Mycobacterium mageritense strain DSM 44476T (CIP 104973), a nontuberculosis species responsible for various infections. The genome described here is composed of 7,966,608 bp, with a G+C content of 66.95%, and contains 7,675 protein-coding genes and 120 predicted RNA genes.We report the draft genome sequence of  Mycobacterium mageritense has been initially reported as a new species of the Mycobacterium fortuitum complex  Middlebrook broth at 37° C. Genomic DNA of M. mageritense was sequenced on the MiSeq Technology  with the two applications: paired end and mate pair, in a 2- × 250-bp run for each bar-coded library. On each flowcell, the index representation for M. mageritense was determined to 5.09 and 7.11%, respectively. The global 1,572,948 reads were filtered according to the read qualities.Genomic DNA was isolated from the M. mageritense consists of six contigs, containing 7,966,608 bp and a G+C content of 66.95%.Illumina reads were trimmed using Trimmomatic , then asNoncoding genes and miscellaneous features were predicted using RNAmmer , ARAGORNMycobacterium mageritense strain DSM 44476T genome sequence has been deposited at EMBL under the accession numbers CCBF010000001 through CCBF010000006.The


Neuronal oscillations support cognitive processing. Modern views suggest that neuronal oscillations do not only reflect coordinated activity in spatially distributed networks, but also that there is interaction between the oscillations at different frequencies. For example, invasive recordings in animals and humans have found that the amplitude of fast oscillations (>40 Hz) occur non-uniformly within the phase of slower oscillations, forming the so-called cross-frequency coupling (CFC). However, the CFC patterns might be influenced by features in the signal that do not relate to underlying physiological interactions. For example, CFC estimates may be sensitive to spectral correlations due to non-sinusoidal properties of the alpha band wave morphology. To investigate this issue, we performed CFC analysis using experimental and synthetic data. The former consisted in a double-blind magnetoencephalography pharmacological study in which participants received either placebo, 0.5 or 1.5 mg of lorazepam  in different experimental sessions. By recording oscillatory brain activity with during rest and working memory (WM), we were able to demonstrate that posterior alpha (8–12 Hz) phase was coupled to beta-low gamma band (20–45 Hz) amplitude envelope during all sessions. Importantly, bicoherence values around the harmonics of the alpha frequency were similar both in magnitude and topographic distribution to the cross-frequency coherence (CFCoh) values observed in the alpha-phase to beta-low gamma coupling. In addition, despite the large CFCoh we found no significant cross-frequency directionality (CFD). Critically, simulations demonstrated that a sizable part of our empirical CFCoh between alpha and beta-low gamma coupling and the lack of CFD could be explained by two-three harmonics aligned in zero phase-lag produced by the physiologically characteristic alpha asymmetry in the amplitude of the peaks relative to the troughs. Furthermore, we showed that periodic signals whose waveform deviate from pure sine waves produce non-zero CFCoh with predictable CFD. Our results reveal the important role of the non-sinusoidal wave morphology on state of the art CFC metrics and we recommend caution with strong physiological interpretations of CFC and suggest basic data quality checks to enhance the mechanistic understanding of CFC. Traditionally brain rhythms have been divided within different frequency bands: delta (1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta (15–30 Hz), and gamma (>30 Hz). These bands have been linked to a variety of normal and pathological behavioral phenotypes  of different dosages (0.5 and 1.5 mg) in separate sessions. Pharmacological power modulations during WM have been published elsewhere  recordings during resting state and WM. We performed a double-blind randomized cross-over design pharmacological challenge where healthy participants received either placebo or lorazepam  gave informed consent approved by the local research ethics committee  and were compensated for their participation. They were engaged in a classic delayed-match to sample visuo-spatial WM task . We used the real-time head localizer tool ; an open-source toolbox developed at the Donders Institute for Brain, Cognition, and Behaviour  and custom Matlab code .MEG data was low-pass filtered below 200 Hz and down-sampled to 600 Hz. Subsequently we analyzed our dataset using Fieldtrip . After partial artifact rejection, data segments shorter or equal to 2 s were excluded leaving the rest of the cleaned data for the main analysis. Remaining eye movements and electrocardiogram (ECG) activity were isolated by independent component analysis (ICA). The independent components (ICs) reflecting potential artifacts were semi-automatically identified by correlating its time-course with the respective vertical EOG and ECG bipolar derivations. Given the stable periodicity of the QRS complex sharp edges, spurious CFC could arise  using a fixed window length of 4 s (4–40 Hz in 0.25 Hz steps), 50% overlap, multiplied by a Hanning taper. Prior to spectral analysis, the mean of each epoch was subtracted. Spectra were normalized based on the total power in each sensor within each participant and MEG session before group averaging.x1, x2, …, xN where N is the number of time points acquired at specific sampling rate (Fs). First, the time-course of amplitude v by applying a sliding tapered time-window followed by a Fourier transform:To investigate cross-frequency interactions we chose the CFCoh index . Note that the time-series x can be zero-padded to reduce boundary effects at the beginning and end of the segments. In the present analysis, low frequencies were defined between 5 and 25 Hz and higher frequencies from 5 to 90 Hz in steps of 1 Hz.The function x and y. Data was segmented into a set of S segments 0.6 s long with 50% overlap. Using a standard FFT algorithm, the complex representations were calculated:Next we proceeded to estimate the Fourier spectra of andxs representing the temporal evolution of raw signal of segments s, yv, s representing the amplitude envelope at high frequency v and segment s and nFFT representing the frequency resolution of the x-axis Δf = Fs/nFFT of the CFCoh comodulogram (nFFT = 1024). The output vector ξ of the individual segments was defined as ξv, s = Xs* where “*” denoted the complex conjugate.with 2η):To quantify the CFC, we used the magnitude squared CFCoh :To assess the directionality of CFCoh, we employed the new CFD metric . /std). The authors considered absolute values larger than 2 as significant  behave randomly across S segments and it is close to 1 when the phase of the numerator (bispectrum) remains constant over segments. As for CFCoh, we epoched the data using fixed window length of 0.6 s, 50% overlapping, multiplied by a Hanning taper.The bicoherence is close to 0 when the coherence of Fourier frequency triplets  locked to the alpha peak .To localize the LZP-related oscillatory power modulations during EC and EO epochs, we adopted a beamformer approach as defined in Gross et al. . Alpha bPreprocessing, artifact rejection, and sensor spectral analysis procedures were done as in Lozano-Soldevilla et al. . BrieflyTo statistically assess the drug main effect on oscillatory power, CFCoh, bicoherence, or peak-triggered averages at the sensors space, a cluster-based nonparametric permutation test was applied . In other words, we introduced small temporal variations in its period. The variable t defined the time axis running from 0 to 1 s. To each cycle of the oscillation we add a short 50 ms burst of high frequency (60–80 Hz) white noise that was tapered with a Hanning window. The Hanning window was scaled to a maximum amplitude of 5. The onset of the gamma signal relative to the alpha cycle varied uniformly between 0 and 20°C. Finally, normally distributed random noise with zero mean and standard deviation 0.1 were added.where the α frequency We generated the same alpha-gamma CFC as in (1) but this time the α waveform was generated with non-sinusoidal wave morphology properties. Concretely, the magnitude of the amplitude of the peaks of the alpha oscillations was higher relative to the troughs:b controlled the degree of amplitude asymmetry of peaks relative to the troughs. The exponent b was chosen to be 3. The signal α was demeaned and rescaled by a factor of 2. Note that the properties of the non-sinusoidal signal morphology described above differ from the amplitude fluctuation asymmetry properties defined by Mazaheri and Jensen  · φ determined the phase of the j-th harmonic. We defined the first two harmonics of the alpha band . The sum of the fundamental and the 1st and 2nd harmonics with the phase term φ = π/2 generated a sawtooth wave whose amplitude raised rapidly decreasing with time in a given cycle (“left-side peak”). This waveform was referred as “negative sawtooth” signal. In contrast, we refered to a “positive sawtooth” (“right-side peak”) a waveform with a constant raising period and a rapidly amplitude decrease (φ = 3π/2). We explored different φ-values  to investigate systematically a variety of periodic wave morphologies. In comparison to Kramer et al.  = 14.48, p < 5 × 10−5; Placebo = 0.5 mg LZP: t(24) = 1.44; p = 0.16; Placebo > 1.5 mg LZP: t(24) = 4.42, p < 0.001; 0.5 mg LZP > 1.5 mg LZP: t(24) = 3.95, p < 0.001; Figure F = 7.66, p < 0.005; Placebo = 0.5 mg LZP: t(24) = 1.24; p = 0.23; Placebo > 1.5 mg LZP: t(24) = 4.24, p < 0.001; 0.5 mg LZP > 1.5 mg LZP: t(24) = 2.45, p < 0.05; Figure Spectral analysis was applied to the MEG axial gradiometers to investigate the power modulations produced by LZP administration during the EO and EC conditions. We found significant differences between drug conditions both during EO  = 12.91, p < 5 × 10−5; Placebo = 0.5 mg LZP: t(24) = −0.88; p = 0.38; Placebo > 1.5 mg LZP: t(24) = −4.12, p < 0.001; 0.5 mg LZP > 1.5 mg LZP: t(24) = −4.23, p < 0.001; Figure F = 38.87, p < 1 × 10−10; Placebo > 0.5 mg LZP: t(24) = −3.98; p < 0.001; Placebo > 1.5 mg LZP: t(24) = −7.06, p < 1 × 10−6; 0.5 mg LZP > 1.5 mg LZP: t(24) = −5.81, p < 1 × 10−5; Figure While LZP decreased occipital alpha power, fronto-central beta power increased as a function of dosage Figure  both durIn conclusion, LZP produced robust power modulations during resting state conditions: while parieto-occipital alpha power decreased with LZP dosage, sensorimotor beta power increased.p < 0.001; Figure F = 8.29, p < 0.001; Placebo = 0.5 mg LZP: t(24) = 1.42; p = 0.17; Placebo > 1.5 mg LZP: t(24) = 2.81, p < 0.05; 0.5 mg LZP > 1.5 mg LZP: t(24) = 3.85, p < 0.005; Figures Given we found a clear alpha power peak in the resting state spectrum, we set out to investigate whether the ongoing alpha phase linked with higher frequency amplitude modulations. Moreover, we also sought whether LZP modulated its amplitude or frequency. First, we calculated the CFCoh metric for all participants and drug sessions during rest. We observed alpha phase (8–12 Hz) to beta-low gamma (20–45 Hz) amplitude coupling over posterior sensors Figure . Moreovep < 0.005; Figures F = 17.14, p < 5 × 10−5; Placebo < 0.5 mg LZP: t(24) = −3.02; p < 0.01; Placebo > 1.5 mg LZP: t(24) = 3.73, p < 0.005; 0.5 mg LZP > 1.5 mg LZP: t(24) = 4.88, p < 5 × 10−4; Figure p = 0.22; Figures It could be that the CFCoh we observe in Figure mg LZP: t4 = −3.02In summary, parieto-occipital sensors display the strongest phase-to-phase coupling between alpha and its first two harmonics . The CFCoh observed in Figure Figure The presence of harmonics in the data Figure  could prSummarizing, event-related oscillatory averages did not allow us to demonstrate clear CFC. This result suggested that the coherence between the alpha band and the beta-low gamma amplitude envelope might be a spurious correlation due to the non-sinusoidal properties of the alpha rhythm.So far, we have focused the analysis on the resting state conditions but it remains to be tested whether similar CFC and drug modulations can be observed under complex cognitive operations. To explore whether the amplitude of high frequency oscillations was phase-locked to the phase of slow rhythms during the WM delay periods, we computed the same CFC measures when participants engaged in a visuo-spatial WM task Figure . Given pp > 0.16). Magnitude squared bicoherence showed the strongest coupling in the first two harmonics over occipital sensors  but they were not significantly modulated by LZP  with very weak modulation in the beta-low gamma band . Figure Significant CFC could be observed in the absence of CFD if the amplitude envelope of the higher frequencies and the analytic phase of the slow frequencies covary in time without phase delays  produced harmonics in the power spectrum and these harmonics were, by definition, phase-to-amplitude coupled Figure . Taken tvariance (not the amplitude) of the peaks of an oscillatory activity minus the variance of the troughs  should not be confused with the alpha asymmetric amplitude fluctuation properties defined by Mazaheri and Jensen . The asyA(t) is a slow signal  that determined the amplitude modulation of the alpha frequency f. Note that, by construction, this synthetic signal will not produce harmonics because it consisted of a DC offset term and a single sine wave. This type of model might not apply to the empirical data presented here because we observed strong coupling between 10 Hz and its first two harmonics using bicoherence . For instance, when assessing the coupling between different frequencies, the potential harmonic contribution to CFC should be taken into account using bicoherence or any other metric sensitive to harmonic.are shown, in simulation and in theory, to depend on the extent to which signal present in the data is matched to the phase estimator” (p. 915). While wavelet phase estimator outperformed Fourier and Hilbert transforms when the signal to be analyzed had a broadband pulse-like wave morphology, the Fourier approach yielded more accurate phase estimates in comparison with the other two methods when the signal to be analyzed showed a stationary sinusoidal-like wave morphology . We suggest a cautionary interpretation of CFC results based on current models that assume that communication between neuronal ensembles are implemented as cross-frequency interactions between gamma and alpha activity (Jensen and Mazaheri, DL designed the study, collected and analyzed the data. DL, Nt, and RO discussed the results and wrote the paper.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.


In animals, low levels of vitamin D are associated with estrus cycle disturbances, but there are virtually no human data. We examined the association of 25-hydroxyvitamin D (25(OH)D) (a biomarker for vitamin D status) with menstrual cycle characteristics.Women aged 35-44 were randomly selected from a Washington D.C. health plan and invited to participate in the Uterine Fibroid Study (1996 – 1999). Our analysis includes 636 women (57% were African-American) who provided a blood sample and completed a telephone interview that included gynecologic history. Women were asked their usual cycle length in the preceding year. Women who reported it was “too irregular to estimate” were classified as having irregular cycles (N = 48). Women were excluded if they currently or recently used hormonal contraception or any other medication that influences menstrual cycles. 25(OH)D was measured by radioimmunoassay in stored plasma samples.The median 25(OH)D level was 12.0 ng/mL . After controlling for age, race, BMI, education, age of menarche, current smoking, alcohol use, and physical activity, a decrease in 25(OH)D of 10 ng/mL was associated with 1.9 times the odds of irregular cycles (Odds ratio (OR) ): 1.9 , p = 0.04). 25(OH)D was not associated with the occurrence of short cycles (OR(CI): 1.08  or long cycles (OR(CI): 1.31 , p = 0.4).Lower levels of 25(OH)D were associated with irregular cycles, but not with short or long cycles. Vitamin D may play a role in regulating ovulatory function. Further investigation of potential mechanisms is warranted. Vitamin D is known for its role in bone health , but itsDiet-induced vitamin D deficiency causes dramatically reduced fertility in both rats and mice, with a 45-70% reduction in the probability of becoming pregnant and a 67-100% reduction in the number of viable pups . Knock-oThe promoter region for the gene encoding anti-Müllerian hormone (AMH) contains a domain for the vitamin D response element, suggesting that vitamin D can regulate AMH expression . AMH in Our objective was to examine the association of vitamin D with menstrual cycle characteristics including long and short cycle length and cycle regularity in a population of randomly-selected members of a prepaid health plan.The National Institute of Environmental Health Sciences (NIEHS) Uterine Fibroid Study, 1996 – 1999, enrolled participants identified from a large health plan in Washington, DC ,9,10. InAll of the women provided informed consent and the study protocol was approved by the NIEHS institutional review board.2 and 25(OH)D3. The intra- and interassay coefficients of variation based on blind replicates were 7.6% and 10.6%, respectively. One hundred and four women were missing a 25(OH)D measurement, mostly due to a lack of available blood sample, leaving 642 D) in stored plasma samples. 25(OH)D is a widely accepted biomarker for vitamin D . 25(OH)DWomen were asked to report their menstrual cycle length in the preceding year. Lower 25(OH)D is hypothesized to interfere with normal follicular development, which might manifest as short, long or irregular menstrual cycles . To investigate this, we separated women into four categories of cycle length: short, long, normal, or irregular. Women who reported that their cycle length was “too irregular to estimate,” and thus could not choose one number to describe their usual cycle length, were classified as having irregular cycles (N = 48). Women who did not report irregular cycles were classified as having “short” cycles if their usual cycle length was 17 - 25 days. We defined “long” cycles as 32-56 days. Given the natural decrease in cycle length among older women, the expected rarity of cycles greater than 35 days , and theData on covariates were collected through self-administered and telephone questionnaires. Body weight was measured at the clinic visit. Potential confounders considered included: age, education, race, body mass index (BMI), alcohol intake, current cigarette smoking, physical activity, and age at menarche.25(OH)D was structured both as a continuous linear variable and as a dichotomous variable of “insufficient” versus “sufficient” based on the Institute of Medicine cutpoint of 20 ng/mL . AssociaEight percent of study participants reported having irregular cycles, 29% were classified as having short cycles and 11% had long cycles (Table Lower levels of 25(OH)D were not associated with short or long cycles (Table Only six women reported a prior diagnosis of polycystic ovary syndrome (PCOS). These women tended to have lower 25(OH)D levels (Mean: 6.6 ng/mL vs. 14.1 ng/mL) and the 25(OH)D level for all 6 women was less than 20 ng/ml. It is unclear whether the biology underlying the association of 25(OH)D with PCOS is reflective of the underlying biology that occurs in women without PCOS. Thus, as a sensitivity analysis, we excluded women with PCOS, however the estimated association was unchanged (OR(CI): 1.8 , p = 0.05).Lower plasma levels of 25(OH)D were associated with an increased odds of having irregular cycles. Plasma 25(OH)D was not associated with the occurrence of long or short menstrual cycles.CYP27B1 gene. Cyp27b1 null mice and mice that lack the vitamin D receptor have shown hypogonadism, arrested follicular development, prolonged estrous cycles, and hypoplastic uteri [Cyp27b1 null mice the effect of vitamin D deficiency was reversible, with estrous cycle length and staging normalized with dietary supplementation with vitamin D3 [25(OH)D is converted to its active form 1,25(OH)D by the enzyme 1α-hydroxylase, coded by the ic uteri ,7,16,17.tamin D3 . The protamin D3 . AMH in tamin D3 . Serum 2tamin D3 . Moreovetamin D3 ,20.Our study is the first to examine the association of vitamin D with menstrual cycle length in a population-based sample of late reproductive-aged women. This analysis is limited by the use of self-reported cycle length and by small numbers of women with extreme cycle lengths, particularly long cycles and irregular cycles. Most of the women in this study had insufficient 25(OH)D levels which, combined with small numbers of irregular cycles, reduced the power to detect an association between the dichotomous 25(OH)D exposure (≤20 ng/ml vs >20 ng/ml) and irregular cycles. This study is based on a cross-sectional design, with women reporting their typical cycle length for the past year at the time of blood collection. It is possible that some of the women in our analysis had undiagnosed PCOS. We did not have other hormonal or clinical markers of PCOS in this sample. Subclinical PCOS might be an underlying factor (or intermediate) in the association of 25(OH)D with irregular cycles.We found that lower levels of 25(OH)D were associated with irregular menstrual cycles in a population-based sample of late reproductive-aged women. Vitamin D may influence cycle regularity through its associations with AMH, insulin, androgens, or a yet to be identified pathway. Further investigation of potential mechanisms is warranted.


