HTT CAG repeat for which genome-wide genetic scans are now revealing chromosome regions that contain disease-modifying genes. We have explored a novel human–mouse cross-species functional prioritisation approach, by evaluating the HD modifier 6q23–24 linkage interval. This unbiased strategy employs C57BL/6J (B6J) HdhQ111 knock-in mice, replicates of the HD mutation, and the C57BL/6J-chr10A/J/NaJ chromosome substitution strain (CSS10), in which only chromosome 10 (chr10), in synteny with the human 6q23–24 region, is derived from the A/J (AJ) strain. Crosses were performed to assess the possibility of dominantly acting chr10 AJ-B6J variants of strong effect that may modulate CAG-dependent HdhQ111/+ phenotypes. Testing of F1 progeny confirmed that a single AJ chromosome had a significant effect on the rate of body weight gain and in HdhQ111 mice the AJ chromosome was associated subtle alterations in somatic CAG instability in the liver and the formation of intra-nuclear inclusions, as well as DARPP-32 levels, in the striatum. These findings in relatively small cohorts are suggestive of dominant chr10 AJ-B6 variants that may modify effects of the CAG expansion, and encourage a larger study with CSS10 and sub-strains. This cross-species approach may therefore be suited to functional in vivo prioritisation of genomic regions harbouring genes that can modify the early effects of the HD mutation.Huntington’s disease (HD) is a dominant neurodegenerative disorder that is due to expansion of an unstable The online version of this article (doi:10.1007/s00335-014-9552-9) contains supplementary material, which is available to authorized users. HTT (previously HD) (The Huntington’s Disease Collaborative Research Group Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder, usually of adult onset, characterised by involuntary choreic movements, cognitive impairment and behavioural changes. It is caused by the expansion of an unstable polymorphic CAG repeat in HTT CAG-initiated disease process and its outcomes in vivo may be needed to reveal modifier effects at each of many implicated modifier loci across the genome but is impractical on a gene-by-gene basis.Amongst other potential modifier loci identified in genome wide linkage scans , while all other chromosomes from the host strain, e.g. C57BL/6J (B6J), are intact and homozygous  and heterozygous mutant mice display phenotypes at a young age that are dominant, Htt CAG repeat length-dependent and manifest in medium spiny neurons (MSNs) . These mice, commonly referred as CSSQ111/+ knock-in mice to generate progeny with one copy of an AJ chr10 and heterozygous for the HD mutation in an otherwise B6J background. Comparing these F1 mice with B6/J HdhQ111/+ knock-in mice tests if chr10 AJ-B6J genetic variants are of sufficient effect size to alter early, dominant and CAG length-dependent phenotypes, such as Htt CAG somatic instability  of HD onset. Since the human chromosome 6 is in synteny with the mouse chromosome 10 (chr10), we used a cross between CSS10 mice and B6/J HdhA/J/NaJ (CSS10) mice that carry both chr10 from strain AJ on an otherwise C57BL/6J (shortened here to B6J) background  and to the precise size of the expanded CAG repeat allele, which varies in any given progeny due to intergenerational CAG repeat instability. Thus, the actual genotyped Htt CAG repeat size of the parental HdhQ111 mice was 139 CAG repeats. As shown in Fig. Q111 knock-in and wild type (CSS10 and B6J) mice were made in one direction—HdhQ111/+ male × B6J and CSS10 females—in order to control for possible parental effects of the Htt mutant allele. Briefly, male heterozygous HdhQ111/+ knock-in mice were crossed with CSS10 female mice in order to generate wild type Hdh+/+.C57BL/6J.Chr10A/J (Hdh+/+B6J.AJ10) and mutant HdhQ111/+.C57BL/6J.Chr10A/J (HdhQ111/+B6J.AJ10) mice that carried one chr10 from strain AJ on an otherwise B6J background. Subsequently, the same male heterozygous HdhQ111/+ knock-in mice were crossed with B6J female mice in order to generate wild type Hdh+/+.C57BL/6J (Hdh+/+B6J) and mutant HdhQ111/+.C57BL/6J (HdhQ111/+B6J) mice on a complete B6J background. Animal experiments were performed to minimise pain and discomfort, under an approved protocol of the Massachusetts General Hospital Subcommittee on Research Animal Care.The C57BL/6J-Chr10n = 119), we selected two different sets of mice for phenotypic somatic instability (set 1) and immunohistochemistry assays (set 2), with groups consisting of both female and male, Htt mutant and wild type mice from both B6J.AJ10 and B6J crosses (Supplementary Table 1). Set 1 (n = 20) consisted of ten 5-month-old Htt mutant mice per genotype (HdhQ111/+B6J.AJ10 and HdhQ111/+B6J) matched by gender  and Htt CAG repeat size. Set 2 (n = 40) consisted of ten 5-month-old mice per genotype  matched by gender  and Htt CAG repeat size. All Htt mutant mice used in this study were heterozygous for the Htt CAG mutation. As we could not generate data for all the animals in all assays, the number of mice included into the analyses of each test is given in the legends.From the F1 offspring mice  ad libitum and weighed at weekly intervals from the time of weaning (3 weeks) until 5 months (~24 weeks). The following body weight traits were analysed: initial (IW: weight in grams at ~25 days of age), medium (MW: weight in grams at ~98 days of age) and final weight (FW: weight at ~169 days of age). We also calculated the mean weight gain/day in grams for the first (EWG) and last (FWG) ~72 days and the weight gain per day in grams/day (WG), as previously described  amplification assay, with fluorescently labelled primers  and GeneMapper version 3.7 software. All runs included the same control DNAs of known Htt CAG repeat size. Somatic instability was quantified from GeneMapper traces as described previously  using DNeasy Blood & Tissue kit . Genotyping of the HdhCoronal sections of paraformaldehyde-perfused and post-fixed, gelatin-embedded hemisphere brains were performed at Neurosciences Associates  using MultiBrain Technology. With this technology, all mouse brain hemispheres were embedded in one single block that was freeze-sectioned at 35 µm in the coronal plane (throughout the striatum) with a sliding microtome, resulting in free-floating sections. Designated sections across the striatum were stained with either EM48 and thionine counterstain or DARPP-32 at 210-µm intervals. Bright field microscopy was performed with an Olympus BX51 microscope equipped with a Qcolor5 Olympus camera and Qcapture image acquisition software. Images that were to be quantified and compared were taken with the same exposure times. For the EM48 sections, three micrographs (×40 objective) were taken from the dorsal medial striatum in three consecutive sections from each mouse. The number of nuclear huntingtin inclusions was quantified in three striatal areas per mouse and normalised to the number of EM48-positive cells. For the DARPP-32 sections, micrographs (×4 objective) in three consecutive sections were taken from each mouse and DARPP-32 intensity was quantified using ImageJ image analysis software . Statistical analyses were performed using GraphPad Prism 6.00 . Sample size estimation  was calculated using the R package samplesize (http://CRAN.R-project.org/package=samplesize).Pairwise comparisons were determined using an unpaired Mthfd1 l, 1700052N19Rik, Katna1, Sash1, Samd5, Epm2a, Utrn, Stx11, Hivep2, Nhsl1, Tnfaip3 and Ifngr1.A genome wide HD modifier linkage scan has provided strong evidence for a QTL, between markers D6S1009 and D6S2436, at the human 6q23–24 interval, that modulates age at HD neurological onset , homozygous males had a lower body weight than wild type and heterozygous mice at 28 weeks , we analyzed body weight at three different ages . As shown in Fig. p = 0.0008, mean IW ± SD: 10.02 ± 1.89 vs 13.03 ± 1.74 for females and p < 0.0001, mean IW ± SD: 9.22 ± 1.72 vs 13.92 ± 2.29 for males). However, they tended to gain more weight per day than B6J.AJ10 mice , resulting in similar body weights between B6J and B6J.AJ10 mice by week 24 . Therefore, the presence of the AJ10 chromosome was associated with less rapid weight gain, thereby confirming an earlier phenotypic effect on an otherwise B6J background. However, there was no significant difference in weight at any time point between HdhQ111 mutant and wild type mice, for either of the two different chr10 genetic backgrounds.We examined the rate of body weight gain as a primary endpoint because it has been reported, from phenotyping across the members of the entire CSS panel, that dominant AJ chr10 variants