells. This disease may be acute or chronic. In the former type, most cancer cells remain in a more immature and frequently dividing “blast” form, which leads to a range of complications and is fatal without proper treatment .Acute leukemia can be further divided in two subtypes by lineage. Of these, acute lymphocytic leukemia (ALL), which is characterized by the abnormal proliferation of lymphocytes , is the most common type of pediatric cancer. Acute myeloid leukemia (AML) involves the abnormal proliferation of immature myeloid progenitors . Both subtypes can progress quickly but differ considerably in terms of survival  [Currently, the chemotherapeutic agents vincristine (VCR) and doxorubicin (DOXO) are often used to treat acute leukemia. However, the clinical use of these drugs is often limited by the risk of side effects such as cardiomyopathy  and neurHistone deacetylases (HDACs) enzymatically regulate DNA expression by removing acetyl groups from histones. As a result, DNA can more tightly wind around histones, thereby decreasing gene expression . MammalsThe HDAC family member HDAC6 resides mainly in the cytoplasm, where it plays a unique role in regulating non-histone proteins without affecting gene expression . Many tyIn the present study, we evaluated the effects of a novel, selective HDAC6 inhibitor, MPT0G211, in acute leukemia cells when administered alone or in combination with chemotherapy drugs. We found that in AML cells, MPT0G211 potentiated the cytotoxic effects of DOXO by impairing DNA repair machinery and activating Bcl-2-associated X protein (BCL-XL)-dependent cell apoptosis. Additionally, when combined with VCR, MPT0G211 disrupted microtubule dynamics to induce mitotic arrest in ALL cells. These data suggest that HDAC6 inhibition represents a novel opportunity in the treatment of acute leukemia.v/v) fetal bovine serum  and 1% of a mixture of penicillin-streptomycin-amphotericin B . For HDAC6 overexpressed cells, HL-60 cells were transfected with HDAC6-FLAG  by using Turbofect . All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2.The human acute myeloid leukemia cell line, HL-60, human acute lymphoblastic leukemia cell line, MOLT-4, and human umbilical vein endothelial cells (HUVEC) were obtained from Bioresource Collection and Research Center (Taiwan). Cells were maintained in RPMI-1640 medium supplemented with 10% , and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. , and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical . Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling . α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex . PARP and CDC2 were from Santa Cruz Biotechnology .6 cells/well and then treated with various concentrations of MPT0G211, TBA, DOXO, VCR or CTX alone or in combination treatment for 48 h. MTT solution  was then added to the 24-well plate in the dark, and the plate was incubated at 37 °C for 2 h. For HL-60 cells, 10% SDS were added in the wells to dissolved the crystal, and for HUVEC cells, MTT-containing medium were removed and DMSO were added to each well to lyse cells; the absorbance was spectrophotometrically analyzed at 570 nm.Cells were seeded in a 24-well plate at a density of 1 × 10Cells were treated with indicated condition and then lysed in lysis buffer (10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM sodium orthovandate, 1 mM NaF, and 1% Triton X-100) for 30 min and centrifuged at 13,000 rpm. The supernatants were quantified by BCA Protein Assay Kit . Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were blotted with different antibodies overnight at 4 °C and conjugated with appropriate secondary antibodies.After treated with MPT0G211, TBA, or DOXO, cells were lysed in lysis buffer containing Halt™ protease and phosphatase inhibitor . Meanwhile, Protein A Magnetic Beads  were incubated with capture antibodies and rotated 10 min at room temperature. The mixture was rinsed with PBST three times to remove unbounded antibodies and incubated with cell lysate for 1 h. The complexes captured by magnetic rack were then washed three times using PBST to remove unbound proteins. All immunoprecipitation samples were suspended in Laemmli sample buffer and boiled for 10 min. The complex proteins were then analyzed by Western blotting.v/v) alcohol at 4 °C overnight. After centrifugation, cells were incubated in DNA extraction buffer (0.2 M NaHPO4–0.1 M citric acid) for 20 min at room temperature. Then, the cells were centrifuged and resuspended with 0.5 mL PI solution . Evolution of the cell cycle histogram was analyzed with the FACScan and CellQuest software (Becton Dickinson).After the treatment of vehicle (0.1% DMSO), MPT0G211, TBA, or chemotherapy