However, the biosynthetic pathway and physiological role of this gas under anaerobic conditions remains largely unknown. In the present study, we demonstrate that cysteine desulfurase (IscS), not 3-MST, is the primary source of endogenous H2S in E. coli under anaerobic conditions. A significant decrease in H2S production under anaerobic conditions was observed in E. coli upon deletion of IscS, but not in 3-MST-deficient bacteria (ΔmstA). Furthermore, the H2S-producing activity of recombinant IscS using L-cysteine as a substrate exhibited an approximately 2.6-fold increase in the presence of dithiothreitol (DTT), indicating that H2S production catalyzed by IscS was greatly increased under reducing conditions. The activity of IscS was regulated under the different redox conditions and the midpoint redox potential was determined to be −329 ± 1.6 mV. Moreover, in E. coli cells H2S production from IscS is regulated under oxidative and reductive stress. A mutant E. coli (ΔiscS) strain lacking a chromosomal copy of the IscS-encoding gene iscS showed significant growth defects and low levels of ATP under both aerobic and anaerobic conditions. The growth defects could be fully restored after addition of 500 μM Na2S (an H2S donor) under anaerobic conditions, but not by the addition of cysteine, sodium sulfite or sodium sulfate. We also showed that the addition of 500 μM Na2S to culture medium stimulates ATP synthesis in the mutant E. coli (ΔiscS) strain in the logarithmic growth phase but suppresses ATP synthesis in wild-type E. coli. Our results reveal a new H2S-producing pathway in E. coli under anaerobic conditions and show that hydrogen sulfide from IscS contributes to sustaining cell growth and bioenergetics under oxygen-deficient conditions.Endogenous hydrogen sulfide (H In addioduction . In mamm2.6.1.3) .2S production derived from mammalian cells, the bacterial production of H2S was described in the 19th century but was considered to be a byproduct of sulfur metabolism that lacked physiological roles  grown in Luria-Bertani (LB) broth, H2S is primarily generated by 3-MST under aerobic culture conditions and can defend against oxidative stress induced by antibiotics (2S production in 3-MST-deficient E. coli (ΔmstA) grown in minimal medium was not significantly altered compared with that in wild-type E. coli  sufficient to release persulfide-bound sulfur from IscS as H2S  is a pyridoxal phosphate (PLP)-containing enzyme that catalyzes the conversion of cysteine to alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue, and this enzyme is highly conserved throughout all kingdoms of life . Cysteinthreitol . In the S as H2S . In planogenesis . Based o2S production in E. coli under anaerobic conditions was primarily generated from cysteine desulfurase but not 3-mercaptopyruvate sulfurtransferase. A mutant E. coli (ΔiscS) strain lacking IscS activity was observed to display a remarkable decrease in H2S production under anaerobic conditions, but not under aerobic conditions. H2S generated by purified recombinant IscS exhibited an approximately 2.6-fold increase in the presence of DTT. Furthermore, the addition of exogenous Na2S (an H2S donor), but not cysteine, sodium sulfite or sodium sulfate can stimulate ATP synthesis in the mutant E. coli (ΔiscS) strain under anaerobic conditions, indicating that H2S from cysteine desulfurase in E. coli contributes to sustaining cell growth and bioenergetics.In this study, we report that endogenous HE. coli DH5α  was used to amplify recombinant plasmids, and E. coli BL21(DE3) pLysS (Tiangen) was the host strain for IscS expression. E. coli BW25113 and the mutant strain BW25113 (ΔiscS) were kindly provided by Jingdan Liang from Shanghai Jiao Tong University (cyuA) was kindly provided by Sheng Yang from Chinese academy of science. Sodium phenylpyruvate (PPNa) and diamide were obtained from TCI . Dithiothreitol (DTT), trans-4,5-dihydroxy-1,2-dithiane (DTToxi), 7-Azido-4-methylcoumarin, lead nitrate and L-cysteine were obtained from Sigma-Aldrich . The other materials used in this study were purchased from Sangon . Unless otherwise specified, all chemicals were used as they were received.The plasmid pET-28a  was used for IscS expression. iversity . The mutE. coli cysteine desulfurase iscS gene GenBank (Gene ID: 947004) was cloned using E. coli DH5α genomic DNA as template for PCR. All the primers used in this study are listed in E. coli, the iscS gene was inserted into the BamHI and XhoI sites of the expression vector pET-28a in the correct reading frame and was transformed into E. coli BL21(DE3) pLysS.The E. coli cells with the IscS expression constructs were grown in LB medium containing kanamycin (50 μg/ml) and chloramphenicol (50 μg/ml) until the optical density at 600 nm (OD600) reached 0.6–0.8. The expression of IscS was induced by adding 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cells were further cultured for 16 h at 30°C. The harvested cells were resuspended in lysis buffer containing 50 mM phosphate buffered saline , 300 mM NaCl and 30 mM imidazole. The resuspended cells were then sonicated using an Ultrasonic Cell Disruption System . Subsequently, the supernatant was collected by centrifugation at 12,000 rpm for 30 min at 4°C and was loaded onto a HisTrap FF column  pre-equilibrated with lysis buffer. The recombinant IscS was eluted with 300 mM imidazole in 50 mM PBS, pH 7.4, and the collected fractions were desalted using a HiTrap Desalting column  that had been equilibrated with 50 mM Tris-HCl, pH 8.0. The fractions containing IscS protein were pooled and stored at −80°C. Protein concentration was determined by the BCA protein assay reagent kit , and bovine serum albumin was used as a standard.Recombinant 2S production in the reaction is trapped by Pb(NO3)2 to produce lead sulfide (PbS). Subsequently, in acid solution PbS reacts with N, N-dimethyl-p-phenylenediamine to produce methylene blue in the presence of FeCl3. The reaction mixture  contained 50 mM Tris, 1 mM DTT, 0.4 mM [Pb(NO3)2], pH 8.0, and 5 μg IscS. The reaction was initiated by the addition of 1 mM L-cysteine and was incubated for 30 min at 37°C, after which the reaction was terminated by the addition of 25 μl of 20 mM N, N-dimethyl-p-phenylenediamine in 7.2 M HCl and 25 μl of 30 mM FeCl3 in 1.2 M HCl. After incubating for 15 min, the samples were centrifuged at 12,000 rpm for 10 min. The supernatants were then transferred to 96-well plates, and the absorbance was determined at 670 nm in a multifunctional microplate reader. The H2S concentration was calculated using a standard curve that was prepared using different concentrations of sodium sulfide.The activity of IscS was quantified using the methylene blue method as previously described, with the following modifications . First, oxi (0.1–9.975 mM), pH 7.4. The total DTT concentration in the redox buffers was 10 mM, and all solutions were de-oxygenated by bubbling with nitrogen for 30 min. The DTT/DTToxi redox potential was calculated according to the Nernst equation (Eq. 1),The effect of the redox potential on the activity of IscS was determined in solutions with various redox potentials as previously described . Redox bE0 = −352 mV at pH 7.4, R is the gas constant, T is the absolute temperature, and F is Faraday’s constant, n = 2, and [DTToxi] and [DTT] are molar concentrations of oxidized and reduced DTT, respectively. Subsequently, recombinant IscS (5 μg) was preincubated in solutions with different redox potentials for 1 h at 37°C, and the reaction was initiated by the addition of 10 mM S-methylcysteine. After incubation for 30 min at 37°C, the H2S-producing activity of IscS was measured using the methylene blue method.Where L-cysteine in the presence 1 mM DTT, and the kinetic constant Km was calculated using non-linear regression in GraphPad Prism version 7  and Tris-HCl (pH 8.0–10.5) in the presence of 1 mM DTT. The kinetics of IscS activity was investigated using varying concentrations of ersion 7 .2S production in the wild-type and mutant E. coli cells as previously described (3)2] was affixed to the culture bottle mouth above the level of the liquid culture. Overnight cultures were diluted 1:100 in LB medium and incubated for 8 h at 37°C. To anaerobically culture the wild-type and mutant E. coli strains, the culture medium was deoxygenated by nitrogen bubbling for 10 min. Stained paper was scanned and quantified using Image-Pro Plus 6.0, and the results were normalized by OD600.A lead nitrate detection method was used to monitor Hescribed . Filter 2S probe 7-azido-4-methylcoumarin (AzMC) was used to measure H2S production in cells (E. coli strains (ΔmstA and ΔiscS) were cultured in a sealed culture bottle at 37°C under anaerobic conditions. After 3 h of culture, H2O2 and DTT were injected into the sealed culture bottles at a final concentration of 1 mM and 5 mM respectively followed by incubation for 1 h at 37°C. Subsequently, the cultures were collected by centrifugation at 4,000 rpm at 4°C for 10 min followed by washing 3 times with ice-cold PBS. The precipitate was resuspended in PBS containing 10 μM 7-Azido-4-methylcoumarin (AzMC). The cell suspension was lysed by five cycles of repetitive rapid freezing in liquid nitrogen and thawing in a 37°C water bath. The supernatant was collected by centrifugation at 15,000 rpm at 4°C for 5 min, and the fluorescent intensity was measured with a F-4500 fluorescence spectrophotometer (λex = 365 nm and λem = 450 nm).A commercially available fluorescent Hin cells . The mutmstA, ΔiscSΔmstA and ΔsufS mutant derivatives of strain BW25113 were created using the gene knockout method described by Sheng Yang . For anaerobic conditions, all the cultures were deoxygenated by nitrogen bubbling for 10 min, and E. coli was cultured in a sealed culture bottle at 37°C with shaking (100 rpm). The OD600 values were determined using a spectrophotometer at specific times. To determine the effect of Na2S on the growth of the wild-type and mutant E. coli strains, different concentrations of Na2S were injected into the sealed culture bottles.Fifty microliters of overnight culture  was inoculated into a culture bottle containing 5 ml of LB medium with the appropriate chemicals as described in the text or figure legends. For aerobic conditions, E. coli (ΔiscS) grown under anaerobic conditions with or without 500 μM Na2S was determined using an SDH activity assay kit . One unit of SDH activity was defined as the amount of enzyme catalyzing the removal of 1 nmol 2,6-dichlorophenol indophenol per minute at 37°C under the specified conditions. The activity of aconitase (ACO) was measured according to the method described by The activity of succinate dehydrogenase (SDH) in the wild-type and mutant E. coli cells was determined using the luciferin/luciferase reaction with