dampen the rapid rate of body weight gain of B6J mice, in F1 B6J.AJ10 mice  and constitutional (i.e. in tail DNA) CAG repeat length (139 CAG repeats). Therefore, as expected, we observed a paternal expansion bias (74 % in crosses of HdhQ111/+ mice with B6J and 82 % in crosses with CSS10 mice), but no significant repeat length difference in the transmissions of the Htt 139 CAG allele to HdhQ111/+B6J (mean ± SD = 3.04 ± 3.18) and HdhQ111/+B6J.AJ10 mice (mean ± SD = 2.25 ± 2.77) was detected. Figure Q111 knock-in allele in both HdhQ111/+B6J and HdhQ111/+B6J.AJ10 progeny used in this study.Previous studies have shown that in HdhQ111/+ knock-in mice also exhibit CAG length, age dependent and tissue specific somatic instability, with significant accumulation of expansions in striatum and liver that becomes apparent by 5 months of age . As shown in Fig. Q111/+B6J and +12.71 ± 1.03 for HdhQ111/+B6J.AJ10) followed by the striatum (+8.15 ± 1.51 for HdhQ111/+B6J and +8.40 ± 1.27 for HdhQ111/+B6J.AJ10). There was no significant difference for the instability indices, or for the expansion and contraction indices (data not shown) when comparing the two groups of HdhQ111 mice. As previously described  diffuse-immunostaining nuclear mutant huntingtin and later (6–12 months) intranuclear inclusions of mutant huntingtin amino-terminal fragments, using the anti-huntingtin antibody EM48  and some nuclei exhibited intranuclear inclusions , although the difference did not reach statistical significance . Moreover, the number of intranuclear inclusions was found to depend on the constitutive CAG repeat length of HdhQ111/+B6J.AJ10 mice, with longer CAG lengths resulting in an increased percentage of EM48 nuclei with inclusions (and higher when compared with HdhQ111/+B6J mice with similar CAG repeat length)  in the striatum (and nucleus accumbens) of HdhQ111/+B6J (134.2 ± 7.1) by 14 % when compared with Hdh+/+B6J control littermates (156.0 ± 3.7)  mice showing a 9 % reduction (p = 0.0003) when compared with control littermates (150.7 ± 7.0). However, we did not find any significant effect of the chr10 background in the levels of DARPP-32 when comparing B6J with B6J.10 mice, for both wild type (p = 0.0920) and mutant (p = 0.2521) Htt genotypes. It should be noted, however, that Hdh+/+B6J.AJ10 showed a 3.5 % decrease of DARPP-32 levels when compared with wild type B6J mice so that the similar DARPP-32 immunoreactivity signal in HdhQ111/+B6J.AJ10 and HdhQ111/+B6J striatal sections may reflect a slower rate of decrease in DARPP-32 staining in the former than the latter genotype.The striatum is critically affected in HD patients, where there is a selective and progressive loss of MSNs , may act as dominant modifiers (of strong effect) of early outcomes of the dominant CAG expansion-dependent disease process in heterozygous of HdhQ111/+ mice. We selected disease phenotypes shown to be modifiable by genetic background (B6 vs 129)  and genetic background (B6J), except for the chr10 in the B6J.AJ10 mice that carried one chromosome from the AJ strain.We set a high bar in exploring a novel approach with CSS and HdhA/J variants, whose effects did not require the presence of the Htt CAG repeat allele to be observed, were segregating in our F1 progeny. We replicated a previously reported metabolic effect of chr10A/J variants in moderating the rapid B6J weight gain associated with an A/J obesity-resistance QTL at chr10 . Therefore, the cohorts for the CAG-dependent phenotypes (n = 10) were half the size of the cohorts assessed for body weight. Based on our current data, we would need at least 19 mice per group to detect the small effect of the chr10A/J variants in the traits approaching significance at 5 months of age (somatic instability in the liver and intranuclear inclusions), perhaps fewer at a more advanced age where each phenotype is more robustly detected  evidence for modifying effects of chr10 variants on some of the HdhQ111 CAG disease phenotypes, in particular those involved in liver somatic CAG repeat instability and striatal neuron nuclear-aggregate formation. Indeed, we found that the different genetic background does not seem to have an effect on striatal instability but the AJ chr10 might have a mild detrimental effect on the levels and pattern of repeat instability in the liver, as the same trend was observed in all traits analyzed for this tissue . Moreover, even though the effect of the AJ chr10 on striatum CAG instability was not statistically significant, the HdhQ111/+B6J.AJ10 mice seemed to have a slightly higher number of neuronal inclusions than HdhQ111/+B6J, despite similar levels of neuronal change in the striatum (DARPP-32 levels). An improved high-resolution quantitative method for detecting EM48-aggregation foci may be required to better assess the impact of modifiers of this phenotype. It is noteworthy that the reduction of DARPP-32 levels from wild type to Htt mutant mice was not as accentuated in HdhQ111/+B6J.AJ10 as the observed for HdhQ111/+B6J, suggesting that the effect of the Htt CAG mutation in decreasing the DARPP-32 stain intensity is greater than the effect of the difference in genetic background (of a single copy of chr10) on this phenotype. Indeed, supporting this interpretation, Htt CAG alleles of about 140 repeats (comparable to the mean CAG size of our DARPP32 cohort) dramatically decrease Ppp1r1b  mRNA levels in 6-month heterozygote B6J Htt knock-in mouse striatum, compared to wild-type littermates , as revealed by inspection of summary whole genome RNA sequencing data from a B6J Htt CAG repeat knock-in mouse allelic series available from the CHDI Foundation website (http://chdifoundation.org/datasets/).There are several possible reasons that clearly statistically significant modifying effects on HdhHTT CAG repeat mechanism in HD subjects .These trends, therefore, suggest that chr10 variants may modify some CAG-associated phenotypes but with effect sizes too small to be reliably detected at the ages and cohort sizes that we utilised. If the trends that we have observed do reflect true modification, with cohorts of sufficient size and age, validation and fine-structure mapping of potential QTL(s) at chr10 could be achieved by using sub-strain chr10 consomic mice with the region syntenous to 6q23–24. In general, therefore, this unbiased cross-species CCS mouse strain approach may offer an efficient in vivo route to prioritise (conserved) genes within chromosome regions that contain dominant modifier loci of relatively strong effect that conform to the genetic criteria of the Supplementary material 1 (DOCX 98 kb)Below is the link to the electronic supplementary material.


Bombyx mori (L.) (Lepidoptera: Bombycidae), was used for the detection of B. mori infectious flacherie virus (BmIFV). A combination of nested and reverse transcriptase polymerase chain reaction was used for detection. Although BmIFV has been reported in almost all the sericultural regions of the world, there had been no reports of BmIFV incidence in India. Therefore, the confirmation of the presence of BmIFV in Karnataka, India, is of great significance. The present method is advantageous because it can be used to detect the virus by using samples from infected midgut tissues, thus simplifying and avoiding laborious genome isolation procedures. This method could help in early detection of BmIFV disease pathogens and help reduce crop losses.In this study, a method for detection of an ssRNA viral pathogen that causes viral flacherie in the silkworm,  Bombyx mori (L.) (Lepidoptera: Bombycidae), economically important (B. mori Infectious flacherie virus (BmIFV) is a single stranded RNA virus that causes flacherie-like symptoms, is the most frequent virus in B. mori, and is known to cause crop losses of up to 40%  PCR is a sensitive technique used for mRNA detection, is semi-quantitative, and is employed for converting mRNA to complementary DNA (cDNA) via reverse transcriptase . The applications of PCR are various, manifold, and well-documented (Lo 1998). While the PCR technique is basically a primer extension reaction used for amplifying specific nucleic acids criptase . It is mcriptase .The nucleic acid of BmIFV is an ssRNA virus , and sinwww.takara-bio.com), and Trizol was purchased from Invitrogen (www.invitrogen.com). Isopropyl alchohol and other routine chemicals used for agarose gel electrophoresis were procured from local manufacturers.TaKaRa One-Step RNA PCR Kit (AMV) DRR024A was purchased from Takara Bio , nuclear polyhedrosis virus (with milky hemolymph), and cytoplasmic polyhedrosis virus  were discarded, and the ones with