agents for the indicated time courses, the cells were fixed with 75%  was used to separate cytosol and nuclear. Briefly, cells were collected and centrifuged at 600g for 5 min, supernatant was removed, and cytosol extraction buffer was added for 10 min. Cells were homogenized in an ice-cold Dounce tissue grinder and transferred homogenate to a new tube. The mixture was centrifuged at 700g for 10 min, supernatant was collected into a fresh tube and centrifuged at 10,000g for 30 min to acquire cytosolic fraction. The pellet was resuspended in mitochondrial extraction buffer and vortex 10 s to obtain mitochondria fraction.Cytochrome c Releasing Apoptosis Assay Kit  was used to separate mitochondria and cytosol. Briefly, cells were centrifuged at 600To observe microtubule distribution, cells were treated with MPT0G211, TBA alone, or in combination with vincristine for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min then permeabilized with 0.1% Tritin X-100 for 10 min. After washing with PBST for several times, 4% BSA were used to block non-specific proteins for 1 h then washed with PBST again and incubated with primary antibody α-tubulin for 2 h. FITC-conjugated anti-mouse IgG antibody were then used for another 2 h. Finally, cover slides were recovered to the slides with mounting gel containing DAPI stain. Images were detected and captured with the ZEISS confocal microscope.7 cells in 0.2 ml PBS) were subcutaneously injected into the flanks of the mice. When tumor sizes reached 200 mm3, mice were randomized into four groups with an indicated dosage of DOXO, VCR, and MPT0G211 alone or in combination treatment. All mouse tumors were allowed to reach an endpoint volume of 1200 mm3.Seven-week-old male severe combined immunodeficiency mice were fed ad libitum water and Pico-Lab Rodent Diet. All procedures were performed in accordance with the NIH guidelines on laboratory animal welfare approved by the Animal Use and Management Committee of Taipei Medical University (IACUC No. LAC-2015-0163). HL-60 or MOLT-4 cells .All data were expressed as mean values ± S.E.M. and were done independently three times. The significance of differences between the experimental groups and controls was assessed by Student’s In our previous study, we showed that MPT0G211 is a selective HDAC6 inhibitor with more potent activity than the currently available HDAC6 inhibitor ACY-1215 . In this50 = 5.06 ± 0.12 and 3.79 ± 0.36 μM, respectively), compared with TBA (IC50 > 10 μM for both cell types), but had no cytotoxic effect on normal human umbilical vein endothelial cells (HUVECs) (IC50 > 90 μM). Subsequently, we used a flow cytometric assay to investigate the effect of MPT0G211 on cell cycle distribution in cells that had been treated with MPT0G211 and TBA for 24 and 48 h. Compared with TBA, treatment with 10 and 3 μM MPT0G211 increased the proportions of HL-60 and MOLT-4 cells, respectively, and in the sub-G1 phase at both 24 and 48 h, the effect in the latter cell type was slight but statistically significant . The combination index (CI) was used to evaluate the effects of these two-drug combinations, with CI values of 1, > 1, and < 1 indicating addiction, antagonism, and synergism, respectively [Anthracycline drugs such as DOXO are considered standard treatment options for AML. Therefore, we evaluated the effect of a combination of MPT0G211 and DOXO in HL-60 cells. Flow cytometry revealed that this combination significantly induced cell cycle arrest in the G2/M phase, with the subsequent accumulation of cells in the sub-G1 phase , mice were randomized into control (vehicle) and treatment groups. As shown in Fig. Furthermore, combined MPT0G211 and TBA treatment induced the acetylation of Ku70 Fig. c, which To validate that the synergistic effect of MPT0G211 and DOXO is truly mediated by the inhibition of HDAC6, we examined whether HDAC6 overexpression could rescue cells from apoptosis triggered by the combined treatment Fig. a. Here, We also tested the combined effects of MPT0G211 plus DOXO, VCR, or CTX on MOLT-4 cells (ALL). As shown in Fig. VCR exerts its action by binding to tubulin to inhibit mitosis during metaphase and cause cell apoptosis . We foun50 = 5.06 and 3.79 μM in HL-60 and MOLT-4 cells, respectively; Fig. Although pan-HDAC inhibitors have been approved for the treatment of cancers, currently, they can only be administered to subset of patients with selected hematological malignancies. The use of these drugs is further limited by the frequent clinical observation of negative side effects such as electrocardiographic changes, characterized by T-wave flattening, ST segment depression, and QT interval prolongation . In cont50 value, it potentiated DOXO-induced G2/M arrest  The viability of cells treated with MPT0G211 and different doses of doxorubicin (DOXO) or vincristine (VCR) for 48 h was tested on CCRF-CEM and MV4-11 cell lines. The synergistic effects of these drugs were evaluated using the combination index (CI), with values of > 1.0, 1.0, and < 1.0 indicating an antagonistic, additive, or synergistic interaction, respectively. (B) Cell cycle distribution was determined in cells treated with various concentrations of MPT0G211 and DOXO for 48 h on MV4-11 cells. (DOCX 197 kb)