an ATP Assay Kit  following the manufacturer’s instructions. The wild-type E. coli, mutant E. coli (ΔiscS) and E. coli (ΔmstA) strains were growth at 37°C under aerobic or anaerobic conditions as mentioned above. Next, 50 μl of overnight culture was inoculated into a culture bottle containing 5 ml of LB medium. To determine the effect of Na2S on the synthesis of ATP in the wild-type and mutant E. coli strains, and Na2S solution was injected into the sealed culture bottle at a final concentration of 500 μM. After incubation for 2.5, 5, 7.5, and 10 h, the cultures were centrifuged at 4,000 rpm at 4°C for 10 min to collect the precipitate, which was followed by washing 3 times with ice-cold PBS. The precipitate was resuspended in 500 μl of a 50 mM HEPES buffer containing 500 mM NaCl, pH 7.4. The cell suspension was lysed by 5 cycles of a rapid freeze-thaw procedure that involved freezing in liquid nitrogen followed by thawing in a water bath at 37°C. Subsequently, the supernatant was collected by centrifugation at 15,000 rpm at 4°C for 5 min, and 20 μl of the lysate was added to the reaction solution. Luminescence was measured using a bioluminometer, and the results are presented as nmol ATP/mg protein 2]-soaked paper, which specifically reacts with H2S to form a brown lead sulfide precipitate, where the H2S concentration is directly proportional to the change in the rate at which the paper is stained  strain and a double mutant (ΔiscSΔmstA) strain were constructed. These mutant E. coli strains were confirmed by agarose gel electrophoresis and sequencing  strain was constructed. The mutant E. coli (ΔcyuA) strain was confirmed by agarose gel electrophoresis and sequencing  strain which was verified by agarose gel electrophoresis and sequencing  strain and the double mutant (ΔiscSΔmstA) strain greatly decreased under aerobic conditions, but not under anaerobic conditions. Conversely, H2S biogenesis in the E. coli (ΔiscS) mutant and (ΔiscSΔmstA) mutant dramatically decreased under anaerobic conditions, but not under aerobic conditions (iscSΔmstA) strain produced the same level of H2S as the mutant E. coli (ΔmstA) under aerobic conditions and the same level of H2S as the mutant E. coli (ΔiscS) under anaerobic conditions. However, the mutant E. coli (ΔcyuA) strain grown in LB medium generated wild-type levels of H2S both aerobically and anaerobically (E. coli (ΔcyuA) grown with cysteine generated substantially less H2S under anaerobic conditions (2S product between the mutant (sufS) strain and a wild type strain both aerobically and anaerobically  of 0.8 ± 0.05 mM at pH 8.0  using 1 mM L-cysteine as a substrate in the absence or presence of DTT. The activity of IscS was determined to be 28 nmol⋅min–1⋅mg protein–1 in the presence of 1 mM DTT, 2.6-fold higher than that of IscS in the absence of DTT  was 6.3-fold higher than that of IscS in the absence of DTT  strain under reductive stress exhibits a 2.5-fold higher than that under oxidative stress. However, H2S production in an IscS-deficient E. coli (ΔiscS) strain was not affected by the addition of 1 mM or 5 mM DTT. These results indicated that H2S production from IscS is redox regulated in E. coli cells under anaerobic conditions.We next investigated the effect of the redox potential on the activity of IscS. The activity of IscS in solutions with various redox potentials was determined using the methylene blue method. As shown in E. coli is greatly altered under aerobic versus anaerobic conditions. Therefore, we speculated that H2S production from IscS would promote the growth of E. coli under anaerobic conditions. In the present study, Na2S was used as a source of exogenous H2S. Indeed, the addition of exogenous Na2S significantly increased the intracellular content of H2S in E. coli cells (2S on the growth rates of the wild-type and the mutant E. coli (ΔiscS) strains under anaerobic conditions. As is shown in 2S at final concentrations of 20–200 μM did not significantly affect the growth of wild-type E. coli, whereas 500–2000 μM Na2S greatly inhibited its growth (E. coli (ΔiscS) strain was significantly promoted by the addition of exogenous Na2S at final concentrations of 100–500 μM (2S (2S on the growth rates of the wild-type and the mutant E. coli (ΔiscS) strains under aerobic conditions was also determined. As shown in 2S significantly inhibited the growth of wild-type E. coli (2S did not significantly promote the growth of the mutant E. coli (ΔiscS) strain under aerobic conditions (2S promotes the growth of the E. coli (ΔiscS) mutant under anaerobic conditions, but not under aerobic conditions.The energy metabolism pathway in li cells , but didli cells . First, s growth . Expecte0–500 μM , and the0 μM , sodium sulfite (0.01–5 mM) and sodium sulfate (0.01–5 mM) on the growth of the wild-type and the mutant E. coli (ΔiscS) strains grown in LB medium and M9 minimal medium under anaerobic conditions were determined. The results indicated that none of the three sulfur compounds could significantly promote the growth of the mutant E. coli (ΔiscS)  .E. coli, the functions of various Fe-S cluster containing proteins in IscS-deficient mutant E. coli (ΔiscS) are impaired. To investigate whether exogenous H2S rescues Fe-S cluster synthesis in the E. coli (ΔiscS) mutant, the activities of two Fe-S cluster containing enzymes SDH and ACO in E. coli grown with or without 500 μM Na2S were determined. As shown in 2S to culture medium did not significantly increase the activities of these two enzymes in the mutant E. coli (ΔiscS) lysate. These results indicated that growth promotion of the mutant E. coli (ΔiscS) by the addition of Na2S should not be attributed to the rescue of Fe-S cluster synthesis.As IscS is involved in the biogenesis of iron-sulfur clusters in 2S promoted the growth of the E. coli (ΔiscS) mutant, we hypothesized that H2S is involved in cellular energy metabolism by stimulating the synthesis of ATP. After a 2.5–10 h incubation of the wild-type E. coli, mutant E. coli (ΔmstA) and E. coli (ΔiscS) strains under anaerobic conditions, the ATP content in cell lysates was measured. As shown in E. coli cells decreased from 10.1 to 4.8 nmol mg–1 protein after the addition of 500 μM Na2S to the culture, and the ATP content in the mutant E. coli (ΔmstA) cells decreased from 10.9 to 4.6 nmol mg–1 protein. These results suggested that 500 μM H2S is toxic to wild-type E. coli and mutant E. coli (ΔmstA) under anaerobic conditions. In contrast, the ATP content in the mutant E. coli (ΔiscS) cells increased ∼2.3-fold (from 2.6 to 5.9 nmol mg–1 protein.) in the presence of 500 μM Na2S. However, exogenous Na2S did not significantly affect ATP content in all three strains in the stable growth phase after incubation for 10 h (2S promotes ATP synthesis in the mutant E. coli (ΔiscS) during the logarithmic growth phase under anaerobic conditions.As exogenous Hfor 10 h , 6A. TheE. coli cells cultured under aerobic conditions. As shown in E. coli (ΔmstA) cells was greatly decreased after the addition of 500 μM Na2S to the culture. Exogenous Na2S (500–2000 μM) significantly inhibited the growth of wild-type E. coli under aerobic conditions (2S inhibited the biosynthesis of ATP in the wild-type and the mutant E. coli (ΔmstA) cells. Interestingly, unlike under anaerobic conditions, the ATP content in the mutant E. coli (ΔiscS) cells cultured under aerobic conditions was not significantly affected by the addition of 500 μM Na2S. These results indicated that hydrogen sulfide from IscS sustains cellular bioenergetics in E. coli under anaerobic conditions.We next measured the ATP content in the wild-type and the mutant nditions . It is, E. coli (ΔiscS) strain under anaerobic conditions. A previous study indicated that incubation with NaHS (an H2S donor) markedly augmented the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in both purified protein and HEK293 cells  under anaerobic conditions.Finally, we explored the mechanism of hydrogen sulfide promoting ATP synthesis in the mutant 93 cells . As the analysis . Recombiactivity . Howevered above . FurtherL-alanine using L-cysteine as a substrate  significantly promoted the growth of the mutant E. coli (ΔiscS) in the logarithmic growth phase under anaerobic conditions, but not under aerobic conditions (E. coli (ΔiscS) increased ∼2.3-fold in the presence of 500 μM Na2S under anaerobic conditions, but was not altered under aerobic conditions (2S might promote the growth of the mutant E. coli (ΔiscS) by directly or indirectly rescuing these defects.IscS acts as a sulfur donor and is involved in the biogenesis of iron-sulfur clusters in the model bacterium ynthesis . Under onditions . Accordinditions . It shounditions . Exogeno2S is toxic to wild-type E. coli under both aerobic and anaerobic conditions, whereas it promotes the growth of the mutant E. coli (ΔiscS) under anaerobic conditions. The mechanism of the opposite effects of Na2S on the wild-type and the mutant E. coli (ΔiscS) is not known but could result from altered energy metabolism pathways. Previous studies showed that the toxicity of H2S has been attributed to its ability to inhibit cytochrome oxidase of the electron transport chain, resulting in the inhibition of ATP production and metabolic suppression (2S (500 μM) would inhibit cytochrome oxidase of wild-type E. coli, and consequently inhibited cell growth and ATP production (E. coli (ΔiscS), however, the function of electron transport in respiratory chain complexes was severely impaired due to defects in iron-sulfur cluster synthesis. Therefore, dysfunctional cytochrome oxidase or respiratory chain complexes may be insensitive to H2S treatment, resulting in enhanced H2S tolerance in the mutant E. coli (ΔiscS).Additionally, in the present study it was observed that 500 μM Napression . In the oduction . In the 2S in mammalian mitochondria stimulates the production of ATP, which is based on the mechanism by which H2S donates electrons to the electron transport chain through H2S oxidation catalyzed by sulphide:quinone oxidoreductase (SQR)  under aerobic conditions  . Additionditions . Howevernditions . Third, 2S in E. coli under anaerobic conditions, as evidenced by assays using chemical inhibitors and iscS, mstA, cyuA and sufS knockout strains. Hydrogen sulfide from IscS sustains cell growth and bioenergetics in E. coli under anaerobic conditions.In summary, our results clearly demonstrated that IscS, not 3-MST, is the primary source of HAll datasets generated/analyzed for this study are included in the manuscript/WN conceived and designed the experiments. JW, XG, HL, HQ, JQ, SY, and WN performed the experiments. WN, JW, and JS analyzed the data. JW and WN wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In 2016, the Government of India introduced the oral rotavirus