GC, CYP2R1, and DHCR7/NADSYN1 were replicated, while nine GWAS SNPs in GC and CYP2R1 were replicated in EAs. A CYP2R1 SNP, rs12794714, exhibited the strongest signal of association in AAs. In EAs, however, a different CYP2R1 SNP, rs1993116, was the most strongly associated. Our models, which take into account genetic and environmental variables, accounted for 20 and 28 % of the variance in serum vitamin D levels in AAs and EAs, respectively.Vitamin D deficiency is more common among African Americans (AAs) than among European Americans (EAs), and epidemiologic evidence links vitamin D status to many health outcomes. Two genome-wide association studies (GWAS) in European populations identified vitamin D pathway gene single-nucleotide polymorphisms (SNPs) associated with serum vitamin D [25(OH)D] levels, but a few of these SNPs have been replicated in AAs. Here, we investigated the associations of 39 SNPs in vitamin D pathway genes, including 19 GWAS-identified SNPs, with serum 25(OH)D concentrations in 652 AAs and 405 EAs. Linear and logistic regression analyses were performed adjusting for relevant environmental and biological factors. The pattern of SNP associations was distinct between AAs and EAs. In AAs, six GWAS-identified SNPs in The online version of this article (doi:10.1007/s00439-014-1472-y) contains supplementary material, which is available to authorized users. Many chronic diseases are disproportionately distributed in the US populations. Diabetes mellitus, cardiovascular diseases, several malignancies, and other diseases are more prevalent among African Americans (AAs) relative to European Americans (EAs) and Asian Americans. Differences in serum vitamin D [25(OH)D] concentration among racial/ethnic groups are suspected to be one of the sources of health disparities (Grant and Peiris Circulating levels of 25(OH)D are strongly influenced by multiple factors. Various studies have demonstrated that dietary intake, dietary supplement use, season of blood draw, UVR exposure, smoking, sex, age, body mass index (BMI), and race/ethnicity are important predictors of serum 25(OH)D levels , DHCR7 (7-dehydrocholesterol reductase) and NADSYN1 region, and CYP2R1 . Smaller scale replication and candidate gene studies in European and Asian populations also demonstrated the association of variants in these gene regions with serum 25(OH)D levels D levels in AAs D levels. Two meta-analyses of genome-wide association studies (GWAS) in European descent populations found single-nucleotide polymorphisms (SNPs) in vitamin D pathway genes associated with serum 25(OH)D levels  and 405 EA men from Chicago were included for this study. The subjects from Washington, D.C. were recruited at Howard University Hospital D levels <50 nmol/l (<20 ng/ml) and vitamin D insufficiency as 25(OH)D levels between 50 and 75 nmol/l (20 and 30 ng/ml) (Holick Blood samples for 25(OH)D assays and DNA analysis, demographic information, and information on potential modifiers of serum 25(OH)D were collected at the time of recruitment. Research coordinators conducted in-person interviews, administered structured questionnaires, and obtained information on ancestry, medical history, income, education, marital status, and lifetime history of sun exposure  Holick .GC, DHCR7/NADSYN1, VDR, CYP2R1, CYP27A1, CYP27B1, CYP3A4, and CYP24A1), including 19 GWAS-identified variants (Table We genotyped 39 SNPs in eight vitamin D metabolic pathway genes (ts Table . SNP selts Table . Individts Table . All then = 652). We also tested for association adjusting for age, WAA, study site, and environmental predictors . We adjusted for WAA to control for population stratification in the admixed AA populations. For the analyses of EAs, we adjusted for age , and we performed analyses further adjusting for environmental predictors . We treated total vitamin D intake, season of blood draw, and UVR exposure as binary variables. We treated total vitamin D intake as a binary variable , because it was not normally distributed. We defined from June to November as UVR high months and from December to May as low UVR months to account for serum 25(OH)D decay time. Mean serum 25(OH)D levels were not different between individuals with UVR medium and low exposure, so the levels of UVR exposure were categorized into high vs. low/medium exposure. For GWAS-identified SNPs, we considered the P < 0.05 as the threshold for statistical significance. For other SNPs, pointwise empirical P values and empirical P values corrected for multiple testing were obtained using the max(T) permutation procedure  using PLINK D levels and logistic regression models to test the association with vitamin D deficiency (<50 nmol/l). We performed separate analyses for AAs and EAs because of differences in linkage disequilibrium (LD), allele frequencies, and biological and environmental factors contributing to serum 25(OH)D levels in the two populations. For the analyses of AAs, we adjusted for age, West African ancestry (WAA), and study site  and a burn-in length of 30,000 for 70,000 repetitions. Each participant was scored from 0 to 100 % for individual estimates of West African, Native American and European ancestry. Mean WAA in AAs was 0.79, which was similar to previous estimates in the study populations . A much smaller proportion of AAs (14.9 %) had the Institute of Medicine Recommended Dietary Allowance (≥600 IU/day) compared to EAs (25.4 %).Vitamin D deficiency and insufficiency were more common in AAs than in EAs (Table P < 0.001). Skin pigmentation and WAA were not associated with serum 25(OH)D levels. In EAs, age (P = 0.006), season of blood draw (P < 0.001), total vitamin D intake (P < 0.001), and body mass index (BMI) (P < 0.001) were significantly associated with serum 25(OH)D levels. Also, UVR exposure was marginally associated with serum 25(OH)D levels (P = 0.06) in EAs. These significant modifiers of serum 25(OH)D levels were included in the linear and logistic regression models in our genetic association analyses.Before we tested SNP associations, we performed multiple linear regression analyses to identify biological and environmental modifiers of serum 25(OH)D concentrations (Supplementary Table 2). Among AAs, study site, total vitamin D intake and season of blood draw were significantly associated with serum 25(OH)D levels  and rs1989969, were hypervariable loci exhibiting more than two alleles, and we removed them from our analyses. We also excluded rs11568820 (Cdx2) from the analyses in AAs, because it deviated from Hardy–Weinberg Equilibrium (P < 0.001). A CYP27A1 SNP, rs116071925, was excluded in the analyses of EAs, because it was monomorphic.Of the 39 SNPs genotyped, two SNPs in CYP2R1 and 2 in DHCR7/NADSYN1) were significantly associated with serum 25(OH)D levels (Supplementary Table 3). Although the number of individuals included was smaller in Model 2 than in Model 1, five GWAS-identified SNPs (1 in GC and 4 in CYP2R1) and one non-GWAS-identified SNP in GC were significantly associated with serum 25(OH)D levels in Model 2 . These two SNPs were weakly linked (r2 = 0.07) . When these two SNPs were included in the regression model, they showed independent association (P = 0.04 for both rs10741657 and rs12794714). The logistic regression analysis for vitamin D deficiency supports the strong associations of CYP2R1 SNPs, and rs12794714 showed the strongest association with vitamin D deficiency . A previously reported GC SNP, rs115563, was weakly associated with serum 25(OH)D levels (P = 0.048). Although not significant after correcting for multiple testing, another SNP in GC, rs115316390, that was not reported in the GWAS meta-analyses showed stronger association with serum 25(OH)D levels. This SNP was not in LD with rs1155563 (r2 < 0.001), and they were independently associated (P = 0.03 for both rs1155563 and rs115316390), when they were adjusted for each other in the regression model. Although the two SNPs in DHCR7/NADSYN1 were not associated with 25OHD in Model 2 in the linear regression analysis, rs12800438 was significantly associated with vitamin D deficiency  in AAs.In the linear regression models among AAs, we observed stronger associations of SNPs with serum 25(OH)D levels when we additionally adjusted for vitamin D intake and season of blood draw (Model 2) than when only age, WAA, and study site were included Model 1). In Model 1, three GWAS-identified SNPs . After adjusting for rs1993116, no other SNP in CYP2R1 remained significant. The logistic regression analysis confirmed the strongest association of this SNP . A GC SNP, rs7041, showed the second strongest association (P = 0.0007), and after conditioning on this SNP in the regression analyses, other GC SNPs were no longer significantly associated with serum 25(OH)D levels. Figure β) in AAs and EAs for rs7041. However, different alleles were tested in AAs and EAs, because the minor allele in AAs was the major allele in EAs, and rs7041 was not associated with serum vitamin D levels in AAs. In addition, a CYP24A1 SNP, rs73913757, was associated with serum 25(OH)D levels (P = 0.04), but it was not significant after controlling for multiple testing.A contrasting pattern of associations was