Results: iPTH levels decreased in the study group appropriately and were more significantly decreased at 16 weeks. Study group had significantly increased 25(OH)D3 levels. In addition, the study group had significantly increased serum hCAP-18 levels compared with control group. Correlation analysis showed a significant correlation between the percentage increase in serum hCAP-18 and 25(OH)D3 levels. Conclusions: Cholecalciferol, in combination with paricalcitol, additively lowers the iPTH levels in a significant number of patients after 16 weeks of supplementation. A dose of 5000 IU/week of cholecalciferol could maintain serum 25(OH)D3 levels above 30 ng/dL as early as 8 weeks after beginning supplementation. Doubling of serum cathelicidin levels were noted after 16 weeks of cholecalciferol supplementation in 40% of study patients.Background: Active Vitamin D analogues are used clinically for prevention and treatment of secondary hyperparathyroidism (SHPT) in hemodialysis (HD) patients. Nutritional vitamin D supplementation is used for additional local parathyroid (PTH) suppression, with lower incidence of hypercalcemia and hyperphosphatemia. This study evaluates the possible beneficial effects of combined vitamin D treatment . Methods: Sixty HD patients with serum parathyroid hormone (iPTH) >300 pg/mL were enrolled. All patients administered 2 mcg/day of paricalcitol and were randomly allocated into control group (placebo) or study group  for 16 weeks. Serum 25(OH)D The enzyme 24-hydroxylase (24-OHase) is responsible for degradation of the vitamin D analogs 25-hydroxyvitamin D D) and 1,25-dihydroxyvitamin D 2D). With progressive kidney function impairment, end stage kidney disease (ESKD) patients experience a decrease in 1α-OHase activity and in 1,25(OH)2D production, with subsequent hypocalcemia and secondary hyperparathyroidism (SHPT). Active vitamin D analogs  are used clinically to prevent or treat the SHPT and mineral disorders among ESKD and dialysis patients. However, longer use of active vitamin D analogs revealed hypercalcemia, vascular calcification [Vitamin D naturally acquired from the diet or dermal synthesis  is converted into more active vitamin D compounds with the help of liver 25α-hydroxylase (25α-OHase) for 25-hydroxyvitamin D D), and kidney 1α-hydroxylase  for 1,25-dihydroxyvitamin D 3, a circulating vitamin D metabolite which indicates vitamin D status [3 serves as an important substrate for the local generation of 1,25(OH)2D [3 levels [3 in these patients. Ritter et al. [3 in PTH suppression possibly occurs through direct activation of vitamin D receptor (VDR) in parathyroid glands. Furthermore, 25(OH)D3 played less of a role in hypercalcemia and mineral disturbances. These findings explain the possible additive role of nutritional vitamin D  supplementation to active vitamin D analogs in SHPT patients.Circulating parathyroid (PTH) levels have been found to be inversely correlated with serum 25(OH)DD status ,5,6,7. W25(OH)2D ,9. Most 3 levels ,11,12,133 levels  revealed3 levels , and higr et al.  demonstr3 has been associated with inadequate non-classical effects despite higher extrarenal 1α-hydroxylation status among chronic kidney disease (CKD) patients [2D [2D in immune cells is responsible for the production of human cathelicidin (hCAP-18) and enhances the capacity of autophagy through toll-like receptor activation [Similarly, the non-classical actions of vitamin D take place by local active vitamin D in extra-renal tissues  through local 1α-hydroxylation. Deficient 25(OH)Dpatients ,18. Humaents [2D ,20 and htivation ,22,23. Ativation . However3 and hCAP-18 levels effectively in regular HD patients.In our recent study, we hypothesized that cholecalciferol supplementation generates additive SHPT control with active vitamin D analogs  and increases 25(OH)DThis was a prospective, randomized, interventional, efficacy study comparing cholecalciferol plus paricalcitol with a standard vitamin D therapy, paricalcitol plus placebo (control group). Patients were given an oral form of cholecalciferol 5000 IU per capsule  in the study group and cholecalciferol placebo (olive oil) was used in the control group. A baseline visit was performed just before the start of the study, and further study visits were performed at 4, 8, 12 and 16 weeks after baseline. Both patients and/or investigators were blinded to the study intervention. Prior to the study, G*power was used to calculate the required sample size  and effeThe trial was conducted in Taipei Medical University Shuang Ho Hospital from 1 June 2014 to 31 May 2015. The study was approved by the authorities of the Institutional Review Board of Taipei Medical University, and all the patients provided written consent before study enrollment.n = 29) had this medication withdrawn one month before the start of the study. Only non-calcium containing phosphate binders were administered. The patients were excluded if they were pregnant, breastfeeding, or of childbearing potential and not practicing birth control. Those with malignancies, severe malnutrition, and inflammatory or infectious disorders diagnosed more than three months before the start of the study were also excluded. Other exclusion criteria included surgical interventions and vascular diseases, including acute coronary syndrome, unstable angina, cerebrovascular accident, transient ischemic attack, deep vein thrombosis, pulmonary embolism, or congestive heart failure within three months of the study period. The patients were also excluded if they had a history of allergy to cholecalciferol or if the investigator considered one-month withdrawal of paricalcitol unsuitable  > 300 pg/mL). Dialysate Ca was 1.5 mmol/L, and the patients had not been using any form of vitamin D analogs for at least one month before the start of the study. Patients under regular treatment with calcitriol  daily (fixed dose protocol) and were randomly allocated into two groups with placebo or with low-dose cholecalciferol supplementation (5000 IU/week). Follow-up visits were scheduled at week 4, 8, 12 and 16 after the start of the medication. Serum concentrations of 25(OH)DSerum 25(OH)D was determined by ELISA according to the manufacturer’s instructions . The serum samples were measured in triplicate. The coefficients of variation for 25(OH)D measurements were <3% at levels <30 ng/mL. Serum iPTH was measured using a kit from Immutopics Inc., San Clemente, CA, USA. The PTH kit had a detection limit of 13 pg/mL, with intra-assay Coefficient of Variability (CV) of 4.5% and inter-assay CV of 4.8%. Plasma cathelicidin (hCAP-18/LL-37) was measured by ELISA . hCAP-18 measurements were conducted in duplicate after initial thaw of the serum samples, and the calculated mean of the measurements was used in the analyses. The intra-assay CV for hCAP-18 measurements was <10%. Serum levels of Ca, P, and alkaline phosphatase (ALP) were also monitored. Hospital records were obtained and examined by two practicing nephrologists .The primary study objective was to assess the beneficial effects of cholecalciferol combined with paricalcitol in HD patients with concomitant SHPT.Primary Outcome: The primary therapeutic outcome was serum iPTH levels less than 300 pg/mL at the end of the study.3 more than 30 ng/mL and doubling of serum cathelicidin (hCAP-18) level at the end of the study.Secondary Outcome: The secondary outcomes were serum 25(OH)Dt or Wilcoxon tests, respectively, and for between-group comparisons, the Student t, one-way ANOVA or Mann–Whitney U tests were used. Unilateral correlation analysis was performed using Pearson (r) or Spearman correlation (rs), as appropriate. All the tests were two-sided, and p < 0.05 was considered statistically significant. Statistica  was used for calculations.According to data distribution, the results were expressed as mean ± standard deviation or median (interquartile range). Parametric or nonparametric tests were used for analysis; for paired data, the Student n = 31), primary glomerulonephritis (n = 5), hypertension (n = 10), and others  (n = 16). No significant difference was noted regarding the prior calcitriol usage (n = 8 in control group vs. n = 10 in study group). All patients had high serum iPTH levels  and low 25(OH)D3 levels . Serum albumin, ALP, albumin-corrected Ca, P, and hCAP-18 levels were not significantly different between two groups. p < 0.05) vs. from 681.9 ± 173.3 pg/mL to 299.37 ± 74.62 pg/mL in the control group (p < 0.05) (p = 0.00032 (Chi-Square test))  . The lowe test)) .3 levels increased from 19.6 ± 7.26 ng/mL to 30.4 ± 7.7 ng/mL (p < 0.05). However, in the control group, 25(OH)D3 levels did not change significantly (from 19.53 ± 8.2 ng/mL to 19.63 ± 7.56 ng/mL) (3 ≥ 30 ng/dL) was significantly higher in the study group from the 8th week of the study onwards ) D6 ng/mL) . Betweene test)) .p < 0.05) vs. from 24.05 ± 7.99 ng/mL to 26.59 ± 65.68 ng/mL in control group) (p = 0.006 (Chi-Square test)) (Study group had significantly increased serum hCAP-18 levels from baseline compared with control group (from 22.25 ± 6.71 ng/mL to 82.13 ± 68.67 ng/mL in the study group (l group) . Betweene test)) .3 levels (p < 0.05)  in serum hCAP-18 and 25(OH)D < 0.05) .3 levels above 30 ng/dL as early as 8 weeks after start of supplementation. Doubling of serum cathelicidin levels was noted with 16 weeks of cholecalciferol supplementation in 40% of study patients. Our primary and secondary outcomes were met at the end of the study period in the cholecalciferol supplementation group. Additive PTH lowering was found in a