vaccine  in 4 states of India as part of the Universal Immunization Programme, and expanded to 5 more states in 2017. We report four years of data on rotavirus gastroenteritis in hospitalized children < 5 years of age prior to vaccine introduction.Children from 7 sites in southern and northern India hospitalized for diarrhoea were recruited between July 2012 and June 2016. Stool samples were screened for rotavirus using enzyme immunoassay (EIA). The EIA positive samples were genotyped by reverse-transcription polymerase chain reaction.Of the 5834 samples from the 7 sites, 2069 (35.5%) were positive for rotavirus by EIA. Genotyping was performed for 2010 (97.1%) samples. G1P[8](56.3%), G2P[4](9.1%), G9P[4](7.6%), G9P[8](4.2%), and G12P[6](3.7%) were the common genotypes in southern India and G1P[8](36%), G9P[4](11.4%), G2P[4](11.2%), G12P[6](8.4%), and G3P[8](5.9%) in northern India.The study highlights the high prevalence of rotavirus gastroenteritis in India and the diversity of rotavirus genotypes across different geographical regions. Pre- vaccine surveillance data is necessary to evaluate the potential change in admission rates for gastroenteritis and circulating rotavirus genotypes after vaccine introduction, thus assessing impact.The online version of this article (10.1186/s12889-019-6406-0) contains supplementary material, which is available to authorized users. Reoviridae family, and is classified into 7 groups (A-G) , P[6], P[8], P[9], P[10], and P[11] [A commercial enzyme immunoassay (EIA) was used to screen all the stool samples for rotavirus antigen . All EIA positive samples were further tested for VP7 (G type) and VP4 (P type) genes using reverse-transcription polymerase chain reaction (RT-PCR) according to the guidelines of the World Health Organization (WHO) for rotavirus detection and characterization . In bries P[4], P, P[8], Pnd P[11] . For sams P[4], P, P[8], Ps P[4], P, P[8], Pp- value using Fisher’s exact test. A p value < 0.05 was considered to be statistically significant. For evaluating the regional variation in rotavirus positivity, the month-wise proportion of rotavirus positivity by EIA in the 2 north Indian sites and 5 south Indian sites were compared over the study period of 4 years.All sites submitted summary data on all diarrhoea-related hospital admissions. The forms were scrutinized for completeness, and the data were entered into Excel 2003 (Microsoft). The data were analyzed to evaluate the prevalence of rotavirus associated diarrhoea, genotype diversity, as well as temporal and regional variation in rotavirus strain prevalence from 2012 to 2016. To evaluate the prevalence of rotavirus associated diarrhoea, the proportion of rotavirus positive stool samples out of the total samples collected was calculated. SPSS software  was used to calculate The study was approved by the institutional review board of CMC, Vellore, India (IRB Minute. No. 7311 dated 20.10.2010). The study was also approved by the institutional review boards/ethics committees of all the sites, which included St. Stephen’s Hospital (New Delhi), SV Medical College (Tirupati), Christian Medical College (Ludhiana), Pragna hospital (Hyderabad), Child Jesus Hospital (Trichy), and Malankara Orthodox Syrian Church Medical College (Kolenchery). All the study participants were enrolled after obtaining written informed consent from the parents/guardian.During the four years of surveillance (July 2012–June 2016), 6576 eligible children hospitalized for AGE were enrolled at the 7 sites ; of which 1960 (97.5%) were G typed, and 1955 were P typed. Both G and P types were determined for 1947 samples Table . The mosp < 0.001). Mixed infections were approximately 8% of all the genotyped strains, while partially typed and untyped samples comprised 1 and 2.1% of the genotyped samples respectively  of the total strains genotyped. The highest proportion of G12 strains were identified in New Delhi (12.8%) in the north and Kolenchery (10.7%) in the south Table . G3P[8] The year wise distribution of rotavirus strains showed interesting trends. G3P[8] appeared in 2015 (0.4%) and by 2016 the frequency increased to 12.1% , G2P4] (9.1%), G9P[4] (7.6%), G9P[8] (4.2%), and G12P[6] (3.7%) , 6.3%, G2Pp = 0.003). The proportions of partially typed and untyped samples were significantly more common in the southern sites  (p = 0.01).Mixed genotypes were more common in the north (10.6%) than the south (6.6%)  and G2P[4] (9.8%) were the two most common genotypes in this study, which is similar to the previous multi-centre surveillance studies conducted in India between 2005 to 2012 , 4 Other9.9% and The emergence of G3P[8] in our study across southern and northern sites from 2015 onwards emphasizes the rapid temporal changes in circulating rotavirus strains. The circulation of uncommon genotypes, such as G1P[4], G1P[6], G2P[6], G2P[8], G3P[4], G3P[6], G4P[6], G9P[6], G12P[4], and G12P[11], and the high proportion of mixed infections (8%) indicate that the children probably acquire infections from a variety of sources, and could serve as sources of new global strains.We did not find any association between the severity of diarrhoea and genotypes of the rotavirus, unlike previous studies which reported that children infected with G1 strains had a greater risk of developing severe diarrhoea than children infected with other rotavirus strains , 18.The strengths of this study include the use of a standardized protocol across the sites and laboratory confirmation of rotavirus diarrhoea in a single reference laboratory. The difficulty in extrapolating the data from 7 sites to the whole Indian population can be a potential limitation of the study, but previous studies have shown that data tend to be similar across the country .To summarize, this study highlights the ongoing high prevalence of rotavirus disease in India and the change in circulating rotavirus strains across sites. The study demonstrates the continued circulation of G9 and G12 strains and the emergence of G3P[8] from 2015 onwards. With new vaccines and the recent introduction of the indigenous oral rotavirus vaccine into the national immunization schedule in nine Indian states by the Government of India, ongoing surveillance is important to interpret the rotavirus epidemiology and evaluation of vaccine effectiveness against a broad range of serotypes, thus assessing the impact of vaccination.Additional file 1:Table S1. List of excluded cases. The list contains details of reasons for site wise exclusion of cases from final analysis. (DOCX 16 kb)Additional file 2:Table S2. Association between rotavirus genotype and severity of diarrhoea. The file contains details of the six common rotavirus genotypes in this study, and their association with severity of diarrhoea in children < 5 years. (DOCX 21 kb)Additional file 3:Table S3. Year wise rotavirus genotype distribution across 7 sites. The file contains details of year wise distribution of rotavirus genotypes in children with acute gastroenteritis in India across 7 sites. (DOCX 19 kb)Additional file 4:Table S4. Rotavirus genotype distribution in north Indian sites. The file contains details of year wise distribution of rotavirus genotypes in the 2 north Indian sites from July 2012 to June 2016. (DOCX 20 kb)Additional file 5:Table S5. Rotavirus genotype distribution in south Indian sites. The file contains details of year wise distribution of rotavirus genotypes in the 5 south Indian sites from July 2012 to June 2016. (DOCX 20 kb)


COL4A5 gene. The c.2858G>T(p.(G953V)) in COL4A5 gene (rs78972735) has been considered pathogenic previously. However, there are conflicting interpretations of its pathogenicity recently. Here we presented 19 Chinese families, out of which 36 individuals (18 probands and 18 family members) carried the c.2858G>T(p.(G953V)) in COL4A5 gene. The clinical manifestations and genetic findings of them were analyzed. We found there were no clinical features of Alport syndrome not only in six probands with c.2858G>T(p.(G953V)) in COL4A5 plus pathogenic variants in other genes  but also in another six probands with only the c.2858G>T(p.(G953V)) variant. The other six probands with a combination of c.2858G>T(p.(G953V)) and another pathogenic variant in COL4A5 had XLAS. Eleven family members  who had only the c.2858G>T(p.(G953V)) variant were asymptomatic. These two males (at age of 42 and 35 years) had normal result of urine analysis and no more clinical traits of Alport syndrome. We conclude c.2858G>T(p.(G953V)) in COL4A5 gene is not a pathogenic variant for XLAS. Individuals should not be diagnosed as XLAS only based on the detection of c.2858G>T(p.(G953V)) in COL4A5 gene.X-linked Alport syndrome (XLAS) is an inherited renal disease caused by mutations in  COL4A3, COL4A4, or COL4A5 genes encoding α3, α4, and α5 chains of collagen type IV, a component of the glomerular basement membrane (GBM) in the kidney [COL4A5 gene [COL4A5 gene, are severely affected [Alport syndrome (AS) is an inherited renal disease characterized by hematuria, proteinuria, and progressive renal failure, variably associated with hearing loss and ocular abnormalities –3. It is4A5 gene , 7. It iaffected , 9. All affected .COL4A5 gene have been included in the Human Gene Mutation Database (HGMD) and about 36% of them are glycine substitutions. Glycine substitutions in the collagenous domain of the α5 (IV) chain are usually considered pathogenic, since glycine is the smallest amino acid and it is critical for the formation of the triple helical structure [COL4A5 gene are found and the relationship of genotype and phenotype are analyzed [Up to now, 1000 mutations in tructure , 14. Howanalyzed , 16. TheCOL4A5 gene (rs78972735) is considered pathogenic in the HGMD database, while it is considered conflicting of pathogenicity in the ClinVar database [COL4A5 gene and their clinical manifestations, providing more evidence to assess its pathogenicity.The c.2858G>T(p.(G953V)) in database –20. In ahttps://www.nrdrs.org.cn/app/rare/), according to the following two criteria (1) cases with gene mutations tested by targeted NGS; (2) cases with the variant of c.2858G>T(p.