observed in EAs. Compared to AAs, there were more SNPs with stronger signal of association in both Models . We added a CYP2R1 SNP, rs12794714, that showed the strongest association in single SNP linear regression and logistic regression analysis, and a GC SNP, rs115316390, to the linear regression model. The two SNPs together explained an additional 1.1 % of the serum 25(OH)D variation (adjusted R2 = 0.202). Genetic Risk Score was calculated using the top two SNPs by adding the number of A alleles for rs12794714 (CYP2R1) and G alleles for rs115316390 (GC). The Genetic Risk Score ranged from one to four . The regression coefficient (β) estimates for these two SNP were different, so we weighted the Genetic Risk Score using β estimates. In the same linear regression model, the weighted Genetic Risk Score was also significantly associated with serum 25(OH)D levels .To understand the additive effects of the significant vitamin D pathway gene variants on serum 25(OH)D levels, we performed linear regression analysis by adding the top two independently associated SNPs to the linear regression model with the biological and environmental modifiers. In AAs, the model including age, WAA, study site, total vitamin D intake, and season of blood draw explained 19.1 % of the variance in serum 25(OH)D levels  and rs1993116 (CYP2R1) and environmental variables accounted for 28.2 % of variance (adjusted R2 = 0.282). Genetic Risk Score was calculated with sum of the number of C alleles for rs1993116 (CYP2R1) and T alleles for rs7041 (GC). A very small proportion (9.1 %) of EAs who had zero risk allele was vitamin D deficient  in linear regression model adjusting for age, BMI, season, vitamin D intake, and UV exposure. Because the regression coefficient was virtually identical for two SNPs, we did not perform analysis with weighted Genetic Risk Score.Similarly, compared to biological and environmental modifiers, genetic variation explained a small proportion of serum 25(OH)D variation in EAs. SNPs and biological and environmental modifiers, however, accounted for greater serum 25(OH)D variation in EAs than in AAs. Adjusted R2 for rs12794714 and rs115316390, when they were added to the regression model (Model 2) in AAs, were 0.008 and 0.006, respectively. Our AA samples provide only 65 and 56 % power for these two SNPs to detect the effect size at a significance level of α = 0.05. In EAs, increments of R2 for the top two SNPs were larger. When rs7041 and rs1993116 were added to the regression models with the other modifiers, R2 increase were 0.022 and 0.021, respectively. Our EA samples have 90 and 89 % power to detect the observed R2 increment.Increments of GC among AAs and a SNP in CYP24A1 among EAs that were associated with serum 25(OH)D levels, though they were not significantly associated after correcting for multiple testing. The strength of associations and the SNPs that showed the strongest signal of association were, however, different between AAs and EAs.In this study of 652 AAs and 405 EAs, we investigated the association of 37 candidate SNPs in eight vitamin D pathway gene regions, and we successfully replicated GWAS findings in our AA and EA populations demonstrating that six SNPs in three vitamin D pathway genes in AAs and nine SNPs in two genes in EAs were significantly associated with serum 25(OH)D levels or vitamin D deficiency (Supplementary Table 5). We also found a previously unreported SNP in 3 D levels in European populations . Decreased ability to synthesize vitamin D due to older age and/or darker skin pigmentation may explain the inconsistent results between our study and the two GWAS meta-analyses D levels among people of African descent.When human skin is exposed to UVR, 7-dehydrocholesterol is converted to pre-vitamin D3 Holick . Also wiCYP2R1, may impact vitamin D metabolism. In our study, SNPs in CYP2R1 showed the strongest signal of association in both AAs and EAs. The CYP2R1 SNP, rs1993116, was most strongly associated with serum vitamin D status in our EA subjects and also showed the strongest signal of association within this gene in one of the GWAS meta-analyses D levels and vitamin D deficiency in our AA subjects, was not the most strongly associated SNP in either GWAS meta-analyses. Two other studies investigated the association of CYP2R1 SNPs and serum 25(OH)D levels in AAs, but none of the associations were significant , the two SNPs show weak linkage in AAs (r2 = 0.05) . The LD difference suggests that a functional variant exists in the vicinity of these SNPs.Vitamin D that is synthesized in the skin or consumed from the diet is converted to a circulating form of vitamin D, 25(OH)D, in the liver with the enzyme, hydroxylase, and DNA sequence variants in these hydroxylase genes, such as GC or ability of vitamin D binding protein to bind to 25(OH)D. Thus, SNPs within GC may reduce circulating 25(OH)D levels. The two GWAS meta-analyses found the strongest signal of association with SNPs in GC D, 25(OH)D binds to the vitamin D binding protein and is transported to target tissues through the circulation system. Sequence variants may reduce expression of GC creates an Asp → Glu amino acid change in the vitamin D binding protein. In our EA population, rs7041 showed the strongest association with serum 25(OH)D levels. The association of this SNP with 25(OH)D levels was consistently demonstrated in the two GWAS meta-analyses . The SNP, rs115316390, located in the intron region showed the strongest GC association in our AA population (P = 0.03). Although the association was not significant after correcting for multiple testing, this study is the first to report the evidence of association of rs11531690 with serum 25(OH)D concentrations. These data suggest that there could be other functional variants in the vicinity of these SNPs affecting serum 25(OH)D and vitamin D binding protein levels. It should also be noted that while serum vitamin D binding protein levels stay relatively stable over time, serum 25(OH)D levels can seasonally fluctuate D levels in EAs than in AAs (4.1 % increase in R2 among EAs compared to a 1.1 % increase among AAs). Nevertheless, these SNPs account for a very small proportion of variation in serum 25(OH)D levels in both populations compared to behavioral, biological, and environmental predictors.Contrasting patterns between AAs and EAs were also observed when significantly associated SNPs in Several studies have shown that an increasing Genetic Risk Score, or number of risk alleles, is associated with decreased serum 25(OH)D levels or vitamin D deficiency/insufficiency using the candidate SNPs D levels and genetic variations in AA populations. This study also incorporated many biological and environmental modifiers of serum 25(OH)D levels, including objective measurements of skin pigmentation, into the analyses. Combined effect of gene and environment likely explains the difference in prevalence of vitamin D deficiency between AAs and EAs D levels at 50 nmol/l (20 ng/ml), but maintaining levels ≥75 nmol/l (30 ng/ml) likely requires more than 1,500 IU/day of vitamin D D variation than other genes in the vitamin D pathway in our study populations. The contrasting pattern of associations between AAs and EAs suggests that additional studies are warranted to identify the causal variants affecting vitamin D binding and enzymatic activities.In summary, this study successfully replicated several GWAS-identified SNPs associated with 25(OH)D levels in AA and EA populations. The results also provide evidence that Supplementary material 1 (PDF 431 kb)Below is the link to the electronic supplementary material.


Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae” are obligate intranuclear endosymbionts of freshwater amoebae. Here, we present the draft genome sequences of these two bacteria, with total sizes of 2,990,361 bp and 3,626,027 bp, respectively.“ Candidatus Berkiella cookevillensis” strain CC99 was recovered from an infected amoeba from a cooling tower, and “Candidatus Berkiella aquae” strain HT99 was collected from an infected amoeba in an outdoor hot tub. The 16S rRNA gene sequences were 100% (CC99) and 96% (HT99) similar to sequences from uncultured organisms in the NCBI GenBank database. CC99 had 100% identity to an uncultured isolate from a South Korean oyster shell waste pile. Preliminary analyses showed 94% 16S rRNA sequence similarity between the two isolates and <92% similarity to Legionella or Coxiella species . For isolate CC99, 2,598 genes were predicted, with 2,557 predicted coding sequences (CDSs). For HT99, 3,231 genes and 3,189 CDS regions were identified. Using RNAmmer and tRNAscan-SE, the CC99 genome is predicted to contain 3 rRNA and 40 tRNA genes, while the HT99 genome is predicted to contain 3 rRNA and 41 tRNA genes.Prokka and the IGS Annotation Engine were used for structural and functional annotation of the sequences , 6, and dot or icm-type IV secretion system (T4SS) genes in both isolates, which is important for the pathogenesis of both Legionella pneumophila and Coxiella burnetii, bacteria similar to CC99 and HT99 , have been deposited at DDBJ/EMBL/GenBank as Berkiella cookevillensis CC99 under the accession no.