symptoms of viral flacherie were retained. The midguts of the diseased larvae were homogenized to collect the crude extract, and this extract was used for further propagation of the virus.Diseased B. mori were reared on fresh mulberry leaves in the laboratory at optimum conditions of 26° C and 65% RH. The gut extract of the diseased B. mori was incubated at pH 4 for 15 min at room temperature, and then neutralized with 1 mol/L NaOH, and fed to B. mori larvae as leaves smeared with the extract. The densonucleosis virus (DNV)-resistant B. mori race Feng1X54A was used for the propagation of the virus. The larvae were infected during the third instar and reared until the fifth instar, depending on the virulence of the disease; symptoms were usually seen after 4–6 days. Maximum mortality of the larvae was noticed on the 10th day post infection. Multiplication of the virus was performed for several generations until uniform symptoms were seen in the B. mori larvae infected with the virus. B. mori with typical flacherie symptoms were dissected to collect the midguts, which were the site of viral infection and used for further extraction of the virus. The collected infected midguts were stored in a refrigerator at -20° C until further use.B. mori larvae were isolated and purified by using the procedures of Himeno et al. (4)2SO4. Next, the solution centrifuged at 12000 g for 30 min and was suspended in 0.1 mol/L EDTA Tris-HCl buffer (pH 7.2). Then, the supernatant was ultracentrifuged at 130000 g for 2 hr. The sediment thus formed was the crude virus.The virus from flacherie-infected o et al.  and Nakao et al.  as followww.gelifesciences.com) and was used for further RT-PCR experiments.The glassware, equipment, and buffers used for the experiment were immersed in 0.1% DEPC for 12 hr at 37° C and then autoclaved. For extracting RNA, 50 µL of virus was mixed with 1 mL Trizol reagent, and the mixture was then gently shaken and kept at 26° C for 15 min. 200 µL of chloroform was added to the mixture, which was then shaken and incubated at 28° C for 3 min. The virus suspension was then centrifuged at 12000 rpm for 15 min at 4° C. Following centrifugation, the mixture separated into phases, with a colorless aqueous layer containing the RNA. The fluid containing RNA was transferred into Eppendorf tubes. 500 µL of isopropyl alcohol was added to precipitate the RNA, and the samples were then incubated at 26° C for 15 min. This was followed by another centrifugation at 12000 rpm for 10 min at 4° C. Then, the supernatant was removed, and the RNA was washed once with 1 mL of 75% alcohol followed by centrifuging at 7500 rpm for 5 min at 4° C. The supernatant was decanted and the pellet was dried. Finally, the pellet was redissolved in 40 µL of RNAse free water. The concentration of the extracted RNA was measured using an Ultrospec 3100 Pro UV/Visible spectrophotometer . The control primers used were Control F-1 Primer 5´ - CTGCTCGCTTCGCTACTTGGA-3´ and Control R-1 primer 5´ - CGGCACCTGTCCTACGAGTTG-3´The positive control RNA RT-PCR using TaKaRa One-Step RNA PCR Kit (AMV) was performed following the manufacturers protocol. Each reaction mixture was prepared adding the following components in PCR tubes: 10× one-step RNA PCR buffer (5µL), 25 mM MgCl2 (10 µL), 10 mM dNTP Mixture (5 µL), Rnase inhibitor (1 µL at 40 U/µL), AMV Rtase XL (1 µL at 5 U/µL), AMVOptimized Taq (1 µL at 5 U/µL), 20 µM Control F-1 Primer (1 µL), 20 µM Control R-1 Primer (1 µL), Positive Control RNA (1 µL), Rnase Free dHPCR was performed for 30 cycles, with RT reaction at 50° C for 15 min, RTase inactivation at 94° C for 2 min, and followed by denaturation at 94° C for 30 sec, annealing at 60° C for 30 sec, and extension at 72° C for 1.5 min.RT-PCR was performed by using RNA from purified BmIFV and BmIFV-infected tissue, and DNA from purified DNV virus and DNV infected tissue. TaKaRa One-Step RNA PCR Kit (AMV) was used following the manufacturers protocol as described earlier. Virus RNA  with the following primers was used for the first PCR: IF-31 ACTGCTTCATTCCAACATCTCTAT and FV-4 TATCTCTAAACAGGCGGAGC.The PCR was performed for 30 cycles, followed by denaturation at 95° C for 1 min, annealing at 60° C for 1 min, and extension at 72° C for 2 min. For the second PCR, the product of the first PCR was added to the primers FV-5 GCATTCATCGACTTTCCCAC and FV-10 TAAACAGGCGGAGCACTACC.The second PCR was performed by adding 1 µL of PCR product to the reaction mixture for 35 cycles, followed by RT reaction 50° C for 15 min, RTase inactivation at 94° C for 2 min, denaturation at 94° C for 30 sec, annealing at 46° C for 1 min, and extension at 72° C for 1 min.www.thermoscientificbio.com) was used as a marker containing the following 13 discrete fragments (in bp): 1031, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 50. The amplified DNA was detected using an ultraviolet transilluminator.For agarose gel electrophoresis, 10 µL of RTPCR products mixed with loading buffer were analyzed on 1% agarose gel, stained with ethidium bromide (0.5 µg/mL). A GeneRuler 50 bp DNA Ladder (Thermo Scientific, The PCR test proved positive for BmIFV using the RNA from purified BmIFV virus and BmIFV-infected tissues by using the designed primers, and the amplified PCR product of 214 bp could be visualized on Agarose gels stained with ethidium bromide  in both RT-PCR technology has proven to be effective for diagnosing infectious agents , and the


Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, Physcomitrella patens, a model for primitive land plants. Using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from P. patens encoding a 16.4 kDa cytosolic class II sHsp. PpHsp16.4 was also induced by salicylic acid, dithiothreitol (DTT) and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. Expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. PpHsp16.4 is encoded by two identical genes arranged in tandem in the genome. Targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. In vivo localization studies revealed that PpHsp16.4 localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth.In this study we characterized the function of a sHsp in abiotic stress tolerance in the moss PpHsp16.4. The product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. Our study provides the first direct genetic evidence for a role of a sHsp in osmotic and salt stress tolerance, and supports a function for this protein particularly during the stress recovery stage of P. patens.We identified a member of the class II sHsp family that showed hormonal and abiotic stress gene regulation. Induction of the gene by DTT treatment suggests that damaged proteins may act as signals for the stress-induction of  Plants must face and cope with various environmental stresses during their life cycle. Drought, salinity and exposure to extreme temperatures are serious threats to agriculture and have a great impact on plant productivity. Most of these stresses share common consequences as a result from water deprivation, namely osmotic stress and the associated oxidative stress .Tracheophytes have evolved numerous anatomical adaptations to cope with water deficit. These include the presence of vascular tissues, root systems and stomata, which all together help minimizing water loss. By contrast, bryophytes lack these adaptations and must rely on efficient biochemical and physiological mechanisms to survive stress by limiting or repairing the cellular damage resulting from these conditions .Numerous studies have shown that the plant hormone abscisic acid (ABA) plays a crucial role in controlling downstream responses essential for adaptation to abiotic stress. As part of osmotic stress responses, regulation of gene expression occurs in both ABA-dependent and ABA-independent manner . ActivatArabidopsis genome encodes 19 sHsps which are divided into several subfamilies according to their sequence relatedness and their subcellular localization . A search in the P. patens full sequence (v1.6) in the public database Phytozome v9.1 (http://www.phytozome.net)  and PpHsp16.4b [Phypa_428984]. These genes are 100% identical and exist in a tail-to-tail orientation with 7725 bp separating their stop codons, suggesting their origin from a single ancestral gene that most likely had undergone recent events of tandem duplication and inversion. The conserved α-crystallin domain of sHsps was used as query to search the genome of P. patens, revealing the existence of 22 genes encoding sHsps. A list of P. patens sHsp genes, their genomic location, and the deduced proteins with their predicted subcellular localization is shown in