significant number of study patients after 16 weeks of supplementation. A dose of 5000 IU/week of cholecalciferol could maintain serum 25(OH)D2D may aggravate 25(OH)D3 deficiency because of its feedback inhibition of hepatic 1α-OHase and 25α-OHase, which results in 25(OH)D3 shortage among other tissues or organs  [2D also induces the 24-OHase enzyme and autoregulates its own catabolism [2D may not only reduce 25(OH)D3 production but also increase 1,25(OH)2D and 25(OH)D3 catabolism. Clinically, serum 25(OH)D3 levels decline since early CKD and low serum 25(OH)D3 levels might increase serum iPTH levels, suggesting that this event is crucial for SHPT development. Several factors, including aging and comorbidities such as diabetes and hypertension, were consistently associated with low 25(OH)D3 levels in both dialysis and nondialysis CKD [3 deficiency [3 levels higher than 20 ng/mL seem sufficient to control serum PTH in patients with CKD [Active vitamin D compounds were traditionally used as sole agents among SHPT patients for its inhibition effects on PTH gene transcription and chief cell hyperplasia. Growing evidence shows that higher doses of active 1,25(OH)s, etc.) ,27. Actitabolism . Thus, aysis CKD . A previficiency . In addiwith CKD . Studies3 is important for non-classical actions of vitamin D in extra-renal tissues by converting to active 1,25(OH)2D through local 1α-OHase activity. Furthermore, circulating 25(OH)D3 levels also increase 1α-OHase activity in a substrate-dependent manner among CKD patients; a higher concentration of 25(OH)D3 is associated with stronger 1α-OHase activity and vice versa [3 in autoregulation of the PTH glands and probably through direct activation of the vitamin D receptor (VDR) within parathyroid cells [3 requirement among these patients. Although 25(OH)D3 had several hundred times lower VDR binding affinity and lower suppressing PTH activity than 1,25(OH)2D3 [3 levels were a thousand times higher than 1,25(OH)2D levels. These findings explain the critical need for 25(OH)D3 supplementation in SHPT patients in addition to active vitamin D analogs. Indeed, our study results revealed that additional reduction in PTH levels was achieved in almost 76.7% of the study patients with cholecalciferol supplementation.We found that our primary outcome, a iPTH level less than 300 pg/mL was achieved in both control and study group at the end of study, with significantly lower PTH level at week 16 in the cholecalciferol supplementation group . Furtherce versa . Recent id cells ,16. Anotid cells  which fu3 levels significantly increased in the study group, whereas they remained low in the control group (3 levels. Sixty percent of study patients achieved target level (25(OH)D3 ≥ 30 ng/dL) with cholecalciferol supplementation for 16 weeks Dol group . The numol group . Clinica16 weeks . This wa3 levels were found to closely correlated in correlation analysis (p < 0.05)  . Catheliortality . Many inortality ,43,44,45ortality . A high ortality . In critality [3 . Therefo3 target level in dialysis patients remains unknown, however, we supposed some additive PTH lowering effects with 25(OH)D3 ≥ 30 ng/dL. The beneficial role of cholecalciferol in chronic kidney disease-mineral bone disorder (CKD-MBD) requires a multifaceted long-term approach and needs further study in larger clinical trials. The results from our study showed that nutritional vitamin D compounds synergizes with active vitamin D compounds in lowering PTH and should be more broadly considered among HD patients with SHPT.Our study has several limitations. Firstly, we cannot apply our findings or draw definitive conclusions in the general HD population due to relatively small sample size, however the results were indeed promising and clinically important. We used total calcium concentrations rather than ionized calcium concentrations, which could have some effects on vitamin D levels. Furthermore, we excluded patients with severe malnutrition, and inflammatory or infectious disorders who might have benefited the most from the intervention due to vitamin D deficiency. However, our cohort had significantly low vitamin D levels in both arms. Next, we did not determine the morbidity or mortality benefits of combination therapy. Although we performed some questionnaires on acute respiratory tract infections, the results were inconsistent and non-significant due to shorter follow-up duration. The optimal serum 25(OH)D3 repletion with native vitamin D acts additively with active vitamin D analogs for controlling SHPT among HD patients. We further confirm the positive relationship between 25(OH)D3 and cathelicidin levels in these patients. Future large-scale clinical trials are warranted to justify the incorporation of 25(OH)D3 repletion into SHPT treatment recommendations in dialysis patients.In conclusion, our findings provide in vivo evidence that 25(OH)D


A few studies previously suggested that human papillomavirus (HPV) E6 messenger RNA (mRNA) may exist uniformly in all grades of cervical intraepithelial neoplasia (CIN), whereas the detection rate of E7 mRNA may increase with disease progression from low-grade CIN to invasive carcinoma. The aim of this study was to clarify the different roles of E6 and E7 mRNAs in cervical carcinogenesis. The presence of each E6 and E7 mRNA was analyzed in 171 patients with pathologically-diagnosed CIN or cervical carcinoma. We utilized a RT-PCR assay based on consensus primers which could detect E6 mRNA (full-length E6/E7 transcript) and E7 mRNAs (spliced E6*/E7 transcripts) separately for various HPV types. E7 mRNAs were detected in 6% of CIN1, 12% of CIN2, 24% of CIN3, and 54% of cervical carcinoma. The presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with E6 mRNA or high-risk HPV (HR-HPV) DNA . The presence of both E6 and E7 mRNAs was significantly associated with HPV16/18 DNA but not with HR-HPV DNA (p = 0.0079 and 0.21), while the presence of E6 mRNA was significantly associated with HR-HPV DNA but not with HPV16/18 DNA (p = 0.036 and 0.089). The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity (100% and 19%) for detecting CIN2+ by contrast with the positivity for HR-HPV DNA showing low specificity and high sensitivity (19% and 89%). The positive predictive value for detecting CIN2+ was even higher by the presence of both E6 and E7 mRNAs than by the positivity for HR-HPV DNA (100% vs. 91%). In 31 patients followed up for CIN1-2, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in contrast with E6 mRNA, HR-HPV DNA, or HPV16/18 DNA . Our findings support previous studies according to which E7 mRNA is more closely involved in cervical carcinogenesis than E6 mRNA. Moreover, the separate analysis of E6 and E7 mRNAs may be more useful than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our findings. Cervical cancer is the fourth most commonly diagnosed cancer and the fourth most common cause of cancer death in women worldwide . Each yeThe current study comprised two parts: a cross-sectional study of analyzing E6/E7 mRNAs in cervical specimens from patients with CIN or invasive cervical carcinoma and an adjunctive longitudinal study of following up patients with CIN1-2 . Women wGenomic DNA was extracted using SepaGene kit  according to the manufacturer’s instruction. HPV genotyping was performed by L1-PCR and RFLP analyses as described previously  or at a ACC GAA AAC GGT TGA ACC GAA AAC GGT and GAG CTG TCG CTT AAT TGC TC; β2-microglobulin, TGT CTT TCA GCA AGG ACT GG and GAT GCT GCT TAC ATG TCT CG.Total RNA extraction and DNase treatment were performed as described previously . RT-PCR Differences in proportions were evaluated by the Fisher’s exact test. Diagnostic indices of sensitivities, specificities, positive predictive values, and negative predictive values with 95% confidence intervals were calculated for detecting CIN2+, CIN3+, and invasive cervical cancer. Disease progression of CIN1-2 was examined as a surrogate by the Kaplan-Meier method calculating the intervals from E6/E7 sample collection until patients showed upgraded results of Pap test compared with the cytology at E6/E7 sample collection or they were censored, and the difference was statistically evaluated by the log-rank test.We first analyzed the E6 and E7 mRNA expression patterns in human cervical cancer cell-lines CaSki and HeLa by RT-PCR , and conSubsequently we analyzed the E6/E7 mRNA expressions in liquid-based cytology samples from 171 patients. Next, we examined diagnostic accuracies for cervical neoplastic diseases by E6/E7 mRNA analyses. For detecting CIN2+, the presence of both E6 and E7 mRNAs showed high specificity and low sensitivity  –18, 23. In conclusion, our separate analyses of E6/E7 mRNAs demonstrated here that the presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with positive E6 mRNA or HR-HPV DNA. Besides, the presence of both E6 and E7 mRNAs was significantly associated with the positivity for HPV16/18 DNA, while the presence of E6 mRNA was significantly associated with the positivity for HR-HPV DNA. The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity for detecting CIN2+ by contrast with the positivity for HR-HPV DNA. Furthermore, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in the patients followed-up for CIN1-2 by contrast with positive E6 mRNA, HR-HPV DNA, or HPV16/18 DNA. Our findings suggest a closer involvement of E7 mRNAs than E6 mRNA in cervical carcinogenesis. Moreover, the separate analysis of E6 and E7 mRNAs may be a more useful tool than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our proposal.