The periodontal ligament cells were stimulated with various concentrations of NaF added into osteogenic inductive medium. Immunohistochemistry of cell identification, cell proliferation, alkaline phosphatase (ALP) activity assay, Alizarin red S staining and quantitative real-time-polymerase chain reaction (RT-PCR) were performed. Moderate concentrations of NaF (50-500 μmol/L) had pro-proliferation effects, while 500 μmol/L had the best effects. ALP activity and calcium content were significantly enhanced by 10 μmol/L NaF with osteogenic inductive medium. Quantitative RT-PCR data varied in genes as a result of different NaF concentrations and treatment periods. We conclude that moderate concentrations of NaF can stimulate proliferation and mineralization in periodontal ligament cells. These in vitro findings may provide a novel therapeutic approach for acceleration of periodontal regeneration by addition of suitable concentrations of NaF into the medication for periodontitis treatment, i.e., into periodontal packs and tissue patches.Fluoride, which is often added to toothpaste or mouthwash in order to protect teeth from decay, may be a novel therapeutic approach for acceleration of periodontal regeneration. Therefore, we investigated the effects of fluoride on proliferation and mineralization in human periodontal ligament cells  Fluoride is found in teeth, bones, thyroid gland and skin of humans. Since reports of its strengthening effect on bones and teeth at recommended doses, it is officially considered to be a beneficial element for humans. However, chronic excessive intake of fluoride in water, diet and dentifrices may result in fluoride poisoning, which manifests as enamel fluorosis and bone injuries . FluorosPeriodontitis is a highly prevalent inflammatory oral disease. It can lead to the destruction of periodontal tissues and the loss of bone that surrounds the teeth. This disease is a major cause of adult tooth loss. Current treatments mainly aim at removing local stimulus and controlling inflammation. Afterwards, efforts are made to repair periodontal supporting tissues, which means achieving simultaneous regeneration of the lost alveolar bone, cementum and periodontal soft tissues. Unfortunately, in most cases, alveolar bone repair and regeneration following periodontitis treatment is still a challenge presented to dentists .In vitro studies of cultured periodontal ligament fragments, periodontal ligament cells (PDLCs) are described as fiber-cord-like cells. They are reported as having multi-differentiation potential, being able to differentiate into fibroblasts, osteoblasts, and bone forming cells. Much of the research in recent years has confirmed that PDLCs can form bone nodules and express bone-related proteins, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OCN) under certain circumstances  for 15 min with shaking every 5 min in constant temperature in 37°C water bath. After terminating digestion, centrifugation described as before was again performed. The supernatant was removed and the precipitate collected, pooled and plated into a 12.5-cm2 cell culture flask  with 2 mL complete ɑ-MEM medium. The flask was placed vertically into a humidified incubator with 37°C constant temperature, 95% air, 5% CO2 for 4 h; it was then placed horizontally. Medium was replaced every 3 days until cell growth of 70-80% confluency. Digestion and passage was done with 0.25% trypsin protease (Gibco). Then cells were again incubated with complete ɑ-MEM medium with 10% FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin. Cultures between passage 3 and 6 were used in the experiments.PDLCs were obtained from healthy premolar or third molar teeth extracted from patients (11 to 23 years of age) for orthodontic treatment or preventive procedures. Teeth were placed in a minimum essential medium , containing 20% fetal bovine serum , 100 U/mL of penicillin, and 100 mg/mL of streptomycin . Teeth were then washed with sterile phosphate-buffered saline (PBS) containing 100 U/mL of penicillin, and 100 mg/mL of streptomycin as many times as possible until all blood and bone fragments were rinsed away. Single-use sterile scalpel was used to carefully scrap the periodontal ligament tissue from the middle third of the root surface. Tissue was centrifuged at 190 4 cells per well in 24-well plates  with complete culture medium. After 48 h, the cell slides were gently rinsed with PBS three times and fixed with 4% paraformaldehyde  for 20 min. Finally, the chromogenic reaction was performed with a diaminobenzidine kit . The cell slides were incubated with primary antibodies against vimentin and cytokeratins (Beijing Zhongshan Golden Bridge Biotechnology) at a dilution of 1:50 for 18 h at 4°C. At the same time, PBS was used as a substitute for the primary antibodies as a negative control.First, the cells were attached to cell slides at a density of 5×103 cells/well. After 24-h incubation at 37°C with 5% CO2, the plates were treated with 0, 1, 5, 10, 50, 100, 5×102, 1×103, and 5×103 μmol/L NaF for 1, 2, 3, 4, 5, and 6 days. CCK-8 was mixed with serum-free ɑ-MEM medium at a proportion of 1:10 in advance. After removal of complete ɑ-MEM medium, 110 μL mixture was added to each well and incubated at 37°C for 2 h, until the media turned yellow. Groups without cells were used as zero setting. We assessed cell viability by absorbance values in each well, which was measured with a spectrophotometer  at a wavelength of 450 nm. Data were calculated using averages of three wells, and untreated PDLCs were considered as the control group. The concentrations of 0, 10, 5×102 and 1×103 μmol/L were chosen for subsequent experiments.Effects of various concentrations of NaF  on PDLCs proliferation was measured using a cell counting kit . PDLCs were seeded into 96-well culture plates (Eppendorf) with a concentration of 1×10NaF was added to osteogenic inductive culture medium. There were four treatment groups: 1) osteogenic medium , 2) osteogenic medium supplemented with 10 μmol/L NaF, 3) osteogenic medium supplemented with 5×102 μmol/L NaF, and 4) osteogenic medium supplemented with 1×103 μmol/L NaF. The PDLCs were treated the same way in subsequent experiments. The medium was replaced every 3 days.4 cells/well in 24-well culture plates (Eppendorf) and cultured in basal medium. When cells reached 70-80% confluency, plates were treated with different concentrations of NaF. For ALP staining, cells triggered for 14 days were washed three times with PBS and fixed with 4% PFA for 20 min at room temperature. The fixed cells were stained with a BCIP/NBT ALP Color Development Kit . Double-distilled water was added to each well to stop reaction after 30min. The BCIP/NBT method produced an insoluble blue NBT formazan. ALP stained areas were measured quantitatively with Image-Pro Plus 6.0 .PDLCs were plated at a density of 1×105 cells/well, different concentrations of NaF were added as previously described for 7, 14, and 21 days. After treatments, the cells were lysed using RNAisoTM Plus  to isolate total RNAs. Single-stranded complementary DNA (cDNA) was obtained by reverse transcribing 1 μg of total RNA from each sample using PrimeScript® RT reagent kit (TakaRa). Quantitative RT-PCR of Runt-related transcription factor 2 (Runx2), type I collagen α1 (COL1A1), ALP, OCN and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were performed on an equal amount of cDNA using SYBR® Premix Ex TaqTM II (TakaRa) according to the manufacturer's instructions. Reactions were performed in triplicate on LightCycler® 480 System Real-Time PCR . The cycling parameters used were: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s, and a dissociation program of 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. Melting curve analysis was included to assure that only one PCR product was formed. Expression of housekeeping gene GAPDH was amplified as an internal control. The relative amount of RNA was calculated by the 2−ΔΔCt method. Primer and probe sequences for Runx2, COL1A1, ALP, and OCN, and GAPDH are shown in After reaching 70-80% confluency from PDLCs seeded in 6-well plates at a density of 1×10PDLCs were subcultured in 24-well culture plates similarly to the ALP activity assay. Mineralization at 28 days was assessed by Alizarin red S staining, which stains the calcium minerals red. Cells were fixed in 4% PFA for 20 min and then stained with 2% Alizarin red S  solution with 500 µL/well for 30 min at room temperature. The excess dye was washed away with double-distilled water. After photographing, semi-quantitative measurement of mineralization was taken. The Alizarin red S-stained cultures were incubated with 500 µL 10% cetylpyridinium chloride  for 15 min to solubilize and release calcium-bound Alizarin red into solution. The absorbance of the released Alizarin red S was measured at 562 nm in 96-well plates using a spectrophotometer. Groups of CTC were used as zero setting.Data are reported as mean±SD from at least three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) using SPSS software . P values less than 0.05 were considered to be statistically significant.In the course of primary cultured periodontal ligament cells, inverted microscope was used for observation of cell morphology and growth. After 7 to 10 days, adherent cells could be found radially stretching out from the edge of periodontal ligament fragments . When ap2 μmol/L treatments compared to the control, while 5×102 μmol/L NaF caused the highest value at day 6 as shown in 3 μmol/L, the absorbance exhibited a significant decrease compared to the control group (P<0.05), indicating a cytotoxic effect. These data indicate that NaF has dose-dependent effects on cell proliferation.To study the effect of the various concentrations of NaF on PDLCs proliferation, we determined cell viability by CCK-8 assay after 1, 2, 3, 4, 5 and 6 days of culture. As shown in 2 μmol/L treatment compared to the control, while 10 μmol/L NaF caused the highest ALP positive area (P<0.05). However, no significant differences were found between 1×103 μmol/L-NaF-treated and untreated groups at the same time point (P>0.05). This suggests that supplementing with a moderate concentration of NaF could induce further mineralization of PDLCs in the presence of osteogenic inducers.As demonstrated in 2 μmol/L NaF significantly increased expression of Runx2 at 7 days. In 2 μmol/L NaF added for the first 7 and 14 days (P<0.05). However, as the culture time increased, COL1A1 mRNA expression was significantly increased on day 21 (P<0.05). ALP expression also showed the same change trend following 5×102 μmol/L NaF addition (3 μmol/L NaF (P<0.05)  . These d2 and 1×103 μmol/L groups there was a significant reduction of the calcium content . However, in the 5×10 P<0.05; . This imin vitro have not been investigated. In this study, various concentrations of NaF were applied to PDLCs cultured in osteoinductive medium, and the results showed that fluoride could stimulate proliferation and mineralization in vitro.Much work has been done to study the dual effect of fluoride on bone formation. Although chronic excessive fluoride intake can result in skeletal fluorosis, low concentrations of fluoride are normally used to reduce the incidence of caries. In recent years, several investigations have suggested that fluoride could promote osteoblastic differentiation ,15–18.2 μmol/L. At a higher NaF concentration of 5×103 μmol/L, the cell viability declined compared with control group. Our results are similar to those of a human osteosarcoma MG-63 cells in vitro investigation, which demonstrated that a moderate concentration up to 5×102 μmol/L of NaF has a pro-proliferation effect, while 2×103 μmol/L NaF inhibited cell proliferation  on the premise that there was no cytotoxicity on PDLCs during the pilot period.As described before, after supplementation with moderate concentrations of fluoride, a growth of PDLCs was stimulated. However, in order to identify a suitable concentration of NaF for novel therapeutic approaches for acceleration of periodontal regeneration, our experiment tested fluoride concentrations of 10, 5×102 μmol/L NaF showed a significant increase in ALP activity.As known, ALP is an early well-defined marker of osteogenic differentiation, and higher ALP activity represents a greater potential for bone formation. In this investigation, we assessed effects of various concentrations of NaF on ALP activity of PDLCs. Previous studies indicate that the addition of fluoride could affect ALP activity in other cells with a potential for bone forming. Human bone marrow mesenchymal stem cells grown on fluoride-modified titanium implant specimens showed higher ALP activity compared with grit-blasted ones . In prim2 μmol/L NaF increased expression of Runx2 as well as proliferation. As the culture time increased, 1×103 μmol/L NaF promoted COL1A1 mRNA expression. 10 μmol/L NaF only increased COL1A1 expression at 7 days. Similar to the ALP activity assessment, 10 μmol/L NaF increased ALP mRNA level at both 7 and 14 days. However, no significant difference in COL1A1 and ALP mRNA expression with 10 μmol/L NaF at 21 days was observed. PDLCs at this age might have already finished the conversion phrase, proliferation phase and cell aggregation secretory phase. OCN is a specific maker for late stages of osteoblastic differentiation, implying that at this time the cultured cells would present the phenotype of mature osteoblasts (This study investigated the widely used bone formation markers Runx2, ALP, COL1A1 and OCN by RT-PCR against the housekeeping gene, GAPDH –18. PDLeoblasts . Unlike in vitro. The result shows that 10 μmol/L NaF lead to an increased activity compared with the control group. In contrast, and to our surprise, both 5×102 and 1×103 μmol/L NaF presented an effect of inhibition on the calcified nodules formation of PDLCs at 28 days, since cells did not reduce the ALP activity. The exact reason for this finding was unclear; however, it might be explained by the different cell passages and the experimental conditions.To further confirm the bone formation induced by fluoride in osteogenic medium, Alizarin red S staining was used to analyze mineralization in PDLCs In conclusion, the results suggest that moderate concentrations of NaF can stimulate proliferation and mineralization in PDLCs. This information may provide a novel therapeutic approach for acceleration of periodontal regeneration by addition of a suitable concentration of NaF into the medication for periodontitis treatment as for instance, into periodontal packs and tissue patches.


Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization.  With the increase of global environmental awareness, more and more people are actively purchasing goods made from ecologically friendly materials. Unfortunately, many of these materials are intrinsically unrecyclable, including many of the predominantly used polymers. However, many natural fibers such as jute and kenaf possess properties that are comparable to more traditional composites, including stiffness, flexibility, impact resistance, and elasticity  is an annual herbaceous fiber crop. It is found predominantly in Southeast Asia and is the second cheapest and most commercially available fiber crop, whereby it provides a biodegradable and renewable lignocellulose fiber. Jute is a promising and featured fiber material among many value-added industrial products, due in large part to its high luster, moisture absorption properties, ability for rapid water loss, and easy breakdown. Furthermore, jute has much potential for use in the industrial production of packaging materials  and F-box family protein (FBX) in Arabidopsis thaliana; 18S ribosomal RNA (18S rRNA), ubiquitin5 (UBQ5), and eukaryotic elongation factor 1-alpha (eEF1α) in Oryza sativa , actin 4 (ACT4), ubiquitin 14 (UBQ14), F-box family protein (FBX6), clathrin adaptor complexes medium subunit family protein (MZA), and polypyrimidine tract-binding protein homolog (PTB) in Gossypium hirsutum , which prevents further studies on this species at the functional gene expression and transcriptome profile analysis levels.In recent years, an increasing number of stable reference genes have been studied in several systems, including eukaryotic elongation factor 1-alpha (rRNA), actin (ACT), actin 7 (ACT7), chaperone protein dnaJ (DnaJ), eukaryotic elongation factor 1-alpha (EF1α), ras-related small GTP-binding protein (RAN), alpha-tubulin (TUBα), beta-tubulin (TUBβ), ubiquitin-conjugating enzyme like protein (UBC), ubiquitin extension protein . We then sought to reveal which reference gene(s) were the best option for gene expression qRT-PCR analysis of jute under various experimental treatments using three available statistical algorithms, geNorm . The results from this work will facilitate future studies on gene expression as well as foster a better understanding of how novel genes function in the molecular mechanisms of jute biological and/or physiological processes.In the present study, 11 genes were selected as candidate reference genes for evaluation expression stability in jute: 18S ribosomal RNA (18S Corchorus capsularis L.) variety Huangma 108 was used for all experimental treatment groups. To ensure disease-free materials, seeds were rinsed under running water for 10 min and sterilized with 5% sodium hypochlorite for 10 min. They were then washed three times with sterile water before germinated on filter paper that had been saturated with water in complete darkness at 28°C. After 3 days, seedlings were grown in the greenhouse in 1/4 Hoagland solution under a 16/8-h light/dark cycle at 30/26°C (day/night). Seedlings were assessed at the 3–5 leaf stage and the most consistent were used for (i) the abiotic and biotic stress groups or for (ii) harvesting of different plant tissues .Jute (6 spores per ml) of Colletotrichum siamense spores in suspension previously described by Ma et al.  PEG6000, respectively, and harvested at 0, 2, 4, 6, 8, 12, and 24 h. For the biotic treatment, seedlings were inoculated with 2 ml  by following the manufacturer's protocol. All final cDNA samples were diluted 10-fold for subsequent quantitative real-time RT-PCR reactions. Samples were stored at −20°C until use.Total RNA was extracted from approximately 100 mg fresh leaf tissue using the OMEGA isolation kit  according to the manufacturer's instructions. The genomic DNA contamination was eliminated using RNase-free DNase I  and RNA sample quality was then determined using the NanoDrop 2000 spectrophotometer . RNA integrity was assessed by 2.0% agarose gel electrophoresis. Finally, RNA samples with an ArRNA (FJ527599), ACT (GU207477), ACT7 (GH985158), DnaJ (GR463675), EF1α (GH985217), RAN (FK826494), TUBα (GH985177), TUBβ (JK743820), UBC (GR463733), UBI (GH985256), and UBQ (FK826547), were identified after performing a TBLASTN among the jute expressed sequence tags (ESTs) database (http://www.ncbi.nlm.nih.gov/nucest/?term=jute). Primers were designed according to these potential reference gene sequences using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and based on the following criteria: GC content 45−80%, melting temperatures 58−62°C, primer lengths 18−24 bp, and amplicon lengths 100−250 bp  and the amplification product of each gene was verified by electrophoresis on a 2% agarose gel. Real-time amplification reactions were performed with the Applied Biosystems 7500 Real-Time PCR System using SYBR® Premix Ex Taq™ . Reactions were prepared in a 20 μl volume containing the following: 2 μl cDNA template, 0.4 μl of each amplification primer, 0.4 μl ROX Reference Dye II, 10 μl 2x SYBR Premix Ex Taq™, and 6.8 μl dHCt−Δ, where ΔCt equaled each corresponding Ct value minus the minimum Ct value. Then, the relative expression values were imported into geNorm and NormFinder to analysis gene expression stability. For BestKeeper analysis, the Ct value was used as input data to calculate the coefficient of variation (CV) and the standard deviation (SD). The comprehensive rankings of the best reference genes were obtained by integrating the results of three algorithms. To validate the reliability of the selected reference genes, we analyzed the relative expression levels of the transcription factor CcWRKY15 in all tested samples. Additionally, a standard curve was generated from a 10-fold dilution of cDNA in a qRT-PCR assay using Microsoft Excel 2003. The PCR efficiency (E) and the regression coefficient (R2) were calculated using the slope of the standard curve according to the equation E = [10slope)−.To select a suitable reference gene, three publicly available software packages, geNorm (version 3.5), NormFinder and BestKeeper, were used to analyze the stability of each reference gene. All analyses using these packages occurred according to the manufacturers' instructions. For geNorm and NormFinder algorithms, the raw Ct values from the qRT-PCR were transformed into the relative expression levels using the following formula: ErRNA, ACT, ACT7, DnaJ, EF1α, RAN, TUBα, TUBβ, UBC, UBI, and UBQ, were identified and assessed under abiotic stress (NaCl stress and drought stress), biotic stress (Colletotrichum siamense) and in different tissues in this study. For each gene, the specificity of the designed primers was verified using agarose gel electrophoresis and the subsequent presence of a single band with the expected size  to 113.7% (ACT), and the regression coefficient (R2) ranged from 0.988 (TUBα) to 0.999 (DnaJ and TUBβ) , followed by EF1α (18.28 ± 0.94), UBQ (20.13 ± 1.54), UBC (21.20 ± 1.13), UBI (21.44 ± 0.62), DnaJ (22.22 ± 0.75), ACT7 (22.24 ± 0.92), and TUBβ (22.40 ± 1.07). TUBα was found to have the lowest level of expression of any of the genes tested, with a mean Ct ± SD of 28.43 ± 2.15, followed by RAN (26.37 ± 1.29) and ACT (25.75 ± 1.31)  and DnaJ (3.38%) showed the least variation in their expression levels across all tested samples. The ranking of gene stability by CV was as follows: UBI > DnaJ > ACT7 > 18S rRNA > TUBβ > RAN > ACT > UBC > TUBα > UBQ  would be optimal in each of the tested samples sets. As shown in Figure e Figure .n) between normalization factors (NFn) calculated by the geNorm algorithm also determines the optimal number of reference genes for accurate normalization. A cut-off value of Vn∕n+1 < 0.15  were sufficient for accurate normalization. In the tissue subset, four reference genes  were needed for accurate normalization, as the V4∕5 value was lower than 0.15. When the total samples were taken into account, the V3/4 value (0.132) was lower than the cutoff value of 0.15, which indicated that three genes  were suitable for all samples in this study  and UBI (2.39 ± 0.52) had lowest CV ± SD values, and showed remarkably stable expression. In the NaCl- and PEG-treated subset, TUBα had the lowest CV ± SD values of 0.79 ± 0.24 and 0.96 ± 0.29, respectively, and showed the most stable expression. In the total samples subset, UBI (2.32 ± 0.50) and 18S rRNA (3.76 ± 0.63) were identified as the two best reference genes for normalization. These results are inconsistent with those acquired from the geNorm and NormFinder methods  and TUBβ (2.03 ± 0.43). This result is consistent with the results obtained from geNorm and NormFinder analyses  and the standard deviation (SD) of the average Ct values. The most stable genes were identified as those that exhibit the lowest CV and SD (CV ± SD), and genes with SD greater than 1 were considered unacceptable and should be excluded  either singly or in combination, and the least stable reference gene (ACT or TUBα), to perform a qRT-PCR analysis. Results showed that in accordance with the behavior of AtWRKY15 previously described in A. thaliana, the CcWRKY15 functions as a negative regulator and was induced by salt-stress, oxidative-stress, and pathogenic infections (C. siamense) in jute  selected by Ferdou et al.  . For example, the relative expression folds of CcWRKY15 were approximately eight-fold (p < 0.01) higher than that of ACT7, RAN, or their combination at 4 h under NaCl stress, when ACT was used as normalization factor  Figure . Contrasr Figure .rRNA as a reference gene for examining the functions of UDP-glucose pyrophosphorylase and caffeoyl-CoA 3-O-methyltransferase in jute (C. capsularis) . In addition, it has been reported that ribosomal RNA genes are not adequate reference genes due to their high transcription abundance. Ultimately, this could lead to experimental error when normalizing genes with weak expression  were significantly lower in our experiment. For instance, when UBC was used as reference gene, CcWRKY15 had a 5.0-fold higher expression value at 24 h under NaCl stress condition  was consistent with the results obtained in the developmental stage series of G. max . UBI, another stable reference gene, showed relatively small variation in tissues subset, which was also the most stable reference gene in different tissues of C. sativus . By comparing the expression pattern of CcWRKY15 with that reported in A. thaliana  under abiotic  and biotic (Colletotrichum siamense) stress conditions as well as across different tissue types. The expression stability of the 11 candidates was analyzed by the three applications , and their results were furtherly integrated into a comprehensive ranking based on the geometric mean. For gene expression study under biotic stress and NaCl stress, we recommend ACT7 and RAN to normalize the qRT-PCR data. For gene expression study under PEG stress, UBC, and DnaJ are the two most suitable reference genes in C. capsularis. For the study of gene expression in the different tissues, TUBβ, UBI, EF1α, and RAN are recommended as the best reference genes for normalization. In addition, the two least stable reference genes 18S rRNA and ACT should be carefully used for normalization. Furthermore, the feasibility of the reference genes screened was further confirmed by comparing the expression pattern between CcWRKY15 and AtWRKT15, and the selected reference genes can perform significantly better in gene expression normalization. In particular, the reference genes selected in current study will facilitate the future work on gene expression studies in C. capsularis.We present here a systematic attempt to validate a set of candidate reference genes for the normalization of gene expression using qRT-PCR in jute (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.