Background: International consensus statements for resuscitation of newborn infants recommend provision of 100% oxygen once chest compressions are required. However, 100% oxygen exacerbates reperfusion injury and reduces cerebral perfusion in newborn babies.Objective: We aimed to establish whether resuscitation with air during chest compression is feasible and safe in newborn infants compared with 100% oxygen.Methods: Systematic search of PubMed, Google Scholar and CINAHL for articles examining variable oxygen concentrations during chest compressions in term newborns.Results: Overall, no human studies but eight animal studies  comparing various oxygen concentrations during chest compression were identified. The pooled analysis showed no difference in mortality rates for animals resuscitated with air vs. 100% oxygen . ROSC was also similar between groups with a mean difference of −3.8 [−29.7–22] s, I2 = 0%, p = 0.77. No difference in oxygen damage or adverse events were identified between groups.Conclusions: Air had similar time to ROSC and mortality as 100% oxygen during neonatal chest compression. A large randomized controlled clinical trial comparing air vs. 100% oxygen during neonatal chest compression is warranted. High concentrations of O2 lead to generation of oxygen free radicals, which have a role in reperfusion/reoxygenation injury after asphyxia especially to oxyregulator tissues such as myocardium  . During ocardium –8. Thus,2 during cardiopulmonary resuscitation (CPR). The aim of oxygen use during resuscitation is to reactivate mitochondrial activity and energy provision and prevent tissue damage from oxygen deprivation during asphyxia while avoiding adverse effects of oxidative stress on the respiratory system and cerebral circulation; and tissue damage from oxygen free radicals : “infant,” “newborn,” “resuscitation,” “chest compression,” “oxygen,” and “delivery room.” Publications were assessed based on title, abstract, and methods. Studies were included if they compared different oxygen concentrations during CC. Studies were excluded when no CC were performed, or if they did not define the infants as newborn. The initial search was aimed to only identify human trials. However, no human trials were identified and therefore the search was expanded to include animal studies. A manual search through the references of the obtained articles was additional performed.2 during neonatal CPR; and presented the outcomes of either death or ROSC. Our primary outcome measure was mortality during neonatal CPR. Secondary outcomes included time to ROSC, oxygenation, and indicators of organ injury or damage. Full articles for potentially relevant studies were retrieved and independently assessed for their eligibility using a standardized data collection form. We also aimed to identify and if available include multiple publications describing the same study. Authors were contacted for data on return of spontaneous circulation (ROSC) as they were only reported as median (IQR) in the respective articles. No language restrictions were applied. Discrepancies regarding inclusion were resolved with another member of the review team (ALS).Two reviewers (CGH and GMS) independently reviewed citations for selection. Studies were included in the review if they met the following criteria: randomized controlled trial; comparing use air vs. 100% OData were recorded using a standardized data collection form to record study design and methodological characteristics, patient characteristics, interventions, and outcomes thereof, including their RR (95% CI). Data extraction was independently performed by two investigators  and discrepancies were resolved in consultation with another member of the review team (ALS).2 statistic. We summarized RR estimates using random-effects models. Analyses were performed in RevMan version 5.3 . All p-values are 2-tailed.The principal summary measures were RR (95% CI) for dichotomous outcomes. Heterogeneity was explored using a chi-square test, and the quantity of heterogeneity was measured using the I2, base excess, and lactate prior the start of resuscitation  and variable durations of