Brassica napus to determine the precise kinetics of S and Mo uptake and whether sulfate transporters were involved in Mo uptake. Leaf contents of S and Mo were also quantified in a field-grown S deficient oilseed rape crop with different S and N fertilization applications to evaluate the [Mo]:[S] ratio, as an indicator of S nutrition. To test genericity of this indicator, the [Mo]:[S] ratio was also assessed with other cultivated species under different controlled conditions. During S deprivation, Mo uptake was strongly increased in B. napus. This accumulation was not a result of the induction of the molybdate transporters, Mot1 and Asy, but could be a direct consequence of Sultr1.1 and Sultr1.2 inductions. However, analysis of single mutants of these transporters in Arabidopsis thaliana suggested that other sulfate deficiency responsive transporters may be involved. Under field conditions, Mo content was also increased in leaves by a reduction in S fertilization. The [Mo]:[S] ratio significantly discriminated between the plots with different rates of S fertilization. Threshold values were estimated for the hierarchical clustering of commercial crops according to S status. The use of the [Mo]:[S] ratio was also reliable to detect S deficiency for other cultivated species under controlled conditions. The analysis of the leaf [Mo]:[S] ratio seems to be a practical indicator to detect early S deficiency under field conditions and thus improve S fertilization management.Under sulfur (S) deficiency, crosstalk between nutrients induced accumulation of other nutrients, particularly molybdenum (Mo). This disturbed balanced between S and Mo could provide a way to detect S deficiency and therefore avoid losses in yield and seed quality in cultivated species. Under hydroponic conditions, S deprivation was applied to  Natural light was supplemented with high-pressure sodium lamps   for 16 h. The aerated nutrient solution contained: 3.75 mM KNO3, 0.5 mM MgSO4, 0.5 mM CaCl2, 0.25 mM KH2PO4, 0.2 mM EDTA-2NaFe, 14 μM H3BO3, 5 μM MnSO4, 3 μM ZnSO4, 0.7 μM CuSO4, 0.7 μM (NH4)6Mo7O24, 0.1 μM CoCl2, 0.04 μM NiCl2 and buffered to pH 6.6 with 0.91 mM CaCO3. This solution was renewed according to the rate of NO3- depletion monitored daily by using NO3- test strips  in order to maintain optimal nutrition conditions.Seeds of 42-, (ii) S-deprived plants (-S) were grown with 8.7 μM SO42- (3- depletion by monitoring the NO3- level in the tank (at the end of the experiment this was about every two days).After 4 weeks of growth, plants were separated into two batches supplied with a modified nutrient solution chosen in order to achieve S deficiency and to maintain the same concentration of other nutrients: (i) control plants (+S) were grown with 508.7 μM SOμM SO42- . These nFour independent samples each consisting of four individual plants were harvested at day 0 and after 1, 2, 3, 7, 13, 21 and 28 days of treatment. Leaves present at the beginning of treatment applications (d0) were identified and marked, and these organs were referred to as “emerged leaves”, while leaves appearing during treatments were harvested separately as “new emerging leaves”. At each date of harvest, whole roots from control and depleted plants (-S) were also harvested. Thereafter, leaves, petioles and roots were frozen in liquid nitrogen and stored at -80°C for further analysis. It must be pointed out that roots were freeze dried without any prior rinsing and therefore the root analyses include whatever is bound outside the tissue as well as internal concentration of elements. An aliquot of each tissue was freeze-dried for dry weight (DW) determination and ground, using an oscillating grinder , to fine powder for element analysis.Arabidopsis plants  were grown for 17 days weeks on vertical agarose plates with modified Long Ashton nutrient solution with full (0.75 mM) or limited (0.075 mM) sulfate in controlled environment room under a long day 16-h-light/8-h-dark cycle at constant temperature of 22°C, 60% relative humidity, and light intensity of 120 μE s–1m–2. The sel1-8 mutant has been isolated from a genetic screen on selenate tolerance using an EMS mutagenized Col-0 seeds and contains a point mutation in the coding sequence :[S] ratio  and without S had the highest [Mo]:[S] ratio, whereas mature leaves of plants with 36 kg S ha-1 had the lowest [Mo]:[S] ratio and mature leaves of plants with 12 kg S ha-1 had an intermediate [Mo]:[S] ratio. The same trends were also found in young leaves  whereas in plot with 65 kg N ha-1 and 0 kg S ha-1 (N65S0) the biomass was 6.70 ± 0.01 g DW leaf-1 (P<0.01). The supply of N mineral fertilization had a direct negative consequence on S content in leaves as a result of the increased growth rate. Indeed, without S supply, N fertilization significantly reduced the leaf S content after 15 days  and the relative S content in mature leaves was reduced. For example, when comparing -1 was significantly lower (P<0.05) than for plants with 65 kg N ha-1 and for a given level of S fertilization . In contrast, the N fertilization rate had no significant effect on the Mo content of mature leaves  and of S deficient plots , respectively. Such thresholds would allow clustering of the plots into three groups: S sufficient (ratio <1.61), at risk of S deficiency (ratio between 1.61 and 2.83) and S deficient (ratio >2.83). This classification into three groups has been tested on 45 commercial oilseed rape crops from different locations in France and for which mature leaves were harvested before flowering and S fertilization  encoding high-affinity sulfate transporters of group 1 have been studied in order to clarify their ability to also transport molybdate. The increased uptake of Mo in S-deprived plants was strongly correlated with an increase in BnaSultr1.1 expression at least during the first 7 days  as well as :[S] ratio is a practical alternative to detect early S deficiency under field conditions, given that molecular indicators require laboratory analysis and use of control plants, which are not easily performed under field conditions. Alternatively, new breakthrough technologies using X ray fluorescence or laser metal analysis, also available as portable field tools, would allow an easy quantification of the [Mo]:[S] ratio.S1 FigLocation of 45 commercial crops in Francewith crops classified into three S status groups: S deficient in red, at risk of S deficiency in orange and S sufficient in green. Map of France from Institut Géographique National .(TIF)Click here for additional data file.S2 Fig-1 DW),  Mo content (μg g-1 DW) and  the [Mo]:[S] 104 ratio (to simplify reading the [Mo]:[S] ratio is presented with a multiplier factor of 104) in young leaves of B. napus grown under field conditions after  15 and  47 days of fertilization. Plants received no mineral fertilization , or 0 kg S ha-1 (white bar), 12 kg S ha-1 (grey bar) or 36 kg S ha-1 (black bar) with 65 kg N ha-1 (dashed bars) or 125 kg N ha-1 (full bars). Within the same graph, letters when different between fertilization treatments indicate significant differences for P<0.05. S content (mg g(TIF)Click here for additional data file.S3 FigCorrelation circles on the factorial planes (A) and (B) projection of the commercial crops (numbers refer to fields/crops given in (TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 TableNutrient concentrations are expressed in mM.(PPTX)Click here for additional data file.


Higher ALA levels were associated with lower T2D risk only among participants within the lowest GRS tertile, with ORs 0.51 (95% CI: 0.26–1.03), 0.44 (95% CI: 0.22–0.89) and 0.49 (95% CI: 0.25–0.96) for the 2nd, 3rd and 4th ALA quartile, compared with the 1st. This study suggests that higher erythrocyte ALA levels are inversely associated with T2D risk only among participants with low T2D genetic risk, with high genetic risk abolishing the ALA-T2D association.Modulation of n-3 fatty acids on genetic susceptibility to type 2 diabetes (T2D) is still not clear. In a case-control study of 622 Chinese T2D patients and 293 healthy controls, a genetic risk score (GRS) was created based on nine T2D genetic variants. Logistic regression was used to examine the interaction of the GRS with erythrocyte phospholipid n-3 fatty acids for T2D risk. Every 1-unit  increase in GRS was associated with 12% (Odds ratio (OR): 1.12; 95% confidence intervals (CI): 1.04–1.20) higher risk of T2D. Compared with the lowest quartile, participants had lower T2D risk in the 2nd , 3rd  and 4th  quartile of alpha-linolenic acid (ALA) levels. Significant interaction ( PEPD gene  to modulate T2D risk in a Chinese population, with higher n-3 fatty acids abolishing the adverse effect of the risk allele at PEPD on T2D ..C2CD4A-CAfter an overnight fast, venous blood was collected by trained nurses with 21-gauge needles in plain and Ethylenediaminetetraacetic acid (EDTA) vacuum tubes, with red blood cells separated by standardized procedure. Erythrocyte membrane phospholipid fatty acids were measure using standard methods . BrieflyAnthropometric parameters , blood pressures and other lifestyle information were collected by a research assistant trained in standardized procedures after blood collection.t-test. The associations of GRS with T2D risk, and erythrocyte phospholipid fatty acids with T2D risk were examined with a logistic regression model, adjusting for age and sex. In the primary analysis, logistic regression was used to examine the interaction of the unweighted GRS with erythrocyte marine n-3 fatty acids (C20:5n3 + C22:5n3 + C22:6n3) and alpha-linolenic acid (C18:3n3) for T2D risk, adjusting for age and sex. Fatty acids were categorized into two groups according to the population