Table sHsp gene family from P. patens, Arabidopsis and rice was analyzed using ClustalW sequence alignment , Os16.91 I [Os01g0136000], Os16.92 I[Os01g0136200], Os16.93I [Os01g0135900], Os17.4A I [Os03g0266900], Os17.4B I[Os03g0267200], Os17.4C I [Os03g0267000], Os17.4D I [Os03g0266300], Os17.6C II[Os02g0217900], Os17.8II [Os01g0184100], Os17.6B III [Os02g0782500], Os18.8 IV [Os07g0517100], Os22.2 V [Os05g0500500], Os17.6 PX [Os06g0253100], Os22.3 ER [Os04g0445100], Os26 CP [Os03g0245800], Os22 MT [Os02g0758000], Os22.4 MT [Os06g0219500], Os16.9C [Os02g0711300], Os17.6A [Os01g0135800], Os18 [Os11g0244200], Os18.2 [Os02g128000], Os21.2 [Os02g0107100]. Accession numbers of sHsp genes from Arabidopsis were: AtHsp17.4I [At3g46230], AtHsp18.1I [At5g59720], AtHsp17.6BI [At2g29500], AtHsp17.6AI [At1g59860], AtHsp17.8I [At1g07400], AtHsp17.6CI [At1g53540], AtHsp18.5VI [At2g19310], AtHsp22.0ER [At4g10250], AtHsp15.4IV [At4g21870], AtHsp15.7PX [At5g37670], AtHsp21.7 V [At5g54660], AtHsp17.4III [At1g54050], AtHsp17.6II [At5g12020], AtHsp17.7II [At5g12030], AtHsp14.2 [At5g47600], AtHsp26.5 [At1g52560], AtHsp21CP [At4g27670], AtHsp23.5MT [At5g51440], AtHsp23.6MT [At4g25200]. Accession numbers from the deduced sHsp proteins of P. patens are listed in Table Accession numbers of PpHsp16.4 was done using the vector pUBW302  were labeled with [α32P]-dCTP using the Rediprime II random priming labeling system (Amersham Pharmacia Biotech) and used as probe in these studies. Filters were washed twice for 30 min at 65°C with 5× SSC-0.5% SDS, and twice using the same conditions with 1× SSC-0.5% SDS, and exposed in autoradiography films. Ethidium bromide staining was used to ensure equal amounts of loading of RNA in the samples.Total RNA was isolated from control or treated ok et al . MembranBamHI and PstI restriction enzymes. Restricted DNA was separated in 1% agarose gels and transferred into nylon filters  according to Sambrook et al -dCTP using the Rediprime II random priming labeling system (Amersham Pharmacia Biotech) and used as probe in these studies.Genomic DNA was extracted as described by Dellaporta et al  with an ok et al . MembranPpHsp16.4-Citrine in the PpHsp16.4 locus was confirmed by PCR amplification of genomic DNA using the following primers: (g) “ccggtacccatccattgatctaac” and (h) “cgccctcgccggacacgctgaact”, specific for a genomic region upstream the targeting construct and the Citrine gene, respectively. To verify the correct expression of PpHsp16.4:Citrine in P. patens knock-in lines, Western blots were performed with protein samples extracted from transgenic lines untreated controls, or treated with 50 μM ABA, incubated at 37°C or in 300 mM NaCl or 500 mM mannitol containing plates for 24 hours. Furthermore, plants were incubated for 48 h, at 37°C and thereafter transferred to optimal growth conditions for 6 hours, to determine the fusion protein levels during stress recovery. Western blot analysis were performed as described in Saavedra et al  × ml acetone mg-1 fresh tissue; Chlb mg g-1 = [(22.9 × Abs645) – (4.68 × Abs663)] × ml acetone mg-1 fresh tissue.Chlorophyll content, fresh weight and dry weight of plants were determined in three independent experiments using 3 plates containing 5 colonies per genotype per treatment and per time point. Dry weight was measured after incubation of individual plant colonies on cellophane discs for 16 h at 80°C. For the determination of chlorophyll content, each plant was ground up in a mortar containing 5 ml of 80%  acetone and the homogenized plant material was filtered to remove cell debris. Total chlorophyll was calculated as chlorophyll in vitro grown plants and electroporated with the construct 35S:PpHsp16.4-GFP or 35S:GFP, following the procedure described in [Agrobacterium tumefaciens cultures containing the construct 35S:PpHsp16.4-GFP as described [For transient expression experiments, tobacco leaf protoplasts were obtained from ribed in . Protoplescribed . Two dayArabidopsis transgenic lines, overexpressing PpHsp16.4-GFP were grown in vitro for 6 days and root and hypocotyl sections were mounted in tap water and analyzed by confocal microscopy. Confocal imaging was performed using a confocal laser scanning microscope Leica TCS-SP5, with an excitation of 488 nm for GFP.Stable P. patens lines, expressing PpHsp16.4-Citrine, under the control of the native promoter of the target gene, were used to examine the expression and localization pattern of the fusion protein in protonema filaments and in the leafy gametophyte. Stress treatments were done for 24 hours by transferring plants to 500 mM mannitol supplemented media for osmotic stress, or by incubating them at 37°C. Protoplasts from P. patens transgenic knock-in lines were obtained as described above for the generation of P. patens transgenic lines. In vivo fluorescence microscopic observations were made using confocal laser scanning microscope Leica TCS-SP5, using 488 nm for the fluorescence excitation of Citrine.Twenty 20 days old colonies of transgenic The authors declare no competing interests.P. patens transformation and performed phylogenetical analysis. VC generated the subtracted library. LSZ contributed in the generation of Arabidopsis transgenic lines and the subcellular localization studies and revised the manuscript. SV designed and supervised the study, contributed to the analysis of the data and wrote the manuscript. All authors read and approved the final manuscript.CR conducted most experiments. AC participated in Expression profile of P. patens sHsp genes in different developmental stages. Transcript levels of sHsp genes are presented as heat maps generated at Genevestigator based on microarray data. Values are log-scaled to the expression potential of each gene.Click here for fileTemporal induction pattern of PpHsp16.4. Total RNA samples from untreated P. patens wild-type plants (Ctrl) or treated with 100 μM H2O2, 100 μM methyl viologen (MV), 500 mM Mannitol (Mtl), 300 mM NaCl, or 50 μM ABA for 4 and 48 hours (h) were analyzed by Northern blot using a 32P-labeled hybridization probe corresponding to the full-length cDNA sequence of PpHsp16.4. Ethidium bromide staining of ribosomal RNA (rRNA) was used to ensure equal loading of RNA samples.Click here for file


Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka. Citrobacter freundii is an infrequent but established cause of systemic infections in farmed food fish and aquarium fish  fish showing signs of generalized septicemia collected from aquaria in Kandy, Sri Lanka.Two C. freundii CF04 and A41 DNA samples. The reads acquired were assembled separately using MIRA version 4.0.2 (Genomic DNA was extracted with the DNeasy Blood and Tissue kit (Qiagen). Genomic DNA libraries were constructed using the Ion Xpress Plus fragment library kit and sequenced on an Ion Torrent PGM (Life Technologies) platform using the ion 318 chip and the 400-bp kit following standard protocols. A total of 1,328,195 reads equivalent to 340,760,015 bp of data and 1,170,722 reads equivalent to 330,995,850 bp of data were, respectively, generated from C. freundii CF04 strain resulted in 58 contigs over 1,000 bp  with a total length of 5,138,911 bp and 51.49% G+C content. The reads of C. freundii A41 were assembled to 153 contigs larger than 1,000 bp  with a total length of 5,301,254 bp and 51.5% G+C content. The total size of the draft genomes and G+C contents are in agreement with published C. freundii genomes (4.9 to 5.0 Mb and 51.6% G+C) (9The assembly of the 6% G+C) 911.Functional annotation using RAST  revealedC. freundii A41 and CF04 have been deposited in DDBJ/EMBL/GenBank under the accession numbers BDFK01000001 to BDFK01000153 and BDFL01000001 to BDFL01000058, respectively.The draft genome sequences of


Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C) and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme. Caldicellulosiruptor bescii (previously classified as Anaerocellum thermophilum) optimally growing at 75°C is distinguished by its excellent capacity to degrade crystalline cellulose [C. bescii [The thermophilic bacterium ellulose . It is aellulose . These i. bescii ,3,4,5.C. bescii is the existence of a large number of multimodular enzymes. The genome of C. bescii encodes at least six unique multimodular glycoside hydrolases bearing two catalytic domains separated by two to three carbohydrate binding modules (CBMs) [CbCel9A/Cel48A [One striking finding for the PCWP-degrading glycoside hydrolases of s (CBMs) . Notablys (CBMs) ,7. Such CbCel9B/Man5A which has been reported [Escherichia coli. Biochemical analyses were carried out on the purified protein.We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase reported , and expCbMan5B/Cel44A, or Athe_1859, GenBank accession number: ACM60947; CbMan5C/Cel5A, or Athe_1866, ACM60954) or C-terminus  (CbCel9B/Man5A was ligated into pET-28a(+) to obtain pET-CbMan5A. The insertion of cbMan5A was verified by restriction digestion of the recombinant plasmid using EcoRI and NotI  as analyzed by SDS-PAGE  . The genand NotI  and DNA SDS-PAGE .CbMan5A was most active in degrading the galactomannan locust bean gum (LBG), followed by konjac glucomannan  and another galactomannan guar gum (GG)  , demonstgum (GG)  unless tproducts . For GG,products . In contfrom KGM .CbMan5A had an optimal pH of 6.5 . At the temperatures from 75–95°C, the enzyme had relative activities of above 60%  were observed for CbMan5A pretreated at both 70°C and 80°C after 1 h  was 89.9°C, with a ΔH of 6.903×104 cal/mol and ΔHv of 1.812×105 cal/mol, respectively.Fitting of the raw DSC data according to a simple 2-state model generated poor overlay of the two curves, as illustrated in C. bescii [CbMan5A is one of the most thermophilic mannanases that have ever been characterized  was higher, while that on KGM (0.9 mg/ml) was lower, than those of CbCel9B/Man5A . The kcats of CbMan5A on LBG and KGM were 1,043 and 514 s-1, respectively, both lower than those of CbCel9B/Man5A . Notably, the thermal activation property is not observed for the full-length enzyme CbCel9B/Man5A (CbMan5A and the other domains (one GH9 and three CBM3s) into a single polypeptide is not a simple assembly since the missing of the N-terminal CBMs and GH9 catalytic module has complex effects on the enzyme characters of CbMan5A. Our finding further supports the hypothesis that the catalytic and accessory modules in the multi-domain glycoside hydrolases may not be simply tandem-linked but rather functionally coupled [Without any CBM3 modules,  coupled .C. bescii. The rest three among the six multimodular enzymes  all have an identical C-terminal GH48 cellobiohydrolase [CbMan5A into the multimodular CbCel9B/Man5A apparently enhanced its activity in terms of the turnover numbers (kcat); however, the thermal activation character, a property that would be more favorable for adaptation to high temperature environment, is not retained. Since there is no evidence of significant cleavage of CbMan5A from the full-length enzyme CbCel9B/Man5A [CbMan5A is an interesting phenomenon but may be non-biologically relevant. Note, in contrast, there is significant cleavage of GH48 modules from the multimodular cellulases [The recurring of GH5 mannanases is not the only case of gene duplication in multimodular glycoside hydrolases in ydrolase . The recCbCel9B/Man5A as the first glycoside hydrolase with heat activation property from a multimodular bifunctional enzyme. CbMan5A is a highly thermophilic, robust endo-mannanase hydrolyzing a variety of mannans, potentiating it as a candidate for industry application. CbMan5A displays similar but still differing characteristics to those of the full-length CbCel9B/Man5A, suggesting that the multimodular and bifunctional enzymes are more than a simple assembly of several independent domains; rather, the behavior of one module may be affected by the other ones in the full-length enzyme.In conclusion, we identified the C-terminal GH5 mannanase of CbMan5A) of CbCel9B/Man5A was amplified from the genomic DNA of C. bescii DSM 6725  with the primer pairs of Pman5A_F  and Pman5A_R  using the PrimeSTAR HS DNA Polymerase . The PCR products were digested with EcoRI and NotI, and then ligated into the pET-28a(+) plasmid  to obtain pET-cbMan5A.The gene encoding the C-terminal GH5  with an column-equilibration buffer of NTA0 containing 50 mM Tris-HCl, 500 mM NaCl, pH7.5 and elution buffers with the NTA0 containing a series of step gradient concentrations of imidazole (40 to 400 mM).The pET-cbMan5A plasmid was transformed into BL21(DE3)  chemically competent cells. Five to six colonies were inoculated into 200 ml of LB medium and the culture was shaken at 37°C. When the ODCbMan5A was incubated in a McIlvaine buffer containing 200 mM sodium phosphate, 100 mM sodium citrate, pH 6.0 with a series of plant cell wall polysaccharides including 5 mg/ml of locust bean gum (LBG), guar gum (GG), and konjac glucomannan , and 10 mg/ml of beechwood xylan, sodium carboxymethyl cellulose (CMC-Na), Avicel, and filter paper. The reaction was carried out at 70°C for 15 min. For the last four polysaccharides, a prolonged reaction was also carried out at 70°C for 12 h. The released reducing sugars were determined using the 3,5-dinitrosalicylic acid (DNS)method [All polysaccharides, unless otherwise mentioned, were purchased from Sigma-Aldrich . Ten μM of S)method .CbMan5A on different polysaccharides. Two nM of CbMan5A were incubated with 2.5 mg/ml of LBG, KGM, and GG in 5 mM McIlvaine buffer (pH 6.5) in a total volume of 500 μl at 80°C for 12 h. Appropriate amounts of the hydrolysates were applied onto a silica gel  and air dried. The plate was developed using a solution containing n-butanol-acetic acid-H2O with a volumetric ratio of 10:5:1. The plate was air dried, sprayed with a solution containing H2SO4-ethanol with a volumetric ration of 1:19, and heated at 110°C for 10 min until the products could be clearly visualized.The TLC method described by Moon et al.  was emplCbMan5A was incubated with 5 mg/ml of LBG in the McIlvaine buffers with different pHs (pH4.0–8.0) at 70°C for 10 min. For optimal temperature, CbMan5A was incubated with 5 mg/ml of LBG in the McIlvaine buffers at pH6.5 at temperatures ranging from 50°C to 90°C with a gradient of 5°C. The released reducing sugars were determined using the DNS method. To determine the pH-stability, 0.2 μM CbMan5A was first pretreated in the buffers with different pHs  at 37°C for 1 h. To determine the thermostability, CbMan5A was pre-incubated at 70°C, 80°C, and 90°C for different time periods . Thereafter, for both pH-stability and thermostability experiments, the residual activity was determined at its optimal reaction conditions (pH 6.5 and 90°C). The released reducing sugars were measured using the DNS method. The experiments were repeated for three times.For optimal pH determination, 0.2 μM CbMan5A were incubated in the McIlvaine buffer with 0.5 to 5 mg/ml of LBG or KGM at its optimal pH and temperature for 5 min when the released reducing sugars versus time were linear. The Km and Vmax were calculated according to the Michaelis-Menten equation using the software GraphPad Prism 5.01 . The specific activity was determined by incubating 2 nM of CbMan5A with 5 mg/ml of LBG, GG or KGM at its optimal pH and temperature for 10 min. One unit (U) of specific activity was defined as the amount of enzyme that released one μmol of reducing sugars per minute.Appropriate amounts of 2, CoCl2, CrCl3, NiCl2, CuCl2, MgCl2, FeCl3, MnCl2, ZnCl2, Pb(CH3COO)2, and AgNO3 containing different metal ions, and three chemicals including sodium dodecyl sulfate (SDS), ethylene diamine tetraacetic acid (EDTA), and β-mercaptoethanol, were added with a final concentration of 5 mM into the reaction mixture. The mannanase activity of CbMan5A was measured using 5 mg/ml of LBG under its optimal conditions.The salts including NaCl, KCl, CaClCbMan5A) were dissolved in 100 mM McIlvaine buffers (pH 7.5).The samples were degassed for 7 min before measurements in a dewar vessel. Then 400 μl of the sample with a 400 μl reference were loaded into the calorimeter cells. As the baseline, the same amount of buffer was measured and then subtracted from measured scan. During all DSC experiments, a constant pressure of 3 atm was maintained to prevent possible degassing of the solution on heating. The test was repeated twice at temperatures between 20°C and 120°C.DSC was performed on a Nano-DSC  at a heating rate of 1°C/min and a scanning rate of 1°C/min. The sample Click here for additional data file.S2 FigCbCel9B/Man5A was incubated at 70°C, 80°C, and 90°C for 1 h. At different time, samples were taken out and measured for the residual mannanase activity. Locust bean gum was used as the substrate. The activities of CbCel9B/Man5A were represented as relative activity (in percentage) by dividing the activities against the reference activity, which was the activity before treatment.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The transcription factor (TF) Snail1 is a major inducer of the epithelial–mesenchymal transition (EMT) during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC) abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC) for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT) for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157   Specifications table•938 proteins deregulated in 3T3-L1 cells and 319 proteins deregulated by Snail in mMSC were identified and quantified using a false discovery rate (FDR<1%).