(G953V)) in COL4A5 gene. Cases without clinical data were excluded. And all their family members in the registry database were included. The clinical data including gender, age, renal and extrarenal manifestations, and renal histopathology results were collected. The Ethical Committee of Peking University First Hospital approved the project, and informed consent was obtained from the probands and their family members.We selected patients from the online registry database of hereditary kidney diseases in children in China (https://www.ncbi.nlm.nih.gov/projects/SNP/), the 1000 Genomes Project (ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/release), and Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/). The allele frequency of the candidate variants were considered to be <5% in these databases. The functional significance of unpublished variants was predicted using SIFT, PolyPhen 2, and Mutation Taster. The evolutionary conservation of the variant sites was evaluated using the PhyloP Primates tool. Splice site effect prediction was performed using Human Splicing Finder (Version 3.1) [Genomic DNA was isolated from the blood samples using a FlexiGene DNA Kit (Qiagen) according to the manufacturer’s protocol. Genomic DNA from probands was detected for pathogenic variants with targeted NGS  by BGI in China. Data were filtered with the variant databases, such as dbSNP (ion 3.1) . The pation 3.1) . If the Sanger sequencing or real-time quantitative PCR (qPCR) was used to confirm candidate pathogenic variants identified with NGS in probands. Genomic DNA from family members was analyzed by Sanger sequencing or qPCR to determine whether they had the same pathogenic variants as the probands.COL4A5 gene. The clinical features and genetic results of the individuals enrolled in this study were shown in Table Nineteen different Chinese families  were enrolled in this study, out of which 36 individuals carried the c.2858G>T(p.(G953V)) in ADCK4 gene and the c.2858G>T(p.(G953V)) variant. And her parents were asymptomatic heterozygous carriers of the variants in ADCK4. The probands from two families (family number 3 and 6), both of them were female, presented with renal failure at an early age, identified with pathogenic variants in TRPC6 gene and NPHP1 gene respectively, in addition to the c.2858G>T(p.(G953V)) variant. Another two probands (family number 4 and 5) presented with hypospadia and microphthalmus, respectively. Genetic test found de novo pathogenic variants in WT gene and PAX2 gene in these two probands, respectively, besides the c.2858G>T(p.(G953V)) variant inherited from their mothers. The proband from family 7 presented with hematuria at age of 3 years, with a positive family history of hematuria in her mother. Genetic test found a heterozygous nonsense variant in COL4A4 gene and the c.2858G>T(p.(G953V)) variant in both the proband and her mother. The last proband (family number 2) in group 1 was diagnosed with congenital nephrotic syndrome (CNS) at age of 1 month after birth. The mother and father undertook genetic test because the DNA sample was not available for proband 2. Genetic test found the parents were heterozygous carriers of the pathogenic variants in NPHS1. Moreover, the mother had the c.2858G>T(p.(G953V)) variant but she was asymptomatic.There were seven families in group 1 (family number 1–7). The proband from family 1, a 10-year-old girl with ESRD, had a younger sister who died at age of 7 years due to ESRD. Both of the two sisters had focal segmental glomerulosclerosis (FSGS). Genetic test of the proband revealed compound heterozygous variants in COL4A5 gene and the c.2858G>T(p.(G953V)) variant in each of the six probands. Both of the two variants in COL4A5 gene in five probands (except proband 9) were inherited from their mothers. It was proved the pathogenic variant c.937-2A>C in proband 9 was de novo, and the c.2858G>T(p.(G953V)) variant was inherited from her mother.There were six families in group 2 (family number 8–13). All of the six probands in this group presented with hematuria and proteinuria. One of the proband from family 13 presented with hearing loss at age of 14 years. Five of the probands underwent renal biopsy, and four had typical GBM changes of AS. Five of the mothers had hematuria with or without proteinuria, except the mother from family 9. Genetic test found a pathogenic variant in COL4A5 gene in all these six probands. The c.2858G>T(p.(G953V)) variant was inherited from their mothers in five of the probands except proband 19, who inherited this variant from her healthy father.There were six families in group 3 (family number 14–19). Three of the probands in this group presented with steroid resistant nephrotic syndrome. The onset age of nephrotic syndrome in these three probands was 1.5, 4, and 6 years, respectively. The result of kidney biopsy in these three probands was FSGS and mesangial proliferative glomerulonephritis (MsPGN) without anomalies of GBM. Two of the probands presented with ESRD at age of 9 and 5 years, respectively. The proband from family 14 presented with steroid sensitive nephrotic syndrome at age of 2 years. All these six probands had no hematuria and no family history of kidney diseases. Genetic test found the c.2858G>T(p.(G953V)) variant in COL4A5 gene was found in five of them (5/16) and a heterozygous pathogenic variant of COL4A4 gene was found in one of them (1/16). These six females presented with hematuria with or without proteinuria at age of 24–44 years and with normal renal function. Another female identified with a heterozygous p.(Tyr1009*) variant in NPHS1 gene and the heterozygous c.2858G>T(p.(G953V)) variant in COL4A5 was asymptomatic. The other nine females who had the heterozygous c.2858G>T(p.(G953V)) variant were all asymptomatic at age of 20–47 years. And the clinical features of the individuals with only c.2858G>T(p.(G953V)) variant in COL4A5 gene were shown separately in Table There were 18 family members identified with the c.2858G>T(p.(G953V)) variant in this study. They were 2 males and 16 females. In the male group, they were two fathers (family number 6 and 19) with normal result of urine analysis at age of 42 and 35 years, respectively. And both of the two fathers had no extrarenal manifestations. In the female group, in addition to the heterozygous c.2858G>T(p.(G953V)) variant, a heterozygous pathogenic variant of NPHS1 gene c.144dup ), one in TRPC6 gene c.335C>T (p.(Pro112Leu)), one in PAX2 gene c.418C>T (p.(Arg140Trp)), and four in COL4A5 gene c.937-2A>C, c.1813_1814dup (p.(Gly606Leufs*13)), c.4024G>T (p.(Gly1342*)), and c.4510delG (p.(Ala1504Profs*50)). The variant c.81G>A (p.(Ala27=)) in COL4A5 was not found in 1000G and ExAC. The mutation Taster predicted it as disease causing. The c.81G>A occurs in the last base pair of exon 1 of COL4A5. Human Splicing Finder predicted it as broken WT donor site and most probably affecting splicing. The c.2858G>T(p.(G953V)) variant (rs78972735) in COL4A5 gene had a high allele frequency of 0.01 in the 1000G Asian populations. It was included in the ClinVar, HGMD, and LOVD database. The mutation Taster predicted the c.2858G>T(p.(G953V)) variant as polymorphism, while Polyphen 2 and SIFT scored it as damaging.Fifteen pathogenic variants were detected in the 19 families in this study besides the c.2858G>T(p.G953V)) variant (Table V) varianCOL4A5 gene. By analyzing the clinical manifestations of 36 individuals (18 probands and 18 family members) who carried the c.2858G>T(p.(G953V)) variant, we found the c.2858G>T(p.(G953V)) variant in COL4A5 gene was not a pathogenic variant resulting XLAS.In this study, we reported 19 Chinese families with the c.2858G>T(p.(G953V)) variant in COL4A5 gene. Moreover, eleven  of the family members who had only the c.2858G>T(p.(G953V)) variant were asymptomatic. The two males (at age of 42 and 35 years) had normal result of urine analysis and no more clinical traits of AS. This strongly suggested that the c.2858G>T(p.(G953V)) variant in COL4A5 gene was not a pathogenic variant for XLAS. In a recently report [COL4A5 gene was identified in four individuals who had no clinical features of AS, which supported our findings.The 19 families enrolled in this study were divided into three groups for analysis. We found probands in group 1 and group 3 presented mainly with early onset of renal failure (median age of 9 years) or nephrotic syndrome. The proband from family 14 in group 3 presented steroid sensitive nephrotic syndrome without hematuria and got complete remission after treatment. In addition, the available renal biopsies of the probands in these two groups were FSGS or MsPGN without abnormal changes of GBM. And the majority of the families in these two groups had no family history of hematuria and had no hearing loss or typical ocular lesions for AS , 24. AppCOL4A5 gene were included in this study. Two of the 11 females, of the youngest ages, developed ESRD at 9-year old and steroid resistance nephrotic syndrome at 6-year old, respectively. And the other nine females aged from 20 to 47 years old had normal urinary analysis. This is different from the natural history of female patients with XLAS. It has been reported that hematuria, usually microscopic, presented in 96% of females with XLAS and proteinuria developed in 72.6–75% [COL4A5 gene had no hematuria. Furthermore, two of the males had normal result of urine analysis at age of 35 and 42. Therefore, no clues to support diagnosis of XLAS in all of the 17 individuals who detected with only c.2858G>T(p.(G953V)) variant in COL4A5 gene, especially for males. In addition, the variant c.2858G>T(p.(G953V)) in COL4A5 gene has a high allele frequency of more than 0.01 in 1000 Genomes, gnomAD—Genomes, gnomAD—Exomes and ExAC, which implies its benign possibility.Totally 11 female and 6 male individuals with only c.2858G>T(p.G953V)) variant in 72.6–75% . Especia53V) variCOL4A5 gene previously reported [COL4A5 gene was tested by RT-PCR and direct sequencing. And in each of the three patients, c.2858G>T(p.(G953V)) in COL4A5 gene and another pathogenic mutation in COL4A5 gene were detected on the same allele in the same family [COL4A5 gene and a splicing variant c.3817+1G>T in COL4A4 gene were detected [COL4A5 gene. Further information and researches are required to explore whether the variant c.2858G>T(p.(G953V)) in COL4A5 gene would have a modifier effect in the phenotype.By reviewing of patients diagnosed as XLAS and detected with c.2858G>T(p.(G953V)) in reported , 18, 20,e family , 18. OnlCOL4A3, c.2996G>A (p.Gly999Glu) in COL4A4, c.1634G>C (p.Gly545Ala) in COL4A4. Accompanied by the wide application of NGS in diagnosis of AS, we would get a better understanding of the pathogenicity of variants in Alport genes. And in order to make an accurate diagnosis, the gene results must combined with clinical manifestations.Glycine substitutions in a collagenous domain of the a (IV) chain are usually disease causing variants. However, a few of Glycine substitutions have been considered neutral polymorphisms, including c.127G>C (p.Gly43Arg) in COL4A5 gene” to confirm the diagnoses of XLAS [COL4A5 gene (including glycine substitutions), any of the following items including [Therefore, we should carefully use the criteria “a pathogenic mutation in the  of XLAS , 24, 26. of XLAS , 25, 27.COL4A5 gene. We conclude c.2858G>T(p.(G953V)) in COL4A5 gene is not a pathogenic variant for XLAS. Individuals should not be diagnosed as XLAS only based on the detection of c.2858G>T(p.(G953V)) in COL4A5 gene.In summary, the cases we reported here providing evidence to reassess the pathogenicity of c.2858G>T(p.(G953V)) in