median. GRS was categorized as tertiles for the interaction analysis. Stratified analyses by GRS or by erythrocyte fatty acids were conducted if significant interaction was observed. We did not apply Bonferroni correction, as the present study is based on a hypothesis-driven approach to test the interaction with GRS for marine- and plant-based n-3 fatty acids, and actually only two interaction tests have been done in the primary analyses.All the statistical analyses were performed using STATA version 14 . Hardy-Weinberg equilibrium of the genotyped SNPs was examined with a Chi-square test. The difference in GRS values between control and T2D groups was compared with two-sample p-value < 0.05 was considered as statistically significant in the present study.We then performed several sensitivity analyses: (1) We further adjusted body-mass index (BMI) in addition to age and sex in the primary interaction analysis; (2) We examined the interaction of weighted GRS with erythrocyte fatty acids for T2D risk, in order to investigate the influence of different weights/effect sizes of the SNPs on the results; (3) We examined the interaction of original unweighted GRS  with erythrocyte fatty acids for T2D risk to test the influence of genotype imputation on the results. A two-tailed p < 0.001). The population characteristics and erythrocyte phospholipid n-3 fatty acids by the unweighted GRS tertiles were presented in The minor allele frequency of the nine genotyped SNP ranged from 0.29 to 0.44, and all SNPs were consistent with Hardy-Weinberg equilibrium . The meap = 0.003) higher risk of T2D. The result was similar for the weighted GRS .Every 1-unit  increase in unweighted GRS was associated with 12% , 3rd  and 4th  quartile of ALA levels. Similarly, the 2nd , 3rd  and 4th quartile  of marine n-3 fatty acids had lower risk of T2D compared with the 1st quartile.p-interaction = 0.029), adjusting for age and sex. The interaction remained significant when we further adjusted for BMI (p-interaction = 0.038) or when we used the original non-imputed GRS (p-interaction = 0.05). Briefly, high ALA levels, compared with low ALA levels, had significantly lower risk of T2D  only among participants within the low GRS group (unweighted GRS tertile 1), but not for participants within the GRS tertile 2 or tertile 3 group (p-interaction = 0.21), for the weighted GRS. Higher ALA was associated with lower T2D risk among the GRS tertile 2 but not tertile 3 group.There was significant interaction between erythrocyte ALA and unweighted GRS for the T2D risk  in the low ALA group , while there was significant genetic association among the high ALA group  . The resIn the present study, we found that erythrocyte phospholipid ALA interacted with unweighted GRS to modulate the risk of T2D in a Chinese population. Briefly, the inverse association between erythrocyte phospholipid ALA and T2D only existed among individuals with low T2D genetic risk, but did not exist among those with high T2D genetic risk. We did not find interaction of GRS with marine n-3 fatty acids in the present study.T2D is considered to result from the complex interplay between genetic and environmental factors . AccumulRecent evidence suggests that blood marine n-3 fatty acids and plant n-3 fatty acid (ALA) showed heterogeneous associations with risk of T2D, with no significant association for marine n-3 fatty acids, but significantly inverse association for ALA . The eviIn the present study, consistent with the previous meta-analysis , we founWe found significant interaction for unweighted GRS but not for weighted GRS. Weighted GRS takes different effect sizes of each SNP into consideration and is widely used together with the unweighted GRS . We treaThere are several limitations and strengths in the present study. First, we are the first to report the interaction between ALA and GRS on T2D risk, and the results lack replication in other independent studies. Second, sample size of the present study is moderate. Third, GRS is created based on only nine SNPs with high minor allele frequency (>0.2), which reduces the statistical power and is a major limitation of the present study. Much more T2D-related SNPs with lower minor allele frequency need to be considered in future studies with larger sample size. The main strength of the present study included the objective measurement of erythrocyte phospholipid fatty acids. Erythrocyte phospholipid n-3 fatty acids are the most suitable nutritional biomarkers for long-term dietary n-3 fatty acids, compared with plasma/serum lipid fractions ,30.In conclusion, the present study found a significant interaction between erythrocyte phospholipid ALA and T2D GRS to modulate the risk of T2D. The interaction suggests that, in order to prevent T2D, it may be more efficient to increase dietary intake of ALA in participants with lower T2D genetic risk. However, given the solid evidence of the beneficial associations of higher dietary/blood ALA with lower T2D and CVD, participants with low ALA status are recommended to increase dietary ALA intake regardless of their genetic profiles.


HNF4A in liver cancers. Interestingly, three out of seven mutations were clustered in its Zn-finger DNA-binding domain; G79 and F83 are positioned in the DNA recognition helix and the sidechain of M125 is sticking into the core of domain. These mutations are likely to affect DNA interaction from a structural point of view. We then generated these mutants and performed in-vitro promoter assays as well as DNA binding assays. These three mutations reduced HNF4 transcriptional activity at promoter sites of HNF4A-target genes. Expectedly, this decrease in transcriptional activity was associated with a change in DNA binding. RNA-Seq analysis observed a strong correlation between HNF4A expression and expression of its target genes, ApoB and HNF1A, in liver cancers. Since knockdown of HNF4A caused a reduction in ApoB and HNF1A expression, possibly loss of HNF4 reduces the expression of these genes and subsequently tumor growth is triggered. Therefore, we propose that HNF4A mutations G79C, F83C, and M125I are functional mutations found in liver cancers and that loss of HNF4A function, through its mutation, leads to a reduction in HNF1A and ApoB gene expression with a concomitant increased risk of liver tumorigenesis.Hepatocyte nuclear factors (HNF) are transcription factors that crucially regulate cell-specific gene expression in many tissues, including the liver. Of these factors, HNF4A acts both as a master regulator of liver organogenesis and a tumor suppressor in the liver. In our whole genome sequencing analysis, we found seven somatic mutations  of  Chronic liver damage arising from excessive tobacco or alcohol consumption as well as hepatitis B and C viral infections often sets the stage for the oncogenic transformation of liver cells –4. NonetCNNTB1, TP53, ARID1A/ARID2, and TERT [HNF4A. HNF4A is a transcription factor which plays a major role in a variety of developmental events, especially in hepatic organogenesis [HNF4A mutations are linked to maturity-onset diabetes of the young (MODY)-1, which is an atypical form of type 2 diabetes (T2D) [HNF4A and T2D has been extensively studied [et al. and others have further identified HNF4A as potential tumor suppressor [HNF4A mutations in liver cancers, namely HNF4A G79C, F83C, and M125I, which are in the Zn-finger DNA binding domain of HNF4A.In our NGS analysis of virus-related liver cancers, the most significantly mutated genes were and TERT . In addiogenesis –13. HNF4ogenesis –20. HNF4ogenesis . Tissue-ogenesis . Moreoveogenesis . Interesogenesis –23. IDH-ogenesis . A recenogenesis , suggestes (T2D) , 25. The studied . Althougppressor –23, the ppressor . Thus, wHNF4A as a driver gene candidate [HNF4A , there are other HNF4A mutations reported, which are mostly located in the Zn-finger region or the ligand binding domain of HNF4A , which were detected in our report [HNF4A mutants were first compared with the wild-type protein for their ability to trans-activate HNF4A-responsive element containing promoters. These experiments were conducted in heterologous HEK 293 cells lacking endogenous HNF4A expression  and the ligand binding domain  of HNF4A, as well as one mutation (Q32X) in the N-terminal region of HNF4A , which lead to truncation of the ligand binding domain, also reduced their transcriptional activity Figure , suggestA Figure , suggestHNF1A and Apolipoprotein B (ApoB) promoters using EMSA analysis. Along with reduced transcriptional activity, HNF4A G79C, F83C, and M125I mutants show a markedly reduced binding to HNF1A and ApoB promoters compared to the wild-type HNF4A . Additionally, we and others have identified 11 mutations  in the HNF4A protein sequence. Ten of these mutations are located in either the Zn- finger region or the ligand binding domain , known as periportal-type (wild-type CTNNB1) and perivenous-type (mutant CTNNB1), have been identified. These subclasses of hepatocellular carcinoma are associated with negatively correlated gene networks . Interes2 and 37°C.HEK293 and HuH7 liver cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) that was supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 4,500 mg/liter glucose, 40 μg/ml streptomycin, and 40 units/ml penicillin. The cells were stored in a humidified incubator at 5% COThe cells were treated with 20 nM of either control or HNF4A siRNAs and cultured for 48 h in medium without antibiotics, according to the manufacturer's instruction. The sequences of the siRNAs and primers employed in the present study are listed in ® 480 System (Roche). GAPDH expression was utilized for normalization.Total RNA was extracted from cells with