Allomyrina dichotoma larvae (ADL), we previously reported that ADL block adipocyte differentiation on 3T3-L1 cell lines through downregulation of transcription factors, such as peroxisome proliferator-activated receptor-γ (PPARG) and CCAAT/enhancer binding protein-α (CEBPA). In this study, we tested whether ADL prevent obesity in mice fed a high-fat diet (HFD) and further investigated the mechanism underlying the effects of ADL. All mice were maintained on a normal-fat diet (NFD) for 1 week and then assigned to one of five treatment groups: (1) NFD; (2) HFD; (3) HFD and 100 mg·kg−1·day−1 ADL; (4) HFD and 3000 mg·kg−1·day−1ADL; or (5) HFD and 3000 mg·kg−1·day−1 yerba mate . ADL and yerba mate were administered orally daily. Mice were fed experimental diets and body weight was monitored weekly for 6 weeks. Our results indicated that ADL reduced body weight gain, organ weight and adipose tissue volume in a dose-dependent manner. Body weight gain was approximately 22.4% lower compared to mice fed only HFD, but the difference did not reach the level of statistical significance. Real-time polymerase chain reaction (PCR) analysis revealed that gene expression levels of PPARG, CEBPA and lipoprotein lipase (LPL) in the epididymal fat tissue of HFD-fed mice receiving 3000 mg·kg−1·day−1 ADL were reduced by 12.4-, 25.7-, and 12.3-fold, respectively, compared to mice fed HFD only. Moreover, mice administered ADL had lower serum levels of triglycerides and leptin than HFD-fed mice that did not receive ADL. Taken together our results suggest that ADL and its constituent bioactive compounds hold potential for the treatment and prevention of obesity.To clarify the anti-obesity effect of  Obesity is a chronic metabolic disease caused by an imbalance of energy intake and expenditure . The preAllomyrina dichotoma) is recognized in traditional medicine to have anti-hepatofibrotic, anti-neoplastic and antibiotic effects [A. dichotoma larvae (ADL) inhibited in vitro differentiation of 3T3-L1 preadipocytes via downregulation of transcription factors, such as peroxisome proliferator-activated receptor-γ (PPARG) and CCAAT/enhancer binding protein-α (CEBPA) [Recently, the Food and Agriculture Organization of the United Nations (FAO) reported the utility of edible insects as human dietary supplements. These are already used as traditional foods in many parts of world and are a rich source of fats, minerals, vitamins and other nutrients ,12,13,14 effects ,16,17. I (CEBPA) . The mur (CEBPA) .in vitro finding to a live animal model to investigate the effects of ADL on HFD-induced obesity. Obesity studies commonly employ high-fat diets to induce visceral obesity in rodent animal models, in which the pathogenesis of obesity is similar to that in humans [Ilex paraguariensis (yerba mate)—which has known anti-obesity effects in animals via decrease of body weight gain, adipokines mRNA levels, serum triglyceride (TG) and low density lipoprotein (LDL) cholesterol level [Thus, in the present study, we extended our n humans ,21,22. Tol level —was usedA. dichotoma larvae were purchased from Canaan farm . Lyophilized yerba mater (MT) leaf and ADL were ground into powder and suspended in deionized water. The sample concentrations were adjusted to 100 mg·kg−1·day−1 (ADL) and 3000 mg·kg−1·day−1 (MT and ADL).Third instar −1·day−1 ADL, (4) HFD with 3000 mg·kg−1·day−1 ADL, and (5) HFD with 3000 mg·kg−1·day−1 MT. Each treatment condition consisted of seven mice (n = 7). The normal-fat diet  provided approximately 3.85 kcal/g with a macronutrient profile of 20% protein, 70% carbohydrate and 10% fat. The high-fat diet  provided 4.73 kcal/g with a macronutrient profile of 20% protein, 35% carbohydrate, and 45% fat. Mice were fed with NFD or HFD for 6 weeks. ADL (100 mg/kg or 3000 mg/kg) and MT (3000 mg/kg) were administered daily by oral gavage using disposable mouse feeding needle . Body weight was measured weekly and weight gain was calculated as the difference between initial and final body weights, divided by initial body weight. At the end of the experimental period, the randomly selected mice (n = 3) were fasted for 15 h prior to sacrifice. Immediately following cervical spine dislocation, the liver, epididymal adipose tissue and abdominal-to-peripheral adipose tissue were dissected, weighed and frozen at −80 °C.This experimental design was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Academy of Agricultural Science (NAAS-1403). Five-week-old male BALB/c mice were supplied by Central Lab Animal, Inc.  and maintained at ambient temperature (22 ± 1 °C) with 12:12 h light-dark cycles and free access to water and feed. At six weeks of age, the mice were randomly assigned to one of five treatment conditions: (1) NFD; (2) HFD; (3) HFD with 100 mg·kgAfter draining the blood, the liver and epididymal adipose tissue were fixed in 10% neutral formalin solution for 48 h. Tissues were subsequently dehydrated in a graded ethanol series (75%–100%) and embedded in paraffin wax. The embedded tissue was sectioned (8 μm), stained with hematoxylin and eosin (H & E), examined by light microscopy , and photographed. Epididymal adipose cell volume was estimated using the IMT i-Solution Lite .GAPDH) as an internal control to quantify the relative expression of target mRNA according to the cycling threshold (ΔCt) method. All samples were analyzed in triplicate.Total RNA was isolated from epididymal adipose tissue using Trizol reagent  and RNA concentration and purity were measured using a UV/Vis spectrophotometer . Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit . Real-time polymerase chain reaction (PCR) amplification was performed with Power SYBR Green Master Mix using a 7500 Real-Time PCR System (both from Applied Biosystems), according to the manufacturer’s instructions. The PCR program was as follows: an initial temperature of 95 °C for 5 min followed by 35 thermal cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. For detection of target gene transcripts, we designed specific forward and reverse oligonucleotide primers using Beacon Designer software . The primers sequences are listed in Blood samples were obtained from mice at the end of the experimental period after having been fasting for 15 h. Samples were centrifuged at 3000 rpm for 15 min at 4 °C and serum samples were stored at −70 °C. Serum triglyceride (TG), total cholesterol (TC), high density cholesterol (HDL-C) and low density cholesterol (LDL-C) levels were assayed using enzymatic colorimetric assay kits  and modular analytics . Serum concentrations of leptin were measured with mouse enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer’s instructions. Absorbance was measured using the microplate spectrophotometer, as mentioned above.p < 0.05.Data were presented as averages with standard deviations (mean ± SD) from at least three independent experimental replicates. Statistical analyses employed the R statistical software . Differences among groups were evaluated using Duncan’s post-hoc test of analysis of variance (ANOVA). Differences were considered statistically significant at in vivo, mice were fed either a normal-fat diet or a high-fat diet for six weeks. Mice in the NFD and HFD treatment groups also received no supplement or daily doses of 100 mg/kg ADL, 3000 mg/kg ADL, or 3000 mg/kg MT. Body weight was monitored weekly. Results revealed that the final mean body weight gain for HFD mice (receiving no supplement) was about 90% higher than that of NFD mice. Intriguingly, oral administration of ADL reduced body weight gain induced by HFD for all doses. For mice in the HFD + ADL (3000 mg·kg−1·day−1) treatment group, weight gain was approximately 22.4% lower compared to mice fed only HFD (To investigate the anti-obesity effects of ADL only HFD .−1·day−1) than in the HFD group , mice were sacrificed and the livers and epididymal and abdominal-to-peripheral adipose tissues were removed and weighed. Epididymal adipose tissue is an important marker of visceral adiposity in mice . We founFD group . HoweverIn obese individuals, adipocytes expand to store more energy. Recent studies reveal that adipocytes are not simply storage depots for energy, but also secrete endocrine factors such as adipokine, cytokine and growth factor ,28. Once−1·day−1) and HFD + MT mice were 5760 μm3, 4115 μm3 and 4600 μm3, respectively. In addition, fat accumulation and cell size in liver tissue were reduced in groups receiving HFD + ADL compared to the HFD group (−1·day−1)  C.Tenebrio molitor  [Protaetia brevitarsis (white-spotted flower chafer) [T. molitor and P. brevitarsis larvae consisted of 50.3% and 57.9% protein, 33.7% and 16.6% total fat, 3.7% and 8.4% ash, 9.3% and 10.6% carbohydrate, and 2.9% and 6.7% moisture, respectively. It has also been reported that ADL contains 38.1% protein, 32.7% fat, 4.14% ash, 22.7% carbohydrate and 2.3% moisture [In previous studies, we investigated the nutrient profiles of larvae of ealworm)  and Prot chafer) . T. molimoisture . TherefoIn summary, our results confirm that ADL reduces body weight gain, adipose tissue weight and volumes of abdominal-to-peripheral and visceral adipose tissues in mice fed high-fat diets.−1·day−1) was associated with 12.4- and 25.7-fold reductions in mRNA expression of PPARG and CEBPA, respectively, compared with the unsupplemented HFD group (−1·day−1) mice to be 11.2-fold lower than in the unsupplemented HFD group  and HFD + ADL (3000 mg·kg−1·day−1) mice than in HFD mice (Adipocytes in adipose tissue secrete a variety of proteins, known as adipocytokines leptin, tumor necrosis factor-α, interleukin-6, resistin and adiponectin . Among tHFD mice . These rIn this study, ADL administered to HFD-fed mice prevented the typical increases in body weight and organ weight, particularly epididymal adipose tissue weight, a marker of visceral fat ,45,46. T


Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation. Hebanthe paniculata a plant traditionally used in Brazilian folk medicine has drawn attention to its antineoplastic effects on several animal and cell models . Quantification of caspase-3 positive cells was performed with the same method and expressed as caspase-3 activation index.For immunocytochemical analysis, 2.5 × 106 cells/well were plated on 6-well plates and underwent treatment with SF-100%. Cells were washed in PBS and centrifuged for 5 min, at 1200 rpm, 4°C. The pellet was resuspended in a lysis solution  and incubated for 2 h at 65°C. After 2 h, samples were centrifuged  and the supernatant was collected. Ice-cold ethanol  and ice-cold sodium acetate  were added. This solution was then gently homogenized until DNA formed a cluster and then centrifuged at 6000 rpm, 4°C, for 10 min. Ethanol  was used to wash DNA twice and the pellet was dried. Pellets were dissolved in TE buffer  and were conserved at −20°C until use. Full genomic DNA yield was measured photometrically at 260 nm (Eppendorf). 10 μL of this DNA extract with 5 μL of bromophenol blue was applied in each lane onto a 1.5% agarose gel, a well received molecular weight marker, which indicates the presence of breaks in DNA samples. Electrophoresis was performed in 60 V for 1.5 h. Afterward, the gel was stained with ethidium bromide and analyzed under UV light using the Image Master VDS-CL (Amersham Pharmacia Biotech).To verify the effects of the SF-100% treatment on DNA integrity, we performed the DNA smear technique. This technique consists of electrophoresis of full genomic extracted DNA resolved in 1.5% agarose gel. 2 × 107 cells were plated (100 × 20 mm plates) and underwent SF-100% treatment for 48 and 72 h (n = 6/time). After each treatment time, the medium was removed and cells were washed three times with buffer solution. Cultures were lysed in buffer containing 50 mmol/L Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, and protease inhibitors cocktail . Samples were centrifuged at 16,000 g for 15 min to remove insoluble material and stored at −20°C. Protein concentrations were measured using the Bio-Rad Protein Assay. Thirty-five micrograms of protein was resolved on 13% SDS-polyacrylamide gel and transferred to nitrocellulose membranes . Membranes were probed with rabbit anti-mouse antibodies to CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin E, and p27 , overnight, at 4°C. After incubation with peroxidase-labeled secondary antibody bovine anti-rabbit IgG-HRP  the membranes were developed with chemoluminescence system ECL Plus and visualized in a Hyperfilm ECL . Beta-actin  was used as endogenous control. The relative density of the immunoreactive bands was measured by densitometry .Western blot analysis was performed under the same real-time PCR treatment times. 2 × 106 cells/well were plated on 6-well plates and underwent SF-100% treatment for 48 and 72 h. After each treatment time, total RNA was extracted from the cell culture with RNAspin Mini RNA Isolation Kit  and reverse-transcribed to yield cDNA. For reverse transcription (RT), we used Taqman Reverse Transcription Reagents . Primers and probes for real-time PCR were purchased from Applied Biosystems, and mRNA expression of CDK2 (Assay ID Hs00608082_m1), CDK4 (Assay ID Hs00262861_m1), CDK6 (Assay ID Hs00608037_m1), p27KIP1 (Assay ID Hs00153277_m1), cyclin D1 (Assay ID Hs00277039_m1), cyclin D3 (Assay ID Hs00426901_m1), and cyclin E (Assay ID Hs00233356_m1) was determined with Taqman 20 assays-on-demand expression assay mix . The beta-actin gene (Assay ID Hs00242273_mL) was used as housekeeping gene to normalize the results. Each sample was analyzed in duplicate for each gene, and negative controls were enrolled. The real-time PCR machine was an ABI PRISM 7000 . Analysis of relative gene expression data was performed according to the 2−ΔΔCT method [Real-time PCR was performed in treatment times which showed the greatest reduction in HepG2 cells development. 2 × 10T method . t-tests with two-tailed comparisons. A P value of less than 0.05 indicated a significant difference between groups.All results shown represented the mean ± SD. Comparison of parameters was made relative to untreated control groups using Mann-Whitney or StudentP < 0.001) cell viability from 24 to 72 h in comparison to control  decreased and consequently increased in the resting or initial phase (G0/G1) independent of the treatment. However, at 24 h incubation time the SF-100% increased cell populations in sub-G1 (dead or haploid cells) and S phases compared to control treatment. This effect on cells in the S phase was not so clear after 48 and 72 h of SF-100% treatment likely due to the incubation time that also affected cells in this phase .To verify whether SF-100% abrogates DNA replication and delays cell cycle, BrdU was used  to identify replicating cells after treatment. BrdU-positive cells were detected by immunocytochemistry and the proliferation index (PIx) was calculated. As observed in cell cycle analysis, the incubation time reduced DNA replicating cells independent of treatment. However, when compared between treatments, the PIx significantly decreased 22% at 24 h, 62% at 48 h, and 36% at 72 h of incubation with SF-100% . These rTo determine whether treatment with pfaffosidic fraction increased sub-G1 cell population by inducing apoptosis, caspase-3 positive cells were measured by immunocytochemistry and the caspase-3 activation index was calculated. As shown in We also analyzed whether the apoptosis induction shown by HepG2 cells after the pfaffosidic fraction treatment was a result of DNA strand breaks. This hypothesis was investigated by analysis of DNA fragmentation using a classical DNA laddering on agarose gel electrophoresis. The treatment did not affect DNA integrity .KIP1 was investigated by quantitative PCR analysis  and its extracts showed antiproliferative properties in several experimental models in vivo and in vitro. In fact, previous studies from our group showed that diet containing 2% of H. paniculata powdered root (2% PP) has a chemopreventive effect in an infant mouse hepatocarcinogenesis model induced by diethylnitrosamine (DEN) [ H. paniculata on survival and proliferation of a human hepatocellular carcinoma cell line (HepG2) and found that pfaffosides inhibit proliferation and induce apoptosis of HepG2 cells.Roots fromne (DEN) , 6. In tne (DEN) . Nonethe H. paniculata root that are associated with its therapeutic properties [Pfaffosides are triterpenoid saponins found inoperties . Studiesoperties , 10, 15.operties –20, as woperties –24. Accooperties , antitumKIP1, p21CIP1, and p57KIP2 are responsible for negatively regulated cell cycle, so in quiescent cells p27KIP1 levels are high, but once cells enter the cycle, they fall [KIP1 protein is targeted for degradation through its phosphorylation at threonine 187 (T187) by kinases such as cyclin E/CDK2 or cyclin A/CDK2 [KIP1. Considering that high levels of p27KIP1 can cause S phase arrest by preventing activation of cyclin E/CDK2, this could be the explanation by which HepG2 cells undergo cell cycle arrest in the S phase during pfaffosidic exposure. In accordance with our results, a study evaluated the effects of Gatifloxacin, a new antibiotic, under pancreatic cell line Panc-1. The authors reported that Gatifloxacin showed anticancer activity in the pancreatic cell line by S phase cell cycle arrest, due to increased levels of p27KIP1 [It is well known that cell cycle progression is tightly regulated by the activity of protein kinases complexes, each consisting of a cyclin-dependent kinase (CDK) and cyclins. Cyclin/CDK complexes are formed and activated to control the entry into and passage through various phases of cell cycle . Progreshey fall . These ihey fall . p27 is n A/CDK2 , 30. Con p27KIP1 . HoweverKIP1 and in gene expression of cyclin D1 and cyclin D3. Taking into account that cyclin D/CDK4 is required for progression through G1 phase and cyclin E/CDK2 is required for the G1/S transition [ P. paniculata could be considered the mechanism of its antitumor activity. Additional evidence for proliferation inhibition and apoptosis induction by depletion of cyclin E was reported in primary cells obtained from DEN-induced mouse liver tumors where cyclin E silencing allows p53 de novo expression and activity inducing p21 and reducing antiapoptotic Bcl-XL levels [ H. paniculata since previous results from our group using the same mouse hepatocarcinogenesis model induced by diethylnitrosamine (DEN) also evidenced that H. paniculata powdered root reduced proliferation and increased apoptotic cell death [After 72 h of treatment of HepG2 cells with pfaffosidic fraction, we also observed reduction in protein levels of cyclin E, CDK2, CDK4, and p27ansition , we coulansition . Taken tL levels . This stll death .Apoptosis is a highly controlled process of programmed cell death, responsible for the elimination of undesirable and defective cells, and it is one of the mechanisms of action found in antitumoral drugs . The cas H. paniculata roots were due to S phase cell cycle arrest and induction of apoptosis by activation of caspase-3. The increasing knowledge of the antitumor cell cycle based properties of H. paniculata roots can provide the development of new antineoplastic therapies for the hepatocellular carcinoma since these cells proliferate at high rates compared to normal hepatocytes that are mostly quiescent.In conclusion, results demonstrated that antitumoral effects of pfaffosidic fraction obtained from