initial positive pressure ventilation. Alsaleem et al.  in addition to air vs. 100% O2 and assessed time to ROSC and oxidative stress studies used a swine model –15, and al model , while tal model . Dannevial model , 11 assem et al.  resuscita et al.  resuscitr et al.  also tesr et al.  comparedve stress, 15.p = 0.77, I2 = 0%; Figure For the pooled analysis, the study by Solevåg et al.  was excl2 with a mean difference of −3.8 [−29.7–22] s, I2 = 0%, p = 0.77 ] time to ROSC of 135 (113–168) s vs. 150 (115–180) s in the air and 100% group, respectively. A second study by Solevåg et al.  were similar among groups with median (IQR) times of 67 (60–76) s, 88 (76–126) s, and 68 (56–81) s in the air, 100% for three min group, or 100% for 30 min group, respectively.Overall, the individual studies and the pooled analysis showed no difference in the time to ROSC with air vs. 100% O7 Figure  randomlyg et al.  randomizand 2017 , 13 rand2 to be increased to 100%. There was no difference in mean (±SD) time to ROSC 211 (±145) s and 306 (±270) s in the air and 100% O2 group, respectively. Perez-de-Sa et al.  s, 107 (5–182) s and 58 (23–368) s]. These studies demonstrate that air is as effective as 100% O2 to achieve ROSC and circulatory recovery. However, recently Linner et al. demonstrated that when PPV is ineffective ROSC can be faster achieved using 100% O2 compared to air with 60 (11–120) s vs. 845 (611–death) s (p < 0.001)  .2) had increased 0.1 kPa from its nadir. Studies in near-term lambs reported higher PbtO2 with 100% O2, and significantly increased partial pressure of arterial oxygen levels immediately after ROSC compared to lambs who remained in air (165 ± 145 vs. 41 ± 16 mmHg), (p = 0.046) (2 delivery to the brain between groups was observed (2) reaching a maximum of 56 kPa (420 mmHg) in the group initially ventilated with 100% O2 for 30 min, a level only previously seen in hyperbaric conditions(2 for 3 min had significantly lower PbtO2 peaking at 4.2 kPa (31.5 mmHg) while those receiving air peaked at 2.9 kPa (19.5 mmHg) (p = 0.002). The 2009 study by Linner et al. found no difference in time to recovery of brain oxygenation but observed arterial hyperoxemia 34 (30–41) kPa [255 (225–308) mmHg] by 2.5 min and peak PbtO2 values during resuscitation were higher in groups ventilated with 100% O2 [4.2 (3.3–5.4) kPa (31.5 (24.8–40.5) mmHg], 12 (6.4–15) kPa [90 (48–112) mmHg], and 25 (15–36) kPa [187.5 (112.5–270) mmHg], respectively)  . Howeverobserved . Perez-d2 vs. air was used in the resuscitation after asphyxia induced cardiac arrest in newborn piglets. Dannevig et al.  and 0.10 (0.08–0.11) (p = 0.005), respectively.Damage caused by oxidative stress was measured in the piglet study assessing 100% O or CCaV . The oxi2 during CPR, however the most effective oxygen concentration in newborn infants remains controversial  0.71  0.54 to 0.94), risk difference −0.05 (−0.08 to −0.01)]  s, I2 = 0%, p = 0.77   compared to resuscitation with air  and 0.10 (0.08–0.11) (p = 0.005), respectively  . During with air . However, 0.14 0.–0.22 andd cancer , 30.Limitations of the current review among others include (i) only data from animal studies were included, (ii) the data should not be directly extrapolated to clinical practice, (iii) different animal models , (iv) transitional model (lambs delivered via cesarean section) vs. post-delivery model (piglets 1–3 days old), (v) induction of cardiac arrest by asphyxia or potassium chloride, or (vi) all animals were intubated with a tightly sealed endotracheal tube  to prevent any endotracheal tube leak –18, 31. Our results indicate that air during CC is safe and human trials are urgently needed. Human studies should compare air vs. 100% and examine effects of both oxygen concentrations to reduce exposure to hypoxia and hyperoxia . Alterna2. Human trials comparing air vs. 100% and/or oxygen titration as an alternative to air or 100% oxygen during neonatal chest compression are urgently needed.No human studies were identified and the results obtained are from animal models. The data suggest that using air instead of 100% oxygen during neonatal chest compression had comparable outcomes including time to return of spontaneous circulating and mortality. Hyperoxia and oxidative stress were significantly higher with 100% OGS, P-YC, MO, and AS Conception and design; GS, CG-H, MO, MV, P-YC, AS, and OS Collection and assembly of data, analysis and interpretation of the data; GS, CG-H, MO, P-YC, AS, MV, and OS Drafting of the article; GS, CG-H, MO, P-YC, MV, AS, and OS; Critical revision of the article for important intellectual content, final approval of the article.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.