the RNAeasy mini kit (Qiagen), and subjected to cDNA synthesis with random primers and superscript II reverse transcriptase (Invitrogen), according to the manufacturer's protocol. RT-qPCR was performed using the KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) and a LightCycler® 7-Minute Blotting System (Invitorgen). Membranes were blocked with 5% skim milk and then incubated with the antibodies: anti-Flag , anti-α-tubulin , anti-HNF4A , anti-HNF1A , and anti-Lamin B1 . The blots were treated with a horseradish peroxidase-conjugated secondary antibody (Invitrogen) and were developed using an enhanced chemiluminescence (ECL) kit . The signal was detected by ImageQuant LAS 4000 mini .HuH7 cells were treated with different types of HNF4A siRNAs at a concentration of 20 nM for 48 h and subjected to preparation as whole-cell extracts with lysis buffer . Cellular debris was removed by centrifugation for 5 minutes at 13000 g and 4°C. Protein concentrations were determined using the Pierce BCA Protein Assay kit. Protein samples were loaded on a 10% SDS-polyacrylamide Wako Super Sep Ace precast gel (Wako), separated, and transferred to a PVDF Membrane using the iBlotCells were fixed by incubating them in 4% paraformaldehyde for 15 min at room temperature. After washing the cells with PBS 0.1% Tween-20 (PBST), the cells were blocked in 1% skim milk for 20 min at room temperature. Next, the cells were rinsed once with PBS 0.1% Tween-20 (PBST), treated with PBS 0.5% Tween-20 (PBST) for 10 min, incubated with mouse monoclonal FLAG-antibody followed by extensive washes and incubation with Alexa546-conjugated anti-mouse IgG antibody (Life Technologies) for 1 h. After being washed with PBST three more times, the cells were stained with DAPI and examined using a fluorescence microscope.HEK293 cells, expressing the genes indicated in the Figure Oligonucleotides synthesized by Sigma-Aldrich were used for DNA binding assays. Sequence information is provided in the HNF4A expression, the “high” expression group of HNF4A made up the 75th percentile and the “low” expression group encompassed the 25th percentile. Overall survival of informative 210 liver cancer patients was compared using the log-rank test.We analyzed RNA-seq of 210 liver cancer, which was a subset of previously published data candidate . Sequenct-test; a p-value < 0.05 was considered significant.Statistical analyses were performed using the Student's


Here, we show that DREAM also binds to E2F sites of S phase genes in quiescence and upon p53 activation. Furthermore, we describe a novel class of promoter sites, the CHR-like elements (CLE), which can support binding of DREAM to E2F elements. Activation of such S phase genes is achieved through binding of E2F1-3/DP complexes to E2F sites. In contrast, the activating MuvB complexes MMB and FOXM1-MuvB bind to CHR elements and mediate peak expression in G2/M. In conclusion, data presented here in combination with earlier results leads us to propose a model that explains how DREAM can repress early cell cycle genes through E2F or E2F/CLE sites and late genes through CHR or CDE/CHR elements. Also p53-dependent indirect transcriptional repression through the p53-p21-Cyclin/CDK-DREAM-E2F/CLE/CDE/CHR pathway requires DREAM binding to E2F or E2F/CLE sites in early cell cycle genes and binding of DREAM to CHR or CDE/CHR elements of late cell cycle genes. Specific timing of activation is achieved through binding of E2F1-3/DP to E2F sites and MMB or FOXM1-MuvB complexes to CHR elements.A central question in cell cycle control is how differential gene expression is regulated. Timing of expression is important for correct progression through the cell cycle. E2F, CDE, and CHR promoter sites have been linked to transcriptional repression in resting cells and activation during the cell cycle. Further, the DREAM complex binds CHR or CDE/CHR elements of G MuvB co mitosis , 4. We h mitosis –10. In t mitosis . The binin vitro , and E2Fin vitro , 13. CDEin vitro . More imin vitro . Thus, C0/G1 but also for activation in late cell cycle phases. This further underlines the significance of the CHR as the central element in cell cycle-dependent regulation of late cell cycle genes [2/M [The CHR is necessary not only for repression in Gle genes , 9. Durile genes . In S phnes [2/M , 16. Botnes [2/M , 9, 17.Having analyzed binding of DREAM, MMB, and FOXM1-MuvB to CHR and CDE sites of late cell cycle genes previously, we aimed for a detailed analysis of DREAM binding and cell cycle-dependent regulation of genes expressed with a maximum in S phase. It has long been recognized that cell cycle-dependent transcription of such genes is regulated through E2F binding sites via activating (E2F1-3) and repressing (E2F4-5) E2F factors together with the pocket proteins pRB, p130, and p107 , 19. p130 upon knockdown or knockout of DREAM components [2/M promoters, we reasoned that the complex may mainly bind to E2F sites of early cell cycle genes but that binding could be supported via interactions of LIN54 with CHR-like elements (CLE) located 4 bp downstream from E2F sites. To verify this hypothesis, we identified and analyzed candidate genes harboring different potential DREAM binding sites in their core promoters: TTK (CDE/CHR); INCENP (CHR); ORC1, Atad5, Gins1 (E2F); RAD51, CDC45, Mybl2 (E2F/CLE). The genes and their promoters were tested for cell cycle-dependent expression and protein binding. Furthermore, DREAM binding to promoters also appears to be central for p53-mediated transcriptional repression [E2F binding sites were found to be enriched in DREAM target genes that are mainly expressed in S phase , 8. Furtmponents , 24. Baspression , 25. Thepression . This papression . Here, wTaken together, we provide a comprehensive model for the control of cell cycle- and p53-dependent transcriptional regulation by mammalian MuvB-derived complexes.KIAA1731/CEP295. Several genes harbor elements that are related to functional CHR sites but differ in one or two nucleotides from the consensus. These genes include CDC6 (TGGGAA), CDC25A (TAGGAA), CDC45 (CTTGAC), MCM3 (TCGGAA), RAD51 (TCTGAA), and TCF19 (TGTGAA).We hypothesized that DREAM binds to the promoters of S phase genes through E2F binding sites and that additional CHR-like elements (CLE) support recruitment of the complex. To test this hypothesis, we screened DREAM target genes with an expression maximum in S phase containing phylogenetically conserved E2F elements in a region of +/−200 bp around the transcription start sites  for CLEsORC1 (Origin recognition complex subunit 1) harbors an E2F binding site  and CDC45 were tested as candidates for E2F/CLE tandem sites and Atad5 (ATPase family AAA domain-containing protein 5) as well as Gins1 as examples for single E2F elements (TTK and INCENP (inner centromere protein) as potential CDE/CHR and CHR genes, respectively, to analyze and compare promoter regulation and protein-DNA interactions with the potential E2F and E2F/CLE sites  or CDE (INCENP) sites did not deregulate the promoters  clearly contributes to binding of DREAM. In addition to the observation that DREAM components bind to the RAD51 probes with a higher affinity than to the ORC1 probe, mutation of the CLE site results in a reduction of DREAM binding. Importantly, binding is diminished to background level when the E2F site is mutated  for 60–72 h. For cell cycle analyses, NIH3T3 and HFF cells were stimulated to re-enter the cell cycle with 20% FCS after the serum-deprivation phase. HCT116 cells were treated with doxorubicin at a final concentration of 0.2 mg/ml for 48 h. The MDM-2 inhibitor Nutlin-3  was applied at a final concentration of 5 mM for 48 h. DMSO served as solvent control for Nutlin-3 treatment.NIH3T3, HFF, T98G, SaOS-2 cells from DSMZ  as well as wild-type and HCT116 /− cells  were culTTK, INCENP, RAD51, and CDC45 were amplified from genomic DNA extracted from human foreskin fibroblasts by standard PCR. Promoters of Mybl2, Gins1 and Atad5 were amplified from mouse DNA extracted from NIH3T3 cells. DNA fragments were cloned into the pGL4.10 luciferase reporter vector (Promega). Site directed mutagenesis was performed following the QuikChange protocol (Stratagene). Sequences of primers are provided in Promoters of DNA affinity purifications were performed as described earlier . ProteinChIPs and quantification of promoter fragments by semi-quantitative RT-PCR were performed as described , 9. SequSDS-PAGE and western blot were performed following standard protocols as described . The fol−/−p5 and SaOS-2 cells were plated in 24-well plates  and transfected by GeneJuice (EMD Millipore) with 100ng of promoter reporter plasmids (pGL4.10) along with 50ng of constructs expressing wt or mutant p53.Cell cycle-dependent promoter activities were analyzed by luciferase reporter assays with extracts of transfected serum-starved and re-stimulated NIH3T3 cells as described before . For meaThe DNA content of serum starved and re-stimulated HFF and NIH3T3 cells was analyzed by staining with propidium iodide (PI) followed by flow cytometry as described earlier .1/S bound by DREAM were identified as described earlier [Evolutionary conserved E2F binding sites in core promoters of cell cycle genes with peak expression in S phase or G earlier . Potenti


Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveillance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools) were 5.8% (CI 5.4–6.0). Subtyping was performed on 468 pooled swabs with M gene Ct < 26. No influenza H7 was detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interaction between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11 partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 distinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Vietnam are warranted to assess H9 endemicity elsewhere in the country. •healthy chickens in Live Bird Markets of Vietnam. Because all breeds of domestic chickens are extremely susceptible to HPAI, we speculate that HPAI detections from market chickens may reflect infections that occur after arrival in the market. Alternatively, shedding of HPAI from healthy birds may reflect vaccine-induced protective immunity that mitigates disease but does not block viral infection.We report detection of highly pathogenic avian influenza (HPAI) from •As many as 49% of all pooled surveillance swabs were positive for influenza A virus, corresponding to an overall Influenza A prevalence of 5.45% .•Low pathogenicity avian influenza (LPAI) H9N2 accounted for the vast majority of all influenza A detections in market chickens sampled from 9 northern provinces.•To date there is no evidence to suggest an interaction effect between circulation of H5 and H9 viruses; however sampling strategies that involve pooling of surveillance swabs from multiple birds greatly complicates the assessment of co-infection rates or evaluation of epidemiological associations. From 2010 to 2015, surveillance focused primarily on monitoring genetic diversity and ongoing evolution of HPAI H5 subtype viruses within duck populations . StartinLow pathogenicity avian influenza (LPAI) H9N2 is the most prevalent influenza subtype circulating endemically in chickens worldwide. H9N2 viruses are endemic in poultry across Asia, the Middle East and Northern Africa , and desResearch and surveillance activities in Vietnam from 2003 to 2013 previously identified a number of LPAI subtypes circulating in duck populations , includi22.1TM Nucleic Acid Automatic Extraction System . Detection of influenza A and subtype-specific monoplex real time RTPCR were performed using standardized protocols (Supplementary T2). Allantoic fluid from inoculated embryonated chicken eggs were clarified by centrifugation for 10 min at 4 °C, and presence of virus was determined by hemagglutination assay as previously described  swabs of 35 chickens were sampled individually from 7 different vendors per market, and aggregated into pools (5 swabs per pool). The pooled OP swabs were stored in 2 mL viral transport medium , transported by cold chain to the National Center for Veterinary Diagnostics (NCVD) and stored at -80 °C prior to further processing. RNA of pooled swab samples were extracted using a tacoescribed .2.2de novo assembly. Genomic sequences and variant frequencies were generated in CLC genomics workbench v. 8.5.1 (http://www.clcbio.com/).We used NGS protocols for influenza sequencing adopted from St Jude's Center for Excellence in influenza Research & Surveillance (SJCEIRS). Briefly, fresh nucleic acid extracts were prepared and samples were pre-amplified to enrich for influenza genomes using MBTUni12/13 primers as previously described . DNA lib2.3www.megasoftware.net). Alignments were trimmed to yield the maximal sequence coverage per segment as follows: 260-2289 for PB2; 78-2242 for PB1; 232-1981 for PA; 160-1599 for HA; 49-1507 for NP; 24-1386 for NA; 26-996 for MP; and 56-859 for NS. Phylogenetic trees were created using the neighbor-joining algorithm and the Tajima-Nei model, with 1000 bootstrap replications. Bootstrap values of < 70 were excluded from trees. Genotyping was performed using 2% nucleotide difference cutoff to indicate genetically divergent segments.Data used for phylogenetic analyses comprised the novel whole genome sequences generated by this study, as well as additional publicly available sequences of closely related viruses identified by Blastn analysis of NCBI and GISAID databases. Full datasets for each gene segment were aligned using the MUSCLE algorithm in Mega software version 6.06 .33.1A total of 1207 of 2450 pooled swabs (49%) screened positive for influenza A by matrix gene real time RT-PCR. Using a maximum likelihood modeling approach that accounts for pooling (Suppl Appendix A), this detection level corresponded to an overall influenza A prevalence of 5.45% . 3.2We utilized next generation sequencing using the Illumina Nextera XT Sample Preparation Kit and the MiSeq platform as previously described  using boEach of the 17 whole genomes of H9N2 possessed the G57-like internal gene cassette, similar to zoonotic H7N9 and H10N8 viruses in mainland China. For example, the genes most closely related to the PB2 and PB1 segments of H7F-14-BN4-437 belonged to the human H10N8 and H7N9 isolates, A/Jiangxi/IPB13/2013(H10N8) and A/Guangdong/04/2013(H7N9) with percentage nucleotide homologies of 99.6% and 99.5% respectively. Additionally H7F-14-CB4-2, H7F-14-CB4-29, H7F-14-CB4-31 and H7F-14-CB4-34 possessed PB1 and NS genes closely related to human and avian H7N9 isolates from 2013 with homologies of over 99.9%, and their PA, NP and MP genes were related to H10N8 viruses with homologies > 99.3%. Using an > 98% nucleotide difference cutoff for each segment, we determined patterns of clustering at the sub-genotype level, and identified 9 distinct sub-genotypes, all of which appeared to be reassortants between closely related H9N2 viruses . For re.Of the 18 surveillance swabs initially identified by RT-PCR as containing both H5 and H9, only 8 were subjected to MiSeq , and 1 yielded sequence results indicating presence of both subtypes (H7F-CB4-35). Additionally, there was one sample that tested positive for H5 but not H9 on initial RTPCR screening (H7F-HG4-604), however it yielded H9HA, H5HA, H9-like PB2, PA, NP, NA, MP and NS genes, and an H5-like PB1. The status of these samples as intersubtypic reassortants cannot be established as they represented pooled swabs from multiple birds. Of the samples designated as ‘influenza positive, unknown subtype’ by RT-PCR, 3 of 9 samples sequenced by MiSeq yielded partial sequence data of H9N2, and the remaining did not yield sequences of sufficient coverage for analysis.3.33.3.1The 19 H9 sequences generated retained the classical BJ94/Y280-like ‘low-pathogenic’ dibasic RSSR cleavage motif that has previously shown to allow cleavage by epithelial proteases such as matriptase and is thought to facilitate systemic infections in poultry . The rec3.3.2The N2 genes of the Vietnamese viruses fell into two distinct clades  within t3.3.3Similar to divergence of NA, segment 8 also segregated phylogenetically into two groups within the G57-like lineage, with a major group represented by H7F-14-CB4-2 and a minor group represented by H7F-14-BN4-315 . Interes3.3.4In addition to the 10 well characterized and almost universally expressed core proteins of influenza, some virus strains also code for accessory proteins such as PB1-F2 and PA-X, which are thought to be important in modulating virulence . All seq3.3.5The polymerase subunits of each of the H9N2 isolates, PB2, PB1, PA, and NP, displayed multiple mammalian adaptation markers  Table S. However3.3.6The M1 and M2 proteins, encoded as alternative splice products of segment 7 of the influenza genome, similarly to the other segments, contained a number of known markers of mammalian adaptation  Table S.4Our results confirm the hyperendemicity of avian influenza viruses in chickens sampled in LBM of northern Vietnam, where influenza H9N2 viruses are by far the dominant subtype in circulation. Our detection rates are comparable to those reported from China, where overall prevalence of AIV in chickens has ranged from 21 to 36% in Hubei, 34–46% in Zhejiang province , and 15.intersubtypic reassortment between H5 and H9N2 viruses, the genomic analysis suggested high levels of intrasubtypic reassortment between different subclades of H9N2  complicates the inference of co-infection rates (and definitely decrease its power), and complicates analysis of intersubtypic reassortment. Among the deep sequencing data generated directly from surveillance swabs, although we could not assess  of H9N2 .in situ evolution of H9N2 viruses within Vietnam.The genomic data demonstrated that most gene segments were closely related to Chinese viruses, which likely reflects proximity of our sampling sites to the Chinese-Vietnam border. Importation of live chickens from China has been illegal since May 2013; hence, these findings suggest that despite regulations to curtail illegal traffic in live poultry, there remains potential for cross-border spread. The pattern of phylogenetic clustering of the H9N2 sequenced here revealed two distinct clusters for each gene segment , suggesting there may have been separate introduction events. However, H9N2 viruses are known to be highly transmissible between chickens and wild terrestrial birds (e.g. sparrows) , and thuGiven the evidence that prevalence and diversity of AIV in neighboring China continues to increase at alarming rates , and given that Vietnam ranks as one of the countries with highest clade diversity for HPAI H5 , the pubRegarding the human health implications of LPAI H9N2 circulation, previous studies of human seroprevalence conducted in northern Vietnam in 2001 documented HI titers to H9 and presence of H9 neutralizing antibodies in 3–4% of people tested, but with no significant differences between the general population and those with occupational exposures to poultry . We specIn conclusion, our findings suggest that circulation of H9N2 in Vietnamese chickens is sufficiently widespread to be influencing the overall ecology and emergence potential of AIV, and we emphasize the need for further research to better understand possible interactions between co-circulating LPAI and HPAI subtypes. We fully support the call for continued vigilance in monitoring ongoing AIV evolution.No competing financial interests exist.