Oryza sativa) have been reported. Here, we identified a core autophagy gene in rice, OsATG8b, with increased expression levels under N starvation conditions. It was investigated the function of OsATG8b by generating three independent homozygous 35S-OsATG8b transgenic Arabidopsis thaliana lines. The overexpression of OsATG8b significantly enhanced autophagic flux in the transgenic Arabidopsis plants. It was also showed that over-expressing OsATG8b promoted growth and development of Arabidopsis, in which the rosette leaves were larger than those of the wild type (WT), and the yield increased significantly by 25.25%. In addition, the transgenic lines accumulated more N in seeds than in the rosette leaves. Further examination revealed that overexpression of OsATG8b could effectively alleviate the growth inhibition of transgenic Arabidopsis under nitrogen (N) stress. N partitioning studies revealed that nitrogen-harvest index (NHI) and nitrogen use efficiency (NUE) were significantly increased in the transgenic Arabidopsis, as well as the 15N-tracer experiments revealing that the remobilization of N to seeds in the OsATG8b-overexpressing transgenic Arabidopsis was high and more than WT. Based on our findings, we consider OsATG8b to be a great candidate gene to increase NUE and yield, especially under suboptimal field conditions.Nitrogen (N) is an important element required for plant growth and development, which also affects plant yield and quality. Autophagy, a conserved pathway in eukaryotes, degrades and recycles cellular components, thus playing an important role in N remobilization. However, only a few autophagy genes related to N remobilization in rice ( All samples were kept in a dry oven at 80°C for 7 days, then dry mass was weighted and ground to fine powder to assess N remobilization using an elemental analyzer/isotope ratio mass spectrometry system , and unlabeled plants were also collected to determine the natural 15N abundance. The 15N abundance of each sample was expressed as a percent of total N and calculated as atom% sample (A%sample), A%sample = 100×(15N)/(15N+14N). The natural abundance of 15N (Acontrol%) obtained from unlabeled samples were considered using the average value 0.365% to calculate 15N enrichment of labeled samples (E%), E% = A%sample-A%control. NRE in this experiment were estimated as described by Guiboileau et al (2012) .The au et al 12 20] d d15N-LabP < 0.05 and **P < 0.01.The experiments were repeated three times, and data from one representative experiment were shown here. The statistically significant differences of all data were calculated based on Student's t-test at significance levels of *S1 Fig(A) Seeds in germination; (B) 1-week-old seedlings; (C) 2-week-old seedlings; (D) 4-week-old of mature seedlings; (E) Early inflorescence; (F) Mature inflorescences and siliques. The left is transgenic Arabidopsis before staining, the right is that after staining. (A) and (B) Bars = 1 mm; (C) and (F) Bars = 5 mm.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigPCR identification of genomic. DNA of transgenic lines. M, Molecular marker DL2000; 1–9, Independent transgenic lines; +, Positive vector containing 35S-OsATG8b plasmid; -, Negative control.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


DP, RB-like, E2F4 and MuvB) cooperate to repress all cell cycle genes during G1 and inhibit entry into the cell cycle. DNA damage activates p53 leading to increased levels of p21 and inhibition of cell cycle progression. Whether the G1/S and G2/M genes are differentially repressed by RB and the RB-like proteins p130 and p107 in response to DNA damage is not known. We performed gene expression profiling of primary human fibroblasts upon DNA damage and assessed the effects on G1/S and G2/M genes. Upon p53 activation, p130 and RB cooperated to repress the G1/S genes. In addition, in the absence of RB and p130, p107 contributed to repression of G1/S genes. In contrast, G2/M genes were repressed by p130 and p107 after p53 activation. Furthermore, repression of G2/M genes by p107 and p130 led to reduced entry into mitosis. Our data demonstrates specific roles for RB, p130-DREAM, and p107-DREAM in p53 and p21 mediated repression of cell cycle genes.Cell cycle gene expression occurs in two waves. The G1/S genes encode factors required for DNA synthesis and the G2/M genes contribute to mitosis. The Retinoblastoma protein (RB) and DREAM complex ( In response to DNA damage, cells can slow cell cycle progression to enable DNA repair. Repression of cell cycle regulated genes during the DNA damage response contributes to the proliferation arrest. Loss of the tumor suppressor proteins RB and p53 perturb the response of the cell cycle genes to DNA damage in cancer . ImportaRB1 retinoblastoma tumor suppressor gene, binds to the activator E2Fs during G0 and G1 and represses G1/S genes (RBL2 gene) binds the repressor E2F4 and the MuvB (multivulva class B) core consisting of LIN9, LIN37, LIN52, LIN54 and RBBP4 to form the DREAM complex  (Cell cycle gene expression occurs in two distinct waves . The ear/S genes . In addind MuvB) . The repnd MuvB) ,13,14. Tnd MuvB) .MYBL2) and then FOXM1 to CHR elements in G2/M gene promoters (MYB-MuvB (MMB)-FOXM1 complex . During G0 and G1, the DREAM complex binds and represses G2/M genes by MuvB specific binding to CHR elements present in their promoters ,13–15. Dromoters . G2/M ge complex  upon act complex ,20,21.Relief of repression by RB and DREAM of G1/S gene expression during cell cycle entry is dependent, at least in part, on Cyclin-CDK phosphorylation of p130 and RB. During G1, RB is phosphorylated by Cyclin D-CDK4/6 and Cyclin E-CDK2 ,23. CyclRBL1 gene) is structurally similar to p130 and has been reported to bind repressor E2F and MuvB in some cell lines  is activated and contributes to cell cycle arrest (reviewed in (CDKN1A) that, in turn, can inhibit Cyclin E-CDK2 and Cyclin A-CDK2 kinase activity . p53 meiewed in . Emerginiewed in . Cellulaactivity ,32. The activity . In addiactivity . Whether2. SaOS-2 cells with inducible p21 were a gift from Panagiotis Galanos and grown in DMEM supplemented with 10% fetal bovine serum, 1% Pen/Strep and 1% GlutaMAX , and 1% GlutaMAX (Gibco) at 37°C in 5% COGlutaMAX . Where iDESeq2 was used to normalize counts (mean-ratio method), calculate total reads, and determine differential gene expression , and quality control checked using Qubit, TapeStation, and qPCR. Libraries were prepared using Kappa stranded mRNA Hyper Prep and single-end sequenced using Illumina NS500. SaOS-2 total RNA was isolated using Qiagen RNAeasy plus kit, quality checked and quantified using the Agilent Bioanalyzer 2100 in combination with the RNA 6000 Nano Kit (Agilent Technologies). Libraries were constructed from 500 ng of total RNA using TruSeq stranded mRNA Library Preparation Kit (Illumina) following the manufacturer's description. Quantification and quality check of cDNA libraries was performed using the Agilent Bioanalyzer 2100 in combination with the DNA 7500 Kit. Libraries were pooled and sequenced in one lane on a HiSeq2500 in 51 cycle/single-end/high-output mode. Reads were mapped to the Hg19 genome by STAR . HTseq wpression . MA ploteatmap.2 .Predicted G1/S and G2/M genes were extracted from an earlier meta-analysis . A list Cells were transfected with 5 pmol siRNA using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies). Specific siRNA oligos are reported in Supplemental Table S2.For RT-qPCR in HFF samples, total RNA was isolated using Qiagen RNeasy plus kit. cDNA was synthesized using High-Capacity reverse transcription kit (Thermo). qPCR was performed using Brilliant III SYBR Master Mix using Aria Mx3000 real time PCR machine (Agilent Genomics). Ct values were normalized to ACTB and B2M. For RT-qPCR in SaOS-2 p21 samples, RT-qPCR was performed on a Quantstudio 5 (ThermoFisher) using the Power SYBR Green RNA-to-CT 1-Step Kit (ThermoFisher). Ct values were normalized to U6. Primer sequences are reported in Supplemental Table S2.To measure S phase, cells were incubated with BrdU for 1 h before harvest, permeabilized with 2N HCl and blocked with Super Block Buffer (Thermo) in 0.1 M sodium borate for incubation with anti-BrdU-FITC antibody (Fisher Scientific) and propidium iodide with RNase I before analysis by flow cytometry (BD FACS Canto II). To measure mitosis, cells were harvested and fixed in 4% formaldehyde and then 70% cold ethanol. Cells were incubated with anti-phospho-H3-Alexa488 antibody  and propidium iodide with RNase I before flow cytometry.Whole cell lysates were prepared in EBC lysis buffer, resolved by SDS-PAGE, and immunoblotted on nitrocellulose membranes as previously described . To prefRB1 (RB) or RBL2 (p130) selected sequentially using puromycin (puro) and neomycin (neo) resistance cassettes and confirmed by next generation sequencing .2 fold change and adjusted P value revealed that the most significantly up-regulated genes were predominantly G1/S genes including MCM5, ASF1B, CDT1, E2F1 and FAM111B  and PUMA  and G2/M genes   Figure  and E. In Figure . These d2 fold change of these genes. Loss of RB alone significantly reduced the level of repression on G1/S genes by doxorubicin and the combined loss of RB and p130 nearly eliminated the repression effect  Figure . We perft Figure . Similart Figure . More th6 Figure . Combine6 Figure . These rMCM5 and E2F1 . Pa. Pa43). and E2F1 . In conte status . These d2 fold change of a list of high stringency G2/M genes (104 genes in each cell line) was assessed  after doxorubicin treatment in sgP130 or sgRB1+sgP130 cells with siP107  in repression of G1/S and G2/M genes after p53 activation. By specifically reducing levels of each pocket protein, we were able to test the individual contributions of RB, p130, and p107 without affecting the MuvB complex, a key activator of G2/M gene expression as part of the MMB-FOXM1 complex. Repression of G2/M genes and inhibition of entry into mitosis by the combined activity of p130 and p107 may be relevant in cancer cells that have disrupted G1/S checkpoint. Cells containing a A recent study examined the control of gene expression after p53 activation by knocking out LIN37, a component of the MuvB core . KnockouIn conclusion, we demonstrate p53 mediated down-regulation of cell cycle genes can be functionally separated into the repression of G1/S genes by RB, with contributing effects of p130 and p107, and repression of G2/M genes by p130 and p107 Figure . TogetheSequence data are deposited at GEO: GSE128669; GSE128711; GSE135842.gkz961_Supplemental_FilesClick here for additional data file.