•Most of the proteins were down-regulated, 463 and 294 repressed proteins in 3T3-L1 and mMSC cells, respectively.•The data provide new insights into the ability of Snail1 to repress newly identified transcription factors, cytokines, growth factors and immunomodulators.•This work provides insight into novel proteins deregulated by Snail that play important roles into the differentiation inhibition of 3T3-L1 and mMSC cells into adipocytes.•Most of the deregulated novel proteins by Snail were transcription factors (TFs) as Nr2f6, Trip4 or Prrx1.Value of the data1Fibroblasts are the most abundant cell type in connective tissues, and some subpopulations are able to differentiate to other cell types as adipocytes or osteoblasts. In normal conditions, fibroblasts are in an inactive quiescent state; however fibroblasts activation is induced by several stimuli, including growth factors such as TGF-β in colorectal cancer. Snail1 transcription is up-regulated by this cytokine in fibroblasts. Snail1 levels are modulated by serum and TGFβ. In addition, Snail1 is not only up-regulated by TGF-β, but also induces the transcription of TGFβ, creating a self-activation loop for the production of TGFβ We carried out an in-depth quantitative proteomic analysis of Snail1-transfected 3T3-L1 and mMSC cells to identify cellular and molecular mechanisms associated to the inhibition of differentiation to adipocytes by Snail. Due to different cell growth requirements, two labeling strategies were followed. Therefore, immortalized 3T3-L1 cells were metabolically labeled in SILAC medium A. Howeve2 ratio distribution to zero  following the manufacturer׳s instructions.Preadipocytes 3T3-L1 and murine mesenchymal stem cells (MSCs) stably transfected cells with pcDNA3 Snail1-HA (“snail”) or control pcDNA3 (“mock”) were obtained as indicated l-lysine and l-arginine or heavy [13C6]-l-lysine and [13C6]-l-arginine was purchased from Dundee Cell Products. For metabolic labeling, 3T3-L1 Snail1 or control cells were grown and maintained in DMEM supplemented with 10% dialyzed FBS, 100 units/mL of penicillin/streptomycin at 37 °C in 5% CO2, for 8 duplications to achieve >98% incorporation of the heavy amino acids. We carried out forward and reverse experiments to avoid any labeling bias in the study.DMEM containing 2.2Proteins from SILAC labeled nuclear cell extracts of 3T3-L1/Snail and 3T3-L1/Control cells were mixed at a 1:1 ratio and run at 25 mA per gel in 12.5% SDS-PAGE A. Forwarm/z 400–1200) were acquired in the Orbitrap with a target value of 1,000,000 at a resolution of 60,000 at m/z 400 and the 15 most intense ions were selected for collision induced dissociation (CID) fragmentation in the linear ion trap with a target value of 10,000 and normalized collision energy of 35%. Precursor ion charge state screening and monoisotopic precursor selection were enabled. Singly charged ions and unassigned charge states were rejected. Dynamic exclusion was enabled with a repeat count of 1 and exclusion duration of 30 s. Mass spectra ⁎.raw files were searched against the SwissProt mouse database 57.15  using MASCOT search engine  through Proteome Discoverer (version 1.4.1.14) (Thermo). Search parameters included a maximum of two missed cleavages allowed, carbamidomethylation of cysteines as a fixed modification and oxidation of methionine, N-terminal acetylation and 13C-Arg, 13C-Lys as variable modifications. Precursor and fragment mass tolerance were set to 10 ppm and 0.8 Da, respectively. Identified peptides were validated using Percolator algorithm with a q-value threshold of 0.01 2 protein ratios to 0 2 ratio distribution to zero. Since metabolic conversion arginine/proline can affect quantification accuracy in some cell types, we investigated arginine to proline conversion in 3T3-L1 cells. Using heavy proline as a variable modification, less than 1% of proline-containing peptides were heavy labeled in 3T3-L1 cells.Peptides were trapped onto a C18-A1 ASY-Column 2 cm precolumn (Thermo-Scientific), and then eluted onto a Biosphere C18 column  (NanoSeparations) and separated using a 170 min gradient from 0% to 35% Buffer B  at a flow-rate of 300 nL/min on a nanoEasy HPLC (Proxeon) coupled to a nanoelectrospay ion source (Proxeon). Mass spectra were acquired on an LTQ-Orbitrap Velos mass spectrometer (Thermo-Scientific) in the positive ion mode. Full-scan MS spectra . Briefly, desalted and dehydrated samples were reconstituted in OFFGEL solution. Focusing was performed on an IPG dry strip  set up with a 12-well frame at 20 kV h with a maximum of 50 μA and power of 200 mW. The 12 fractions collected were desalted on a Vidac C18 microspin column, dried under speed-vacuum and stored at −20 °C until LC-MS/MS analysis.2 pre-column and separated on a home-made 5 µm 100 Å Magic C18 AQ (Michrom) 0.75×150 mm2 column with a gravity-pulled emitter. The analytical separation was run for 65 min using a gradient of H2O/FA 99.9%/0.1% (solvent A) and CH3CN/FA 99.9%/0.1% (solvent B). The gradient was run as follows: 0–1 min 95% A and 5% B, then to 65% A and 35% B at 55 min, and 20% A and 80% B at 65 min at a flow rate of 220 nL/min. For MS survey scans, the OT resolution was set to 60,000 and the ion population was set to 5×10E5 with an m/z window from 400 to 2000. A maximum of 3 precursors were selected for both collision-induced dissociation (CID) in the LTQ and high-energy C-trap dissociation (HCD) with analysis in the OT. For MS/MS in the LTQ, the ion population was set to 1×7E3 (isolation width of 2 m/z) while for MS/MS detection in the OT, it was set to 2×10E5 (isolation width of 2.5 m/z), with resolution of 7500, first mass at m/z=100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD.ESI LTQ-OT MS was performed on a LTQ Orbitrap velos from Thermo Electron  equipped with a NanoAcquity system from Waters as previously described p value was 1×10E−2 for LTQ-Orbitrap data. False-positive ratios were estimated using a reverse decoy database. All datasets where searched once in the forward and once in the reverse database. Separate searches were used to keep the database size constant. Protein and peptide score were then set up to maintain the false positive peptide ratio below 1%. This resulted in a slight overestimation of the false-positive ratio. Only those proteins matching two different peptide sequences were kept for all analyses.Peak lists were generated from raw data using the embedded software from the instrument vendor (extract_MSN.exe). After peak list generation, the CID and HCD spectra were merged for simultaneous identification and quantification Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifier PXD002157


Curcuma longa has emerged as a potent multimodal cancer-preventing agent which modulates multiple cell signaling pathways and also been described as an inhibitor of DNA methylation[The identification of targets and new drugs to fight cancer is one the greatest challenges for Biotechnology researchers. Not only humans, but also animals are affected by this disease. It is estimated for example that one in every 3-4 dogs will develop some type of cancer during its lifetime, twice as much as the human being ,2. DNA m2, DMEM medium was supplemented with 10% Bovine Fetal Serum. To estimate DH82 viability and curcumin cytotoxicity, the MTT protocol was performed at a concentration of 2 × 105 cells/well cultured in 96 well plates. After each treatment, the cells were incubated during four hours with a 5 μg/mL MTT  -2,5-diphenyltetrazolium bromide) solution. Lastly the MTT salt conversion to formazan crystals was quantified by spectrophotometry.DH82 cells grew under 37°C and 5% COA curcumin therapeutic dose curve was established at 24, 48 and 72 hours treatments.50 was 21.88µg/mL (59.4µM), in 48 hours was 14.26µg/mL (38.7µM) and in 72 hours was 12.17µg/mL (33.0µM). A range of values were obtained and the dispersion, especially on IC50 coordinate coincides with the trend line, showing good positive correlation.In 24 hours, the ICThe DH82 cell line demonstrated susceptibility to treatments with curcumin, which reflects the importance of this natural product as a tool for the development of new epigenetic drugs which could be also explored to fight cancer in pet dogs.This study was supported by grants from Research Foundation of the State of São Paulo (FAPESP - 2012/23333-2).


Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The Km and Vmax values of PGC3 from FOC4 were determined to be 0.70 mg·mL−1 and 101.01 Units·mg·protein−1·min−1, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3–7 and >50% activity in 10–50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris.Fusarium wilt (Panama disease) caused by  Musa spp.) is the world’s principal fruit and is regarded as the fourth most important crop in developing countries [Fusarium oxysporum f. sp. cubense (FOC) and is widely regarded as one of the most destructive plant diseases in the world [Banana  that play significant roles throughout the fungal life, including acquisition of nutrients and decomposition of plant cell walls, and may be important in pathogenicity . Among tCochliobolus carbonum [Fusarium oxysporum f. sp. lycopersici [et al., (2011) found that the inhibition of endo-PG was higher than the inhibition of exo-PGs by PGIPs extraction from Ralstonia solanacearum-inoculated plants, and noted that through higher expression of exo-PGs the pathogen might effectively escape plant defense responses through late or no recognition by the plant, since potent elicitor-active oligomers were not produced or could be rapidly degraded by the action of exo-PGs [Exopolygalacturonases (exo-PGs) have been studied in the fungal plant pathogen carbonum  and Fusaopersici  concerniopersici  and are opersici . Schacht exo-PGs . Althougpgc3 genes of FOC1 and FOC4 and then expressed in P. pastoris. Both of them had exo-PG activity. Further studies are required to obtain insight into the role of PGC3 in FOC pathogenicity in banana cultivars.In this study, we report the isolation and the purification of an exo-PG (PGC3) from the pathogen FOC4. We cloned the −1·min−1  non-redundant protein sequences (Nr) database, the top match (100%) was exopolygalacturonase from Fusarium oxysporum f. sp. fragariae (BAE97149.1).The purified PGC3 was sent for pgc3 from FOC4 was cloned by RACE PCR according to the N-terminal sequence of PGC3. The specific primers were then designed to amplify the full-length cDNA of pgc3 from FOC1 and the DNA sequences of pgc3 from both FOC1 and FOC4. A full-length 1622 nucleotide DNA sequence was isolated and sequenced from FOC1 and FOC4. Sequencing revealed the presence of an open reading frame (ORF) of 1368 nucleotides, interrupted by five introns of 59, 47, 47, 51 and 50 nucleotides, and encoding a predicted protein of 456 amino. Analysis with SignalP detected a putative N-terminal signal peptide sequence of 21 amino acids that when cleaved would produce a mature protein. The deduced mature protein of pgc3 from FOC1 (FOC1-pgc3) and pgc3 from FOC4 (FOC4-pgc3) had the calculated molecular mass of 47.88 and 47.95 kDa, the calculated isoelectric point (pI) of 5.97 and 5.79, respectively. PG (pgx4) with very similar calculated molecular mass of 47.9 kDa but different pI (8.0) studying the fungus FOL 4287. We are not sure if a predicted program leads to the different pI or some unknown reasons [The full-length cDNA of  reasons .pgc3 shared as low as 41.5% nucleotide sequences identity with FOC4-pgc2, while their amino acid identity was only 28.9%. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3 as following 28, 147, 153, 204, 207, 303, 312, 315, 339, 648, 830, 985, 999, 1004, 1236, and 1300  was the only degradation product detected during enzyme activity and its hydrolysis of the 3% of substrate leading to a 16% reduction of viscosity, demonstrated that PGC3 is an exo-PG. The Fusarium oxysporum f. sp. lycopersici (FOL) were 5 and 55 °C [Aspergillus niveus were 50 °C and 4–6.5, respectively [To identify the stability and optimal pH value and temperature of PGC3, recombinant PGC3 from FOC1 and FOC4 were used and compared. The optimum pH value and temperature of the recombinant PGC3 were investigated with PGA as the substrate. Both purified recombinant PGC3 from both FOC1 and FOC4 exhibited optimum activity at pH 4.5 a, and thnd 55 °C . The temnd 55 °C . An exo-nd 55 °C . The optectively .To calculate the pH stability of recombinant PGC3, samples were incubated in buffers of different pH values at 4 °C for 24 h. The remaining PG activity was assayed. Both recombinant PGC3s retained more than 40% peak activity at pHs ranging from 3 to 7 c. To invPurified PGC3-FOC1 and PGC3-FOC4 were inoculated with banana tissues to test their ability to macerate banana tissue. Surface sterilization of banana stem tissues were incubated with 1 Unit of recombinant PGC3 and 1 mL of 50 mM sodium acetate buffer (pH 4.5), and then macerations were evaluated after 48 h post incubation.Musa AAB was lower than that of r-FOC4-PGC3, while the maceration activity of r-FOC1-PGC3 to Cavendish Musa AAA was higher than that of r-FOC4-PGC3. Both these two enzymes showed higher maceration activity on Musa AAB than Cavendish Musa AAA. Similarly, both r-FOC1-PGC2 and r-FOC4-PGC2 showed higher maceration activity on Musa AAB than Cavendish Musa AAA [Musa AAB and Cavendish Musa AAA, as an important component of cell walls, might differ in polymer structure because Cavendish Musa AAA pectin is a poor substrate for exo-PGs compared to that of Musa AAB  was obtained from Panyu, Guangzhou, China, and F. oxysporum f. sp. cubense race 1 (FOC1) was obtained from Jiangnan, Nanning, China, respectively.w/v) citrus pectin. Citrus pectin was obtained from Sigma-Aldrich .For DNA extraction, mycelium was obtained from cultures grown for five days in potato dextrose broth (PDB) in flasks on a rotary shaker at 110 rpm and 25 °C. For RNA extraction, mycelium was obtained from cultures of PDB supplemented with 1% (w/v), 50 mM sodium acetate buffer (pH 4.5), and various amounts of enzyme solution at 50 °C for 30 min. The number of reducing groups, expressed as galacturonic acid (GA) released by enzymatic action, was determined by the method of previously reported protocols [Exopolygalacturonase (exo-PG) activity was assayed in a mixture  containing 0.5% polygalacturonate (PGA)  membrane filter, and then applied to a gel filtration column Sephacryl S-100 from GE Healthcare Bioscience  equilibrated and eluted with 50 mM sodium acetate buffer (pH 4.5) at a flow rate of 1 mL/min. Fractions containing PG activity were pooled and applied to a cation exchange column  equilibrated with 20 mM sodium acetate buffer (pH 4.5). The column was eluted with a gradient of NaCl (0–0.7 M) at a flow rate 2 mL/min. Each tube of PG activity peak was checked by PG activity and SDS-PAGE after concentration.Culture of FOC4 grown in PDB with 1% citrus pectin was centrifuged at 16,000× w/v) PGA in 50 mM sodium acetate buffer (pH 4.5) and incubated at 50 °C, for 10, 20, 30, 40, 50, 60 min, and then used for PG activity assay. The method was referred to Somogyi’s method [To analyze the hydrolysis products, the samples (0.02 U enzyme in 0.5 mL water) were added to 1 mL of 0.5% (s method . The metKm) and Vmax values were determined from Lineweaver–Burk plots of enzyme activity measured with the PGA as substrates, at concentrations between 0.25% and 1.25% at optimum pH and temperature, and then plotted the results.The Michaelis constant (w/v) PGA as substrates. The consequence of temperature on PG activity was determined in 100 mM potassium phosphate buffer at pH 4.5, between 10 and 90 °C. To calculate the pH stability, the samples were covered in 100 mM potassium phosphate buffer of different pH values at 4 °C for 24 h, and then used for PG activity assay. To assess the thermal stability, the samples were covered in 100 mM potassium phosphate buffer at pH 4.5 for 2 h, between 10 and 90 °C, and then used for PG activity assay. Three replicates were tested per treatment as well as the negative control.To determine the optimal pH, the PG activity was assayed using 100 mM potassium phosphate buffer for pH values between 3 and 10 at 50 °C, and 0.5% 20. Amplification conditions were one cycle of 3 min at 94 °C, 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 90 s, then 10 min for 72 °C. The amplified fragments were purified and sequenced.For Race PCR, cDNA of FOC4 was synthesised by the RNA with murine leukemia-virus reverse transcriptase . Two primers were designed based on the GAATTCATGTTGTTCAATAACGTTCTTT-3') and (5'-TCTAGATCCATTGGTTGTGTCCCTG-3'). The EcoRI and XbaI restriction sites are underlined. Amplification conditions were one cycle of 3 min at 94 °C, 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, then 10 min for 72 °C. The RT-PCR products were purified and then digested with EcoRI and XbaI and subcloned into the same enzymes digested pPICZaA  to generate recombinant eukaryotic expression vector, pPICZaA-pgc3-FOC1 and pPICZaA-pgc3-FOC4. The presence of the inserted pgc3-FOC1 and pgc3-FOC4 genes was verified by sequencing.Based on the sequence of RACE PCR, the gene sequences and coding sequences of FOC1 and FOC4 were obtained by RT-PCR using the primers (5'-pgc3 from FOC1 and FOC4 were amplified and sequenced using DNA of FOC1 and FOC4 as the template and primers as mentioned above.The complete gene sequences of pgc3-FOC1 and pPICZaA-pgc3-FOC4 were linearized by digestion with SacI and transformed into P. pastoris SMD1168 strain by electroporation. The SMD1168 strains transformed with or without pPICZaA plasmid served as negative controls. The cells were incubated in yeast extract peptone dextrose (YPD) plate containing 1% yeast extract, 2% peptone, 2% dextrose, and 100 µg/mL of Zeocin at 28 °C for 48 h. The integration of the pgc3 gene into SMD1168 was determined by PCR using 5'AOX1 and 3'AOX1 primers, which can be found in the instruction of pPICZaA.Yeast transformation was performed according to the manufacturer’s instructions. The recombinant plasmid pPICZaA-−5% biotin and 1% glycerol) at 28 °C for 24 h, and then transferred to 200 mL BMMY medium  at 28°. The samples were collected at 1, 2 and 3 day, followed by 1 mL of 100% methanol added daily to maintain the final concentration of methanol at 0.5%.Yeast transformants were grown in 30 mL of BMGY medium  equilibrated and eluted with 50 mM sodium acetate buffer (pH 4.5) at a flow rate of 1 mL/min. Fractions containing PG activity were collected.To purify the recombinant PGC3, two days induced culture was collected and centrifuged at 10,000× Musa AAA (resistant to FOC1 and susceptible to FOC4), and Musa AAB (susceptible to FOC1 and FOC4) were used. A length of 1 cm tissue (~0.5 g) was taken from the healthy stems of the four leaf stage plants and placed into test tubes. The sterilized plant tissue was treated with single U of purified enzyme in 1 mL of 50 mM sodium acetate buffer (pH 4.5), and then evaluated macerated activity at 48 h post treatment. A negative control contained the same buffer instead of the enzyme. Tissue macerated activity was calculated from standards of PGA after incubation 48 h at 50 °C by the method of Somogyi [To evaluate tissue maceration, Cavendish  Somogyi .For the tissue necrosis assay, one U of enzyme was injected directly to the stems of healthy banana plant. Each treatment had ten replicates. Autoclaved water and 50 mM sodium acetate buffer (pH 4.5) were used as negative controls Plant stems were cut  to observe vascular necrosis at 5 days post treatment.