This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them  was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences  Tumor suppressor p53 contains two DNA binding domains. The central (core) domain (amino acids ~100 to ~300) is evolutionarily highly conserved and is essential for p53 sequence-specific binding to promoters of p53 target genes that take part in cell cycle regulation, apoptosis and DNA repair . The corT and C.G.C in the pyrimidine motif and C.G.G and T.A.A in the purine motif. Triplexes can be either intermolecular, where the third strand originates from a separate DNA molecule, or intramolecular , where the third strand originates from the same DNA molecule as its duplex acceptor [in vivo under suitable conditions  and their involvement has been implicated in several cellular processes, including transcription, replication and recombination [The triple-helical (triplex) DNA adopts a structure characterized by a third pyrimidine-rich or purine-rich DNA strand located within the major groove of a homopurine/homopyrimidine stretch of duplex DNA –16. Stabacceptor , 16. Natacceptor  and are acceptor , 19. Intbination , 16. Thebination . Intermobination , 22. So bination –26. The bination . Despitebination .in vivo [Negative DNA superhelicity is necessary for the formation of intramolecular triplex DNA (H-DNA) and other non-B DNA structures in vivo . Observain vivo , 29, 30,in vivo , 31 havein vivo , 30–33.in vitro and in cells. We show that p53 protein possessing intact C-terminus exhibits high affinity to intermolecular and intramolecular T.A.T triplex DNA. In-silico analysis of human promoters for simultaneous presence of consensus sequence and T.A.T motifs identified a set of candidate p53 target genes. Possible contribution of DNA triplex-dependent binding of p53 for regulation of their transcription is discussed.In this study, we have analyzed for the first time the interaction of the full-length p53 and its deletion variants to DNA containing triplex-forming sequences T triplex (oligo(dT)50.oligo(dA)50.oligo(dT)50) was formed by standard annealing of (dT)50 to labeled (dA)50 and titration of duplex with (dT)50 to molar excess (3–5×) in presence of Mg2+ ions in triplex forming buffer  at 37°C for 60 min. CTGhairpin and TAhairpin were prepared as described in [The sequences of oligonucleotides used in this study are presented in ribed in  with labT triplex forming sequences (pBA50 and pPA50) were prepared by cloning of (dT)50.(dA)50 into the EcoRV site of pBluescript SK II-  and pPGM1 [34 sequences to the same plasmids, for details see 69.(dA)69 (on pUC19 basis [SmaI) was prepared by SmaI restriction enzyme  cleavage of pBSK. Plasmids for luciferase reporter assay . All plasmids were isolated from bacterial strain TOP10 (Stratagene) and verified by sequencing.Plasmids encoding human p53 proteins pT7-7wtp53 , pET-p53CD , pGEX-2TKp53CT , pGEX-2TKp53T  and pGEX-4Tp53CD  were described in , 29. Pland pPGM1  (S1 Tabl19 basis ) and pUCFull length p53 and isolated DNA binding domains p53CD, p53CT, and p53T (with or without GST tag) were purified according to a protocol described previously , 29. The32P-radiolabeled oligonucleotide probes (1 pmol) were mixed with p53 proteins and incubated in binding buffer  in the presence of 5–50 ng pBSK/SmaI competitor DNA for 30 min on ice or at 25°C to reach equilibrium. Samples were loaded onto a 4–5% polyacrylamide gel containing 0.5× TB buffer with 2 mM MgCl2. After 1–3 h electrophoresis (at 4–6 V/cm2) the gels were dried and DNA was detected by autoradiography using Typhoon FLA 9000 . Polyclonal rabbit CM1 and mouse monoclonal (DO1 (aa 20–25), Bp53-10.1 (aa 375–379), PAb421 (aa 371–380) and ICA9 (aa 388–393)) antibodies, kindly provided by Dr. B. Vojtesek, were used in supershift and IP experiments.2 in 1× PBS. Kd were obtained from at least three independent measurements. Details of the procedures are described in [96-well Immuno Plates (SPL LIFE SCIENCES) were streptavidin (PROSPEC) coated and blocked for unspecific binding by BSA (Sigma). Biotinylated oligonucleotides (0.5 pmol) were bound to the plate and then pre-incubated protein-primary antibody mixes (in 2/1 Ab/protein molar ratio) were added. Secondary HRP-labeled antibody was incubated on ELISA plate for 30 min, washed and then TMB substrate was added. Absorbance was measured at 370 nm on Synergy H1 (BioTek) and evaluated in GraphPad Prism using hyperbolic or Hill equation fittings. All wash and incubation steps were done in the presence of 2 mM MgClribed in .2, 0.01% Triton X-100 and 50 mM KCl) for 30 min either on ice or 25°C to reach equilibrium. Samples were loaded onto a 1% agarose gel containing 0.33× Tris-borate-EDTA (TBE) buffer. After 5 h electrophoresis (at 4–6 V/cm2) agarose gels were stained with ethidium bromide (EtBr) and photographed. Intensities of bands of free DNA substrates were quantified using ImageQuant software. Graphs show the evaluation of p53-DNA binding as the dependence of % of bound DNA on the amount of p53 proteins (expressed by molar ratio p53/DNA), more details in [scDNAs  were preincubated in triplex-forming buffer at 37°C for 30 min. scDNAs were mixed with p53 proteins in p53 tetramer/DNA molar ratios 0.25–5 and incubated in binding buffer  were mixed with the given complexes and incubated in the binding buffer for 30 min on ice. Magnetic beads (12 μl of suspension per sample) coated with protein G  were added to DO1-p53-DNA complexes after washing in binding buffer and incubated with the beads for 30 min at 10°C. Finally, after washing in binding buffer with increased salt concentration , DNA was released from the beads by heating at 65°C in 15 μl of 1.0% SDS for 5 min and analyzed by agarose gel electrophoresis, more details in [The DO1-p53-DNA complexes were prepared by mixing the DO1 antibody (400 ng) with the purified protein (50 ng) in binding buffer followed by 20 min incubation on ice. Then, 200 ng of scDNA (preincubated in triplex-forming buffer) and the same amount of linDNA (pBSK/tails in . Intensi2. The luciferase reporter constructs (50 or (dA)20 sequences were used for luciferase assay as described in [Human breast adenocarcinoma MCF7 , human non-small cell lung carcinoma line H1299  and H1299-wtp53 cells  were grnstructs  containi-ΔΔCT algorithm. All reactions were carried out in biological triplicates. The primer sequences used are listed in Total RNA was isolated using NucleoSpin RNA II (Macherey-Nagel) and 2 μg of RNA was subsequently reverse transcribed into cDNA by applying High Capacity RT kit (Applied Biosystems). qPCR was performed using EvaGreen (Solis Biodyne) fluorescent dye in the standard program  running in RotorGene 6000 (Corbett Research). RT-qPCR reactions for each sample were measured in triplicates. GAPDH was used as reference gene. Absolute quantification was performed using standard curve method. Data were then normalized to GAPDH. The housekeeping genes  were used as endogenous controls. Relative quantification of transcript levels with respect to the calibrator  was done based on 2H1299 and Hwtp53  cells were harvested from 10 cm plates and lysed with 1× PLB (Promega), followed by the sonication of cells (Bandelin Sonopuls). Samples  were analyzed on 12.5% SDS-PAGE gels and proteins were detected by the following primary antibodies: DO1 , anti-CDKN1A (Millipore), anti-β-Actin (Sigma), anti-BAX (Sigma), anti-NAT10 (ThermoScientific).Human breast adenocarcinoma MCF7 treated for 4 hours with nutlin-3 (5 μM) or doxorubicine (1 μM) were subjected to chromatin immunoprecipitation (ChIP) assays as previously described  with theHuman regulatory sequences were obtained using Table Browser  and saveCandidate gene transcription was checked in publicly available microarray and sequencing datasets from experiments involving p53-transformed cells originally lacking active p53 or experiments were p53 was activated by nutlin-3, 5-fluoruracil or doxorubicin . We obt2, 5 mM KCl buffer and placed on freshly cleaved mica V4 surface, incubated for 2 min, washed with distilled water and dried with a stream of compressed air.AFM measurements were carried out on MultiMode 8 system (Bruker) with NanoScope 8.15 software or on a custom-built AFM system . 50 A siT triplex was formed in neutral pH in the presence of Mg2+ ions [T triplex was examined by EMSA in the presence of Mg2+ ions. Increasing amounts of p53 . We observed small differences in p53 binding to T.A.T triplex  with a set of biotinylated target oligonucleotides CON, TAT and CTGhairpin as recently described for p53-quadruplex DNA binding [T triplex with higher affinity (Kd = 0.75 ± 0.07 nM), in comparison with CTGhairpin (Kd = 1.88 ± 0.13 nM) (To better characterize the differences in p53 binding to T.A. binding . Incubat0.13 nM) . But as T triplex recognition we analyzed the interaction of isolated p53 core domain . We observed that binding of both p53CT and p53T to T.A.T triplex DNA was stronger than to the used dsDNA or ssDNA substrates. To better characterize differences in affinities of isolated DNA binding domains to T.A.T triplex, we used ELISA with all p53 constructs . p53T construct with CTDBD and lacking the tetramerization domain recognized TAT with lower affinity (Kd = 10.44 ± 0.84 nM) than p53CT which is still better than for dsDNA or ssDNA  formation in the presence of Mg2+ ions in supercoiled plasmids containing homoadenine-homothymine blocks has been described for several vectors [50.(dT)50) and AT-rich cruciform-forming sequence d(AT)34 . Increasing salt concentration 69 is depicted in Furthermore, we applied a competition immunoprecipitation assay and compared binding of p53 to scDNA with and without TFS and CON in the presence of competitor DNA  was a comparable competitor to all plasmids with CON . The control vector pBSK together with pBAT34  were the worst competitors.To probe differences in relative p53 binding affinity to scDNA with/without TFS and CON we used a competition assay proposed previously . BindingT triplex-forming sequences for transcription regulation we carried out a series of in-silico investigations. Within the context of p53 transcription factor functions involving CON recognition, we looked for T.A.T triplex-forming and CON sequence co-occurrence in the human genome to predict new class of p53 target genes. We analyzed the -5000/+2000 bp neighborhoods of 42106 RefSeq gene transcripts (promoters). Of these, 19373 promoters were found to contain at least one CON sequence when 1 mismatch was allowed. T.A.T triplex-forming sequences with a prevailing poly(A) or poly(T) run with score> = 18 were found in 376 sequences. Because of the asymmetry in occurrence of these two patterns we decided to screen the promoters primarily on the predicted length of the T.A.T triplex. There were 43 promoters of candidate p53 target genes with at least one CON and a T.A.T triplex with a poly(A/T) run longer than 40 bp. in-silico analysis shows that most CONs are downstream of the triplex 50, (dA)20 too short for triplex formation and p53 specific sequence CON ence CON . Luciference CON  with linence CON . Only suence CON . For linence CON . In the ence CON . In summin vivo p53 binding to (dA)50 sequence capable to form H-DNA, supercoiled plasmids B50  and P1  were transfected to H1299 cells together with effector plasmid pCDNA3.1p53 and a ChIP assay was performed with p53 specific antibody CM1 in-silico screening  or down-regulation . Interestingly, NAT10 and STAG2 belongs to several genes down-regulated after activation of p53 by drug treatment and up-regulated in p53 overexpression in p53 null cells. Several up or down regulated genes  were limited to the STRING-db-supported functionally associated group of genes. Intact C-terminus was necessary for strong p53-dependent activation of MCC, one of the best candidate p53 target gene, in contrast to well-known target gene MDM2 50.