INK4A is a biomarker for cervical carcinogenesis and reduces proliferation of stem cells. We aimed to investigate the expression and clinical significance of cyclin-dependent kinase inhibitor 2A (P16INK4A), sex determining region Y-box 2 (SOX2), and Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) in cervical cancer treated with radiotherapy and cervical cell line models. The expressions of P16INK4A, SOX2, and ALDH1A1 were performed by immunohistochemical staining of tumor samples from 139 cervical cancer patients with International Federation of Gynecology and Obstetrics stages Ib to IV. The staining showed high expression in 100, 107, and 13 patients with P16INK4A (>80%), SOX2 (≥10%), and ALDH1A1 (50%), respectively. The high-P16INK4A group had a higher five-year overall survival (OS) rate and disease-free survival (DFS) than the low-P16INK4A group . The low-P16INK4A/high-SOX2 and low-P16INK4A/high-ALDH1A1 groups had a worse five-year OS and DFS rate than the high-P16INK4A/low-SOX2 and high-P16INK4A/low-ALDH1A1 groups, respectively. Depletion of P16INK4A promoted chemoresistance and radioresistance of cervical cancer cells increased the expression of SOX2 and ALDH1A1 and exhibited higher self-renewal ability. These results suggest that lower P16INK4A expression associated with higher CSC markers predicts poor prognostic outcomes and is a promising target in patients with cervical cancer.Previous studies have suggested that cancer stem cells (CSCs) resisted radiotherapy and chemotherapy. P16 INK4A, hypoxia, and cancer stem cells (CSCs) may have a major influence on the survival of patients treated with definitive radiotherapy [Cervical carcinoma is the third most common malignancy diagnosed in women worldwide . Treatmeotherapy ,6,7,8,9.CSCs are a small subpopulation of tumor cells that have characteristics of tumorigenesis, multilineage differentiation potential, and self-renewal . Due to INK4A is encoded by the CDKN2A gene and is thought to be a tumor suppressor. It is one of the cell-cycle inhibitors and inactivates the cyclin d-cyclin-dependent kinases (CDK) 4/6 complex. P16INK4A prevents the phosphorylation of Retinoblastoma (Rb) protein by CDK4/6 and therefore maintains Rb in the mode of growth-suppression that arrests cells at the G1 phase of the cell cycle [INK4A expression gradually increases with aging in most mammalian tissues [INK4A accumulated with age and modulated specific functions associated with ageing in stem cells. Increased expression of P16INK4A with age might decrease the self-renewal ability of stem cells [INK4A [INK4A is known to be overexpressed in many cervical intra-epithelial neoplasias (CIN) and invasive cervical carcinomas [P16 tissues . The preem cells ,19. In cs [INK4A . Thereforcinomas ,22.INK4A on altering the stem cell markers, cell behaviors of CCSCs, and clinical prognosis of cervical cancer patients who received radiotherapy. In the present study, we aimed to investigate whether patients with a higher expression of P16INK4A in cervical cancer had a significantly better prognosis. Furthermore, we also warranted exploring whether the depleting P16INK4A expression affected the stem cell markers’ expression profiles, self-renewal abilities, radioresistance, and chemoresistance of cervical cancer cells.Little is known of the impact of P16INK4A of the tumor, immunohistochemical (IHC) staining was performed in 139 cervical cancer samples. According to the expression of P16INK4A, patients’ tumors were scored with grade 0 (negative), grade 1, grade 2, grade 3, and grade 4 expression of P16INK4A, respectively  and high-expression (>80%) groups . However, we did not find that the clinicopathological variables including age, stage, histologic type, histologic grade, tumor size, squamous cell carcinoma antigen (SCC) level, carcinoembryonic antigen (CEA) level, combined chemotherapy, high expression of SOX2, or high expression of ALDH1A1 displayed a statistically significant difference between the two groups  groups . Notablyo groups .INK4A and stem cell markers SOX2 and ALDH1A1 of the tumor samples obtained before radiotherapy and examined the association among these protein expressions with the five-year overall survival (OS) and disease-free survival (DFS) pattern of the patients. The OS and DFS for the entire cohort were 52.5% and 51.6%, respectively. OS and DFS curves of the patients’ tumors with different expressions of P16INK4A are shown in INK4A group had a higher five-year OS rate and DFS rate than the low expression group . The high expression of the SOX2 group had similar five-year OS rates and DFS rates to the low expression group  were significantly poor predictors for DFS.The results of the univariate (log-rank test) and multivariate  analyses of DFS are shown in analysis , stage IINK4A was involved in modulating resistance to cytotoxic agents and irradiation, we knocked-down the expression of P16INK4A by delivering a short hairpin ribonucleic acid (shRNA) specific to P16INK4A in HeLa cells. We selected the stable knockdown cells as HeLa-shP16 cells. The inhibition of P16INK4A mRNA and protein expression reached 88% and 41%, respectively  and DFS  for the patients treated with radiotherapy in our study (INK4A-positive group (grades 1–4) vs. P16INK4A-negative group were 63% vs. 33% (p = 0.07) and 57% vs. 34% (p = 0.09), respectively. Survival outcomes were also compared between patients with P16INK4A >50% vs. ≤50% expression. The five-year specific survival and DFS of the high-P16INK4A group vs. the low-P16 group were 64% vs. 24% (p = 0.003) and 59% vs. 23% (p = 0.0004), respectively. Schwarz et al. used 50% expression of P16INK4A to separate patients into high and low groups and showed a more significant difference in survival outcomes of the two groups. We used the cut-off level of P16INK4A expression as 80% to separate our patients into high and low groups. The patients of low-P16INK4A groups had significantly poor five-year OS  and DFS . Additionally, multivariate analysis showed that lower expression of P16INK4A was one of the independent poor predictors for the DFS of cervical cancer treated with radiotherapy.In agreement with the studies from Lin et al.  and Huanur study A,B. SchwKim et al.  reportedINK4A expression plus stem cell marker (SOX2 or ALDH1A1) was a good predictor of survival outcomes of cervical cancer patients treated with radiotherapy. Our results showed that patients with low P16INK4A/high SOX2 expression had a similar five-year OS rate but worse five-year DFS , SOX-2 , and ALDH1A1 (BioSB Cat# BSB 2443). The slides were further incubated with an indicated secondary antibody. Antigen–antibody complexes were detected by DAB  and counterstained with Gill’s hematoxylin . Staining was graded by two pathologists (I.-C.C. and H.-S.H.) who were blinded to the clinical outcomes and all other data on the patients. Every tumor was given a score according to the extent of stained cells nucleic or cytoplasmic staining of P16INK4A expression  [For immunohistochemical staining, we deparaffinized the sections with xylene rinse, rehydrated with a graded alcohol series , and then rinsed with distilled water. We enhanced antigen retrieval with citrate buffer . Endogenous peroxidase activity was quenched by incubation in a 3% hydrogen peroxide solution. We incubated the slides with primary antibodies, P16taining) . Expresstaining) . Expresstaining) .l-glutamine  in a humidified 95% atmosphere with 5% CO2 at 37 °C and passaged for fewer than six months after receipt or resuscitation.Cervical cancer cell lines, HeLa cells were obtained from the Bioresource Collection and Research Center  and cell line authentication was performed by BCRC. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/mL streptomycin, and 0.4 mM INK4A, the cells were transfected with pTGMP-p16/p19.478. The plasmid of pBaBepuro3 was transfected as control cells. We transfected cells using Lipofectamine 2000 reagent  according to the manufacturer’s instructions. After transfection, the cells were grown in puromycin  at 5 μg/mL for stable clone selection.The plasmids of pBaBepuro3 and pTGMP-p16/p19.478 were acquired through Addgene. For knockdown expression of P162 flasks and incubated in DMEM with 10% FBS at 37 °C with 5% CO2 for 24 h. The flasks were placed on a linear accelerator Clinac 600C/D  with a fixed source skin distance and X-ray irradiation at 4 Gy/min.The 2000 and 1000 HeLa-control and HeLa-shP16 cells were transferred to 25-cm2 flasks for colony formation. After 14 days, colonies were fixed and stained with a mixture of 6% glutaraldehyde and 0.5% crystal violet. Only if a single colony contained more than 50 cells was it scored using a microscope [After irradiation (the 2000 and 1000 cells) and before irradiation (100) cells were seeded separately in 25-cmcroscope . Each asINK4A , anti-CD133 antibody , anti-SOX2 , anti-ALDHA1 , anti-actin , and then incubated with horseradish peroxidase-conjugated secondary antisera . Enhanced chemiluminescence was performed with ECL-Plus .Cells were lysed in lysis buffer supplemented with 1% protease inhibitor cocktail . Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were probed with anti-P16The cellular toxicity assay of the HeLa cells was determined using a cell counting kit-8  according to the manufacturer’s instructions. In brief, HeLa cells were seeded on a 96-well plate at a density of 3000 cells per well. Then, the cells were treated with increasing concentrations of cisplatin  or vehicle (sterile water) for 24 h. CCK-8 was added and the absorbance  at 450 nm was detected using an enzyme-linked immunosorbent assay reader .Single cell suspensions were suspended at a density of 2000 cells/mL in DMEM and seeded into ultra-low attachment 6-well plates . Suspension cultures were continued for 14 days until tumor-spheres were formed.TM Green Master Mix  and Applied Biosystems® 7500 Fast Real-Time PCR Systems . The cycling parameters for all genes were the following: hot-start 95 °C 15 min, 40 cycles of . The PCR primer sequences used are as follows: P16INK4A Forward primer 5′CCC ACC GCA CCG AAT AGT TA3′, Reverse primer 5′ACC AGC GTG TCC AGG AAG3′; OCT-4 Forward primer 5′CCA CAT CGG CCT GTG TAT ATC3′, Reverse primer 5′AGC AAA ACC CGG AGG AGT3′; CD133 Forward primer 5′CAA CCC TGA ACT GAG GCA GC3′, Reverse primer 5′TTG ATA GCC CTG TTG GAC CAG3′; SOX2 Forward primer 5′GGG AAA TGG GAG GGG TGC AAA AGA GG3′, Reverse primer 5′TTG CGT GAG TGT GGA TGG GAT TGG TG3′; ALDH1 Forward primer 5′CTG CTG GCG ACA ATG GAG T3′, Reverse primer 5′GTC AGC CCA ACC TGC ACA G3′; beta-actin Forward primer 5′TCA CCC ACA CTG TGC CCA TCT ACG3′, Reverse primer 5′ CAG CGG AAC CGC TCA TTG CCA ATG3′. We used beta-actin as a control and final data were normalized to it.Quantitative real time polymerase chain reaction (RT-PCR) was performed using Fast SYBRINK4A high-expression (≤80%) and low-expression (>80%) groups for clinicopathological factors were evaluated using the x2 with Fisher exact for categorical variables and the Student’s t-test was used for continuous variables. The median follow-up duration was 73.4 months  for 139 independently validated cases. The endpoint analyzed was disease-free survival (DFS), last visit, or death ). The durations were calculated from the date of operation until the occurrence of recurrence, death, or last follow-up appointment. Univariate survival analyses were conducted using Kaplan–Meier plots and the statistical significance of the difference between the curves were evaluated by the two-sided log-rank test. Furthermore, hazard ratios (HRs) and 95% confidence intervals (CIs), which were computed from univariate and multivariable Cox proportional hazards regression models were used to assess associations between the higher and lower expression of P16, recurrence, and survival. All statistical analyses were performed using the SPSS statistical package. All p values less than 0.05 were considered statistically significant.Comparisons between the P16