(dT)50, especially in promoters containing this sequence in close proximity to specific p53 binding sites (CONs).Regions with the potential to form triplex DNA are generally over-represented in the promoter regions and introns of genes involved in cell signaling as indicated by genome-wide bioinformatics analyses , 19, 62.in vitro and in vivo, CTG and CAG hairpins were determined as p53 bound non-B DNA structures in that repetitive sequence [A number of independent studies have established that p53 recognizes non-B DNA structures including hairpins, stem-loops, cruciforms, mismatches, bulges, G-quadruplexes, three- and four-way junctions , 63–66. sequence . To bestsequence .T type of triplex was chosen with respect to physiological conditions necessary for triplex formation [T triplex than to the CTGhairpin  are necessary for high affinity p53 binding to the T.A.T triplex.In the present study, a range of biophysical approaches was used to analyze the interaction of full-length and isolated DNA binding domains of p53 with intermolecular triplex DNA. The T.A.ormation , 47 and ormation . Both EMGhairpin . Bindingin vivo and in vitro study with p53 proteins mutated in CTDBD (mimicking acetylation/phosphorylation) points to several positive roles of intact unmodified CTDBD in regulating sequence specific DNA binding, p53 protein stability, p53 cellular localization and co-factor recruitment [Although binding of DNA by the C-terminus is usually marked as non-specific, CTDBD has a major role in non-B DNA structures recognition ruitment . Recentlruitment .There is no systematic study to date of the role of DNA binding domains in different non-B DNA structures recognition. CTDBD is necessary for recognition of DNA cruciform and stem-loop structures both formed by CON sequences , 31, as 50.(dT)50 sequence. We verified H-DNA formation under superhelical stress under conditions used for p53 binding using several techniques and visualized them by AFM. scDNA pBA50 was somewhat more weakly bound by p53 than scDNA with CON (pPGM1). In competition assay, pBA50 and pPA50 capable of H-DNA formation were better competitors than pBAT34 forming AT-rich cruciform and comparable in competition to plasmids with CON with CON . Supercowith CON , p53 alswith CON  already T triplex as a novel p53 binding site recognized by CTDBD raises the question of the physiological significance of such interaction. The nM binding/dissociation constant that we observed for p53 binding to intermolecular T.A.T triplex 50.(dT)50 tracts may act as a non-B DNA p53 binding site essential for p53 stability, co-factor recruitment and regulating sequence-specific binding mainly in the case of unmodified C-terminus by phosphorylation and acetylation. Binding of p53 to a significant number of sites within the genome depends on the availability of unmodified CTDBD according to a recent report [T triplex-forming sequence (dA)50.(dT)50 on the stability of the p53-scDNA complex and p53 binding to (dA)50.(dT)50 in scDNA in cells. For this reason, we hypothesize that p53 interaction with T.A.T triplex, primarily by CTDBD, can stabilize p53 protein in both non-B DNA and CON. Additionally, we can discuss the role of the p53-T.A.T triplex recognition in the process of DNA repair. It was shown that triplex-forming oligonucleotides are able to activate DNA recombination and DNA repair in addition to inducing genomic instability [T triplex recognition can eliminate DNA damage caused by triplex formation.We speculate that the T.A.t report . The C-tt report . p53 bint report . Recentlt report . Walter t report , 30, 33.tability . Intact tability , 78, 79.tability . The DNAtability , 81. So Interestingly, the group of proteins specifically recognizing triplex DNA  are alsT triplex-forming sequences (dA)50.(dT)50 in front of CON, enhanced promoter activation by p53. Interestingly, the reporter vector containing only T.A.T triplex-forming sequence (dA)50.(dT)50 was repressed by p53 protein. Both these effects suggested that T.A.T triplex-forming sequences have the potential to influence transcription in both directions. We assume that positioning of p53 on promoter region facilitates p53 recognition and transcription of genes.Using luciferase reporter assay in two different cell systems, we demonstrate that T.A.in-silico analysis with STRING showed that a fraction of promoters containing both CON and a potential T.A.T triplex-forming sequence belong to the functional and structural association network of p53. Although p53 has a large association network, repeated experiments with randomly chosen UniProt Ids have shown that the majority of blind tests had networks with less than 10 interactions while we observed 14, before adding the additional 10 best connected proteins. A medium strength enrichment  was obtained from gProfiler for the most enriched Gene Ontology term: “regulation of signal transduction”. Consequently, the in-silico experiments did not yield results that would have the power of proof for us. Rather, they should be viewed as a tool to narrow down possible candidates for further studies, such as the RT-qPCR experiments carried out here. Several candidate genes from the narrowed-down list that have been tested by RT-qPCR show increased expression in p53 dependent manner in p53 null cell line. The best candidates are ABCG5, ENOX1, INSR, MCC, NAT10, NFAT5 and MAPK9  Full length p53 was bound to 1 pmol of 32P-labeled 50-mer oligonucleotides represented by p53 nonspecific dsDNA , p53 specific dsDNA with CON  and triplex (dT)50.(dA)50.(dT)50  in the presence of DNA competitor . The reactions were separated on 4% 0.5× TBM (2 mM MgCl2) polyacrylamide gel (PAGE), 3h. Radioactively labeled DNA was detected by autoradiography. B,C) Full length p53 was bound to 1 pmol of 32P-labeled 50-mer oligonucleotides represented by p53 specific dsDNA with CON  and triplex (dT)50.(dA)50.(dT)50  in the presence of DNA competitor . The reactions were separated on 5% 0.5× TBM (2 mM MgCl2) PAGE, 1 h. Radiolabeled DNA was detected by autoradiography.(A) SDS-PAGE analysis of p53 proteins used in the study. The purity and appropriate size of each proteins was analyzed by Coomassie blue staining of 12.5% SDS-PAGE gel. (TIFF)Click here for additional data file.S2 Fig(A) Scheme of p53 used in this study, shown as boxes below the map of p53 domains. The evolutionarily conserved domains are indicated: core DNA binding domain , tetramerization domain  and basic C-terminal DNA binding domain  and location of p53 antibodies PA421, ICA9 and DO1 used in our study. (B) Effect of C-terminal modifications of p53 protein by Ab on T.A.T recognition. The antibodies  were bound to p53 (300 ng) in Ab/p53 molar ratio 2/1 at RT for 15 min. Then 1 pmol of 32P-labeled 50-mer oligonucleotides represented by p53 specific dsDNA with p53CON  and triplex (dT)50.(dA)50.(dT)50  were added and mixtures were incubated at 4°C for 20 min. The reactions were separated on 4% 0.5× TBM (2 mM MgCl2) PAGE at 4°C. Radioactively labeled DNA was detected by autoradiography. Mouse monoclonal anti-p53 antibodies (mAb) (DO1 (aa 20–25), Bp53 10.1 (aa 375–379), PAb421 (aa 371–380) and ICA9 (aa 388–393)) and anti-GST Ab  were used.(TIFF)Click here for additional data file.S3 FigScaI digestion. Detection of two fragments indicates one major non-B DNA structure (cruciform or triplex) formation in the polycloning site in the case of pBA50 (A), pPGM2 , pBAT34 , and pAT34 . But also pPGM1 , pBA50 , pPA50  and pBSK  were sensitive to S1 nuclease treatment; two pairs of fragments (black lines) were detected, indicating that all plasmids can form non-B DNA structures with unpaired bases. (C) AFM visualization of intramolecular triplex in pBA50, conditions as described in 4-bipy by dot blot on nitrocellulose membrane with specific antibody against OsO4-bipy-DNA adduct as described in [2 without/with 100 mM NaCl by OsO4-bipy modification followed by primer extension analysis of pBSK , PGM1 , PGM2 , pBA50  plasmid DNA, conditions described in [ Scheme of non-B DNA structures detection by S1 nuclease treatment described in . scDNAs ribed in . pUC19  Detribed in . Primer (TIFF)Click here for additional data file.S4 Fig69(dT)69  in p53/DNA molar ratios 1–5 at 25°C, EMSA was performed at 4°C.Binding of p53 protein to pUC19 and pA69 detected by EMSA in agarose gel. p53 protein was bound to scDNA  and scDNA with (dA)(TIFF)Click here for additional data file.S5 Fig(A) AFM image of scBA50 plasmid mounted in the presence of 5 mM MgCl2. Scale bar represents 200 nm. (B) Image of pA69 complexes with p53, proteins were incubated with DNA in molar ratio 5/1 in DNA binding buffer and then loaded on mica surface in the presence of 5 mM MgCl2. Scale bar represents 500 nm. (C) pA69 plasmid with p53 proteins in 3D projection.(TIFF)Click here for additional data file.S6 Figin-silico study (red and blue), together with p53 (yellow) and 10 most-related proteins (grey) from STRING-db, organized into a network by common properties and interactions. The 16 proteins from our study that are also part of well-connected networks are shown in blue. See The 43 highest-scoring proteins of candidate p53 target genes found in the (TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TablePositions of lower stringency p53CON sequences with 2 mismatches are shown in parentheses. Genome coordinates refer to human genome sequence hg38 annotation.(XLSX)Click here for additional data file.S3 TableIn-silico candidate gene screening of publicly available microarray and sequencing datasets and summarization of results of verification by RT-qPCR. See (XLSX)Click here for additional data file.