Here, we study the presence of NFATc members in human glioma by using U251 cells and a collection of primary human glioblastoma (hGB) cell lines. We show that NFATc3 member is the predominant member. Furthermore, by using constitutive active NFATc3 mutant and shRNA lentiviral vectors to achieve specific silencing of this NFATc member, we describe cytokines and molecules regulated by this pathway which are required for the normal biology of cancer cells. Implanting U251 in an orthotopic intracranial assay, we show that specific NFATc3 silencing has a role in tumour growth. In addition NFATc3 knock-down affects both the proliferation and migration capacities of glioma cells in vitro. Our data open the possibility of NFATc3 as a target for the treatment of glioma.Calcium/Calcineurin/Nuclear Factor of Activated T cells (Ca/CN/NFAT) signalling pathway is the main calcium (Ca Gliomas are among the most severe forms of cancer. Gliomas originate from glia cells  or cancer stem cells, and are classified by the World Health Organization (WHO) into four grades based on malignancy (I to IV). The most common of these fatal tumours is grade IV astrocytoma or glioblastoma (GBM) and the standard treatment is surgical resection followed by radiation and chemotherapy, which does not dramatically improve clinical outcome.2+) dependent signalling pathways are pivotal in the homeostasis of brain tissue. A key element of the cellular response to Ca2+ signals is the action of calcineurin (CN), a Ca2+- and calmodulin-dependent phosphatase firstly discovered in brain tissue, where is highly abundant1 and described later in many other tissues (reviewed in3). CN activation regulates the activation of the Nuclear Factor of Activated T cells (NFAT) family of transcription factors5.Calcium . NFATc members expression has been shown in astrocytoma8, C69 and U251 glioma cell lines10. Inhibition of the Ca/CN/NFATc pathway by cyclosporine A (CsA) and FK506 has a negative impact on the growth/survival rate of rat and human GBM cells and on glioma invasion capacity12. Accordingly, CsA infusion inhibits tumour growth of implanted glioma in vivo11. Although the relevance of the Ca/CN/NFATc pathway in glioma biology has been already described by using pharmacological intervention, the mechanism and contribution of the different individual NFATc members, and their transcriptional program, remain to be fully assessed.NFAT family includes four classic members: c1, c2, c3 and c4 which were first described in immune cells13, but the search for possible cytokine or other immune based therapies remains to be fully explored. Cytokines and cytokine receptors expressed by glioma cells have been shown to modulate cell invasiveness, survival, proliferation and angiogenesis14, and many of these cytokines are over-expressed in high grade human glioma15. Cytokines and chemokines prevalently produced in glioma tissue include Tumour Necrosis Factor alpha (TNF-α)16, IL-8 (CXCL8)19, macrophage chemo attractant protein-1 MCP-1/CCL220, granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2)21 and Chemokine Receptor 3 (CXCR3). In other cellular systems, the expression of these cytokines has demonstrated to be sensitive to the inhibitors of the Ca/CN signalling pathway such as CsA25. The source of these molecules, attributable to glioma cell, surrounding glial cells, tumour-associated macrophages (TAMS), neuronal or lymphocytes, remains to be fully elucidated.Cytokines have been proven to be potent mediators of antitumor immunity, and overall, their use as treatment for glioma has produced mixed results. The initial preclinical successes using systemic or local delivery of recombinant cytokines failed to adequately translate into clinical benefitIn this work, we evaluated the expression of the individual NFATc members and investigated the role of the most abundant, NFATc3, by exogenous activation and knock-down strategies. We showed that NFATc3 has a positive role in tumour growth, since NFATc3 knock-down inhibits both proliferation and migration of U251 glioma cells as a model. Our results suggest that NFATc3 alone is able to modulate a cytokine network required for the normal growth of glioma. Tools limiting NFATc3 might be considered useful to explore future therapeutic approaches against glioma.26. Nevertheless, the expression and the contribution of the different NFATc members in glioma cells have not been completely assessed.CN/NFATc pathway has been previously implicated in the regulation of glioma growth by using pharmacological inhibitors27. First, we evaluated the expression of the individual NFATc members (c1 to c4) by quantitative PCR (qPCR) in U251 cells, together with a collection of human glioblastoma (hGB) cell lines obtained from xenografts. We found that NFATc3 has the highest expression levels in both hGB cell lines and U251. NFATc1 is also clearly expressed, but in relative less amount. NFATc2 transcript was observed only in some hGB cell lines but hardly detected in U251, and NFATc4 expression varied among hGB cell lines  by ionophore A23187 (Io)30. Cells were stimulated with Io (1 μM) for 30 minutes and, as expected, faster migrating bands were detected, corresponding to dephosphorylated forms of NFATc3 and NFATc1 , known to be involved in tumour progression32. We and others have previously shown that RCAN1-4 expression is highly sensitive to NFATc activation. Therefore, it has been used as a sensor for the Ca/CN/NFAT pathway activation in different biological setting34. Thus, we tested if an increase of [Ca2+]i by Io alone was able to induce the RCAN1-4 expression in U251 glioma cells. We also included, as stimulus, the MAPK inducer Phorbol 12-myristate-acetate (PMA) in combination with Io (PIo) to activate other cofactors known to partner with the NFATc proteins to regulate a variety of genes30. We observed that Io alone or in combination with PMA (PIo), increased RCAN1-4 protein levels. This increase was inhibited by CsA pre-treatment   Fig. . When U26), we investigated the individual contribution of NFATc members expressed in U251 glioma cell line. Therefore, we used lentiviral vectors expressing short hairpin RNA (shRNA) to specifically silence each of the two NFATc members mostly expressed in these cells. Lentiviral shRNA vectors targeting NFATc1 and NFATc3, defined as NFATc1 shRNA (KD-c1) and NFATc3 shRNA (KD-c3), and lentiviral vectors encoding for a scramble shRNA (KD-Sc) as control, were previously described and validated29. More than 95% infection efficiency was monitored by flow cytometry of Green Fluorescent Protein (GFP) reporter gene  as a mock control. We have shown in astrocytes that RCAN1-4 depends on NFATc activity alone, while COX-2 (Cyclooxygenase 2) transcription depends on a cooperation between NFATc and cofactors activated by stimulation with PMA30. COX-2 and RCAN1-4 protein expression was used as read out to validate the constitutive activation of this signalling pathway with the Ad NFATc3 CA  NFATc3 (Ad NFATc3 CA) CA Fig. . Cells w CA Fig. ; whereas CA Fig. , coherenlls Fig. . AdenoviTNF-α, CXCR-3, GM-CSF, IL-2 and CCL-2 mRNA expression. As shown in Fig. TNF-α, CXCR-3, GM-CSF, IL-2 and CCL-2 mRNA levels. PMA stimulation was needed to achieve maximal induction of these genes, indicating the requirement of cofactors that are elicited by other signalling pathways, similarly to COX-2 , activating protein (AP-1), CAAT/enhancer-binding protein β , C/EBP homologous protein (CHOP) and cAMP response element binding protein (CREB)38. In our experiments, the presence of PMA alone was sufficient to obtain maximal expression of IL-6 . Next, puromycin-selected mCherry-Luc U251 glioma cells were transduced with KD-Sc (Sc) or KD-NFATc3 (c3) lentiviral vector. Cells were analysed by flow cytometry confirming that 100% of the cells co-expressed mCherry and GFP proteins , when control Sc animals started to show physical distress. Luminescence signal showed that implanted Sc tumour cells were surviving and growing. In contrast, implanted c3 tumour cells was under the detection limit. As a further confirmation that c3 cells were not able to grow in vivo, we visualized by immunofluorescence GFP expression in brains isolated from the two animal groups . Nonetheless, we found that the expression of the individual NFATc members varies in the different human xenografts lines tested, being always NFATc3 the most abundant . The impact of CsA and FK506 as inhibitors of the Ca/CN/NFAT signalling pathway on glioma growth has been published before26. High doses of CsA (40 μM) has been reported to induces apoptosis of rat C6 glioma cells48. However, other authors have not found morphological changes indicative of apoptosis in human glioma cells line such as U251 when using a lower dose, 1 μM49. We have used 1 μM CsA which do not produce significant cell death but inhibit efficiently the activation of Ca/CN/NFAT pathway. Furthermore, KD-c3 and KD-Sc U251 cells present similar cell cycle profiles at 24 hours before injection in the orthotopic mouse model . Our experiments show that even in condition in which NFATc1 and NFATc3 knockdown were efficiently achieved, the majority of the NFATc-directed signalling correlates with NFATc3 presence. Combining the results obtained by silencing or over-expressing NFATc3, this study indicates that this member is involved in RCAN1-4, COX-2, TNF-α, IL-2, GM-CSF and CXCR-3 gene regulation in U251 glioma cells. Although the KD-c3 reduced partially the expression of PIo-inducible IL-8, NFATc3 overexpression did not enhance IL-8 induction above PMA values. It has been shown that IL-8 promoter can be activated by PMA-elicited signals alone in cancer cells38 although, in breast cancer, requires NFATc participation39. Therefore, it is quite plausible that the inhibition of the abundant NFATc3 in U251 might be able to partially compromise the IL-8 transcription in glioma cells.Gene deletion of single NFATc members on immune cells has shown a mild impact, and this fact has been interpreted as the existence of functional redundancy (reviewed in7 (reviewed in6 and references therein) and, in other cell systems, has been related to the response to VEGF34, to SFRP (secreted frizzled-related protein)51 and activated Peroxisome Proliferator Activated Receptor gamma (PPARγ)52. Treatment of glioma cells with CsA inhibited growth of EGFP-transfected glioblastoma cells in organotypic brain slices12. CsA is an inhibitor of the CN, which has different cellular substrates besides NFATc53; if the CsA mediated inhibition of glioma proliferation and migration is NFATc dependent has not been clearly assessed in vivo. Here we show that NFATc3 is required for the normal proliferation and migration capacity of the U251 glioma cells, and for tumour establishment in xenografts.Migration and invasion capacity of glioma cells are main features which account for the aggressive behaviour of brain tumours. Many processed are involved in tumour migration, from cytokine and chemokine expression, regulation of adhesion capacity and changes in the extracellular matrix. A role of NFATc signalling in migration has been described for different cancer cellsCOX-2 and CXCR-3. NFATc dependent COX-2 induction has been observed in different cellular systems such as endothelial cells and breast epithelial cells and is required to promote invasive migration54. High COX-2 expression in tumour cells is associated with clinically more aggressive gliomas and is a strong predictor of poor survival55. More recently, epidemiologic studies have highlighted associations between the regular use of non-steroidal anti-inflammatory drugs (NSAID) and reduced glioma risks in humans and it has been shown that COX-2 pathway promotes gliomagenesis by directly supporting systemic development of myeloid derived suppressor cells (MDSCs) and their accumulation in the tumour microenvironment56. Our results support a specific role for NFATc3 in the positive COX-2 regulation in U251 glioma cells, with clear therapeutic implication for the glioma growth. Interestingly, the opposite role of NFATc1 on COX2 regulation will require to be further explored. The CXCL10/CXCR-3 axis has been shown to be expressed in glioma cells, and NFATc3 dependent regulation of CXCR3 is on line with the role of this receptor in migration and site specific dissemination in many cancerous cells59. Migration is a complex multistep process and we expect that many other factors, besides the genes explored in this study, contribute to cell migration. If these factors are NFATc3 dependent will need further analyses. Preliminary tests by using individual blocking antibodies against target genes were not sufficient to inhibit the migration of control glioma cells (data not shown).We have identified NFATc3-dependent genes that are known to be involved in tumour migration: Inhibition of NFATc-signalling using small molecule inhibitors is predicted to suppress tumorigenesis, hopefully without the secondary effects of the immunosuppressant regimes. Therefore, future cancer therapy targeting NFATc must take into account the different cellular types which participate in glioma microenvironment such as cancer cells, microglia and macrophages, and tools for specific delivery of this regulators will be required.U87 and U251 human glioblastoma cell lines were purchased from ATCC and maintained in DMEM (LONZA), containing 1X Glutamine (LONZA), 10% Fetal Bovine Serum (FBS) (HiMedia), and 100 U/mL PenStrep (LONZA). Cells were genotyped using StemElite ID genotyping system (Promega).Human glioblastoma (hGB) cell lines obtained from xenografts were obtained after injection of small pieces of human tumours  in the flank of immunodeficient mice, as described in60. Samples were obtained from patients treated at Hospital Universitario 12 de Octubre . Informed consent from patients was obtained. None of them was under the age of 18th. The study was performed with the approval and following the guidelines of the Research Ethics Committee of Hospital 12 de Octubre (CEI 14/023).U251 cells were transduced with lentiviral vector (mCherry-Luc) as below. After 48 hours, cultures were monitored for cherry fluorescence. Cherry positive cells were selected with 1 μg/mL puromycin in media and analysed by flow cytometry.30. Total extracts were resolved by 10% SDS-PAGE and proteins transferred to nitrocellulose membranes. The following antibodies were used: anti-NFATc1 , NFATc3 , NFATc2 , NFATc4  α-tubulin (Sigma), β-actin (Sigma), mouse anti RCAN1-4 (as described in30). Antibody was detected with enhanced chemiluminescence (ECL) detection reagent .For whole-cell extracts, cells grown and stimulated were washed twice with cold PBS and lysed for 30 min on ice in 100 μL hypertonic buffer as describedTotal RNA was extracted with Tripure (Roche), and 2 μg were reverse transcribed to cDNA with MMLV-RT (Invitrogen). Real-time quantitative PCR (qPCR) in duplicate was carried out on 100 ng cDNA, using TaqMan probes and SYBR Green system (Applied Biosystems) in ABI Prism 7900 or 7500 Fast Real Time PCR System (Applied Biosystems). Taqman probes used were NFATc1 (cat # Hs 00542678_m1), NFATc2 (cat # Hs 00234855_m1), NFATc3 (cat # Hs 00190046_m1), NFATc4 (cat # Hs 00190037_m1), COX2 (cat #Hs00153133_m1), IL2 (cat #Hs00174117_m1) RCAN1.4 (custom assay hDSCR1.4-E4_5 cat#4331348), Rcan1 (cat# Hs01120954_m1), IL8 (cat# Hs00174103_m1), VEGF (cat#Hs00173626_m1), TNFα (Hs00174128_m1). The following Taqman probes were used as endogenous controls: TFRC (cat #4333770 F), GAPDH (cat Hs99999905_m1) and 18 S rRNA (cat#4319413E). Relative quantification was calculated according to manufacturer’s instructions. Primers used for SYBR Green assays were as follow, TFRC was used as endogenous control.CCL2: Forward: CAGCCAGATGCAATCAATGCCReverse: TGGAATCCTGAACCCACTTCTCxCR3: Forward: TTTGACCGCTACCTGAACATAGTReverse: GGGAAGTTGTATTGGCAGTGGGM CSF: Forward: GTGGCATTCAAGGAGTACCTCReverse: TGATGGCCTTCGATTCTGGATTTFRC: Forward: GCAAGTAGATGGCGATAACAGTReverse: CAATAGCCCAAGTAGCCAATCAT29. The KD sequences employed were as in61.Knockdown (KD) sequences were designed cloned into an EcoRI-NotI LV-Ubq-GFP lentiviral vector (LV) as described previously inKD-Sc (5′-3′): GATCCGGCAACAAGATGAAGAGCACCTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTGTTTAAACGC.KD-c1 (5′-3′): GATCCCCGCCAGTACCAGCGTTTCACTTCAAGAGAGTGCGCTGGTACTGGCTTTTTGGAAC.KD-c3 (5′-3′): GATCCCCGACAGTCGCTACTGCAAGCTTCAAGAGAGCTTGCAGTAGCGACTGTCTTTTTGGAACLentiviruses expressing mCherry and luciferase genes (mCherry-Luc) were produced using PLKO-mcherry-luc-puro-Renilla cDNA (Addgene).et al.62. U251 glioma cells were infected by adding virus (MOI = 6) and incubating for ON at 37 °C in complete medium DMEM 10% FBS and antibiotics. Infection efficiency (GFP or mCherry expression) and cell death (propidium iodide staining) were monitored by flow cytometry.Lentiviruses expressing mCherry-Luc and KD sequences were obtained by transient calcium phosphate transfection of HEK-293 cells with a three plasmid HIV-derived and VSV pseudotyped lentiviral system kindly provided by M. K. Collins . Supernatant containing lentiviral particles was collected 48 h after removal of the calcium phosphate precipitate and was then ultracentrifuged for 2 h at 121,986xg at 4 °C . Viruses were collected by adding cold sterile DMEM and were titrated by qPCR, as described by Scherr 34. Adenoviruses were generated and purified following the standard protocols30. Adenovirus infection was performed on subconfluent U251 glioma cells, and expression of the encoded protein was monitored by immunoblotting.The adenovirus encoding constitutively active NFATc3 (Ad NFATc3 CA) and the control adenovirus (Ad Control) were as describedDivision Index: the average number of cell divisions that a cell in the original population has undergone. Proliferation Index: the total number of divisions divided by the number of cells that went into division. Only takes into account the cells that underwent at least one division. Data was analysed with FlowJo 7.6.3 software .Single cell suspensions were prepared in staining buffer (2% FBS) in PBS. Analysis was performed in a LRS Fortessa X-20 (BD Biosciences) cytometer, using the FlowJo v6.3.4 (TreeStar) and DIVA v8.0 software packages. Proliferation of infected U251 cells was analysed with the Violet Proliferation Dye 450 (BD Horizon). Infected cells were labelled according to the manufacturer’s protocol and stimulated with 2% FBS for three days. Violet staining was determined by flow cytometry in viable cells at day 0 and day 3 after stimulation. Housing and animal experiments were performed according to European Union 86/609/EEC guidelines and approved by the Animal Committee of Instituto de Salud Carlos III. All surgical procedures were carried out under general anaesthesia with 8 μL/g intraperitoneal (IP) anaesthetic solution composed of 10% ketamine  and 9.3% xylazine (Xilagesic) with subcutaneously administration of 10% analgesic solution . After surgery, mice were woken from anaesthesia with 7.7% reverting solution . All of them prepared in PBS.5 of U251 transduced with lentiviral vector as indicated. Cells were resuspended in 2 μL PBS and injection were made into the striatum  using a Hamilton syringe. Animals were sacrificed at the onset of distress or when specify if symptoms were not apparent.gGuided intracranial injections using stereotactic frame (Leica) in athymic nude Foxn1nu mice (Harlan Iberica) were performed by administering 1 × 102/steradian).Mice were injected with 100 μL of luciferin  in PBS (Becton Dickinson), anesthetized with isoflurane and placed in an IVIS Lumina XR Serie III , being kept with the same solution of 2% isoflurane gas in 100% oxygen at a flow rate of 0.5 mL/min during the process. Mice were placed in the IVIS and 5 images were taken every 30 seconds to get the signal peak. Imaging data are reported as , by submerging brains in increasing concentrations of OCT, snap frozen in dry-ice-cooled 2-methylbutane (Sigma) and stored at −80 °C. Coronal sections (30 μm) were obtained with a CM1950 cryostat (Leica Microsystems) and stored at −20 °C until use.For confocal studies, mice were deeply anesthetized by intraperitoneal (ip) injection of a mixture of ketamine and xylazine and transcardially perfused with 25–30 mL of saline solution for 5 min, followed by 10 min with 4% PFA (Sigma), pH 7.4, in 0.1 M phosphate buffer . Brains were obtained and post fixed with 4% PFA for 18–20 h at 4 °C, rinsed in 0.1 M PB and placed in 15% glucose at 4 °C until they sank followed by immersion in 30% sucrose in PB at 4 °C for 72 h. Finally, brains were embedded in tissue freezing medium . Brain maps were imaged using a 20X immersion objective and a 3.0 optic zoom . Map images are presented as maximum projections of z-stacks. Co-localization was measured on one z using the Leica LAS AF program to obtain Pearson coefficient.For wound healing assay, infected U251 cells were plated at 90% confluence in p35 dishes. After 24 h, monolayers were scratched with a p200 pipette tip. Cells were then washed and stimulated with medium containing 2% serum. Wound healing was monitored by time lapse microscopy with a NIKON Ti-Eclipse inverted microscope fitted with a 4X objective lens. Images were acquired with Nikon Nd2 Viewer and cell-free area was calculated with ImageJ software.5/well) were seeded onto 8 µm-pore transwell 24-well inserts (Costar) in DMEM 0.1% BSA (DMEM-BSA). 1 mL DMEM-BSA with or without 2% FBS was added in the lower chamber of the transwell, after 4 hours non migrating cells were removed and the filter was fixed in 4% paraformaldehyde and stained with Hoechst 33342 (Invitrogen). Migrated cells were counted with Image J software in four random fields in 4X pictures using a Nikon Eclipse TE2000-U Microscope in duplicates.For transmigration assay, infected U251 cells . Differences were considered significant at *p < 0.05. For immunoblots, a linear correlation was observed between increasing amounts of input protein and signal intensity.http://cancergenome.nih.gov/) were analysed using GlioVis (http://gliovis.bioinfo.cnio.es/) data portal for visualization and analysis of brain tumour expression datasets63. Data downloaded were analysed using GraphPad Prims 5.0.TCGA expression data (Suplemental material