Efficacy and safety of anticancer drugs are traditionally studied using cancer cell lines and animal models. The thienodiazepine class of BET inhibitors, such as JQ1, has been extensively studied for the potential treatment of hematological malignancies and several small molecules belonging to this class are currently under clinical investigation. While these compounds are well known to inhibit cancer cell growth and cause apoptosis, their effects on stem cells, particularly mesenchymal stem cells (MSCs), which are important for regeneration of damaged cells and tissues, are unknown. In this study we employed umbilical cord derived MSCs as a model system to evaluate the safety of JQ1.Cord derived MSCs were treated with various doses of JQ1 and subjected to cell metabolic activity, apoptosis, and cell cycle analyses using MTT assay, Annexin-V/FITC and PI staining, and flow cytometry, respectively. The effect of JQ1 on gene expression was determined using microarray and quantitative real-time reverse transcriptase polymerase chain reaction analysis. Furthermore, protein expression of apoptotic and neuronal markers was carried out using western blot and immunostaining, respectively.Our results showed that JQ1 inhibited cell growth and caused cell cycle arrest in G1 phase but did not induce apoptosis or senescence. JQ1 also down-regulated genes involved in self-renewal, cell cycle, DNA replication, and mitosis, which may have negative implications on the regenerative potential of MSCs. In addition, JQ1 interfered with signaling pathways by down regulating the expression of WNT, resulting in limiting the self-renewal. These results suggest that anticancer agents belonging to the thienodiazepine class of BET inhibitors should be carefully evaluated before their use in cancer therapy.This study revealed for the first time that JQ1 adversely affected MSCs, which are important for repair and regeneration. JQ1 specifically modulated signal transduction and inhibited growth as well as self-renewal. These findings suggest that perinatal MSCs could be used to supplement animal models for investigating the safety of anticancer agents and other drugs.The online version of this article (doi:10.1186/s13287-016-0278-3) contains supplementary material, which is available to authorized users. Recently, bromo- and extra-terminal domain (BET) protein inhibition has emerged as a therapeutic target for cancer, metabolic disorders, and inflammatory diseases –3. BET pc-MYC which is involved in their pathogenesis .MSCs . Each reaction was subjected to the following conditions: 98 °C for 10 minutes, followed by 44 cycles of 98 °C for 30 seconds, 60 °C for 20 seconds, and 72 °C for 30 seconds in 96-well optical reaction plates (Bio-Rad). The reference gene GAPDH was used to normalize the amplification of the target genes. Each qRT-PCR analysis was performed in triplicate. Primer sequences are listed in Additional file Total cellular mRNA was isolated using the GeneJET RNA purification Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Total RNA was purified by incubation with DNase at 37 °C for 30 minutes using a thermocycler . cDNA was synthesized using the BioRad iScript kit . Quantitative reverse transcriptase PCR (qRT-PCR) was performed using 10 μl reaction volume containing 5 μl Syber green , 3 μl distilled HCells were treated with 0 and 500 nM JQ1 for 72 hours. Protein was extracted by lysing the cells in RIPA buffer with PMSF (Sigma). Then 10 μl total proteins were separated with SDS-PAGE with 12 % resolving gel and 6 % stacking gel. Samples were diluted in sample diluting buffer, heated at 95 °C for 5 minutes, and loaded on to the gel and electrophoresed. Proteins were then electrophoretically transferred onto Polyvinylidene difluoride (PVDF) membrane for 1 hour at 150 mA constant current. The membrane was blocked with 5 % nonfat dry milk for 1 hour at room temperature and then incubated overnight at 4 °C with primary antibodies for Caspase-8, Caspase-9, and β-actin (Santa Cruz Biotechnology) diluted 1:50 in blocking solution. The membrane was washed to remove unbound primary antibody and was incubated with secondary antibody  in blocking solution for 1 hour at 37 °C. Unbound secondary antibody was washed with PBS containing 0.1 % Tween-20. Blots were developed with chemiluminescence system (Bio-Rad), and exposed to X-ray film . Band intensity was quantified with ImageJ software, .p ≤0.05 were considered statistically significant. All analyses were performed using SPSS  version 11.5 .Data are presented as the mean ± standard error of the mean (SEM). Results with MSCs isolated from umbilical cord had fibroblastoid morphology Fig.  and wereChanges in the morphology of the JQ1-treated MSCs were further quantified by flow cytometry. Cell size and granularity increased in a concentration and time-dependent manner Fig. . ExposurTo test the effect of JQ1 on cell cycle progression, cell cycle analysis was performed on the JQ1-treated MSCs and the results depicted in Fig. p ≥0.01   Fig. , b. Furtnes Fig. . In addiCCNA2, CDK1, E2F2, and CCNB2 transcription in comparison with the control, and 500 nM JQ1 led to more than a 9-fold decrease in the transcription of these genes (Fig. CCND1, CDK6, c-MYC, and RB1 were found to be downregulated 4-fold to 9-fold compared with control when examined by qRT-PCR. Among these genes, c-MYC transcription was downregulated 8-fold, while the transcription of CCND1 decreased 5-fold to 6-fold in JQ1-treated cells. CDK6 transcription, however, was downregulated by 8-fold to 9-fold. RB1 transcription also decreased in a dose-dependent manner from 4-fold to 8-fold as shown in Fig. TOP2A, CDCA8, NEK2, and CENPF (Fig. Downregulation of these genes in MSCs treated with 100 and 500 nM JQ1 for 24 hours was further investigated by qRT-PCR. Results depicted in Fig. nes Fig. . FurtherNPF Fig. . Taken tBCL2, proapoptotic BIM, and initiator Caspases 8 and 9. Results showed that mRNA expression levels of BCL2, Caspase 8 and Caspase 9 were reduced significantly by 6-fold to 9-fold while no significant change in the mRNA expression of BIM was observed (Fig. MSCs treated with JQ1 had no observable cell death. To further investigate whether JQ1 induced apoptosis in MSCs, Annexin V-treated cells were analyzed by flow cytometry. These results, as depicted in Fig. ved Fig. . Furtherved Fig. , d. Howeved Fig. . These rOct4, Nanog, Sox2, and Klf4 markers in MSCs treated with JQ1 was confirmed by qRT-PCR (Fig. WNT4, WNT5a, and WNT11 were not expressed in MSCs and were not affected by JQ1. However, the expression of WNT2, a member of the canonical WNT pathway, was decreased by 8.5-fold and 9.5-fold in MSCs treated with 100 nM and 500 nM JQ1, respectively, compared with control. This is in agreement with the microarray analysis, which showed that the expression of FZD2, a WNT receptor involved in the canonical WNT pathway, and WNT2 were also downregulated in JQ1-treated cells (Fig. FZD4 but a 2-fold upregulation of FZD6 and LRP6 compared with the control. In addition, WNT-related proteins CTNNB1 and AXIN were upregulated by 2-fold and 6-fold, respectively. Taken together, these results suggest JQ1 modulated signal transduction by turning off WNT pathway resulting in downregulation of pluripotent genes and induction of differentiation (Fig. Col1 and Runx2, and chrondrogenic markers Col2 and Sox9, were downregulated. Interestingly, Nestin was downregulated but TUJ1, CCL5, INA, and SMURF2 were significantly upregulated (Fig. To test whether JQ1 induces the differentiation of MSCs, microarray data indicated downregulation of genes involved in self-renewal Fig. . SignifiPCR Fig. . This lells Fig. . Furtherion Fig. . These rted Fig. . Microarted Fig. . While eA number of chemicals have been developed and investigated for their anticancer properties, such as cell cycle arrest or cell death of cancer cells , 6. PoteOur study showed that JQ1 affected cell morphology and inhibited proliferation of cord-derived MSCs. This observation was consistent with the growth inhibition caused by JQ1 in various cancer cell lines derived from NMC, multiple myeloma, leukemia, and neuroblastoma –15. JQ1 Previous studies with cancer cells have shown that JQ1 binds to BET proteins and inhibits their interaction with acetylated histones, causing cell cycle arrest in the G1 phase and apoptosis , 12. FurCCNA2, CCNB2, CCND1, CDK1, CDK6, c-MYC, and E2F2, were downregulated. CCNA2 interacts with CDK2, and their activity starts late in the G1 phase and lasts through the S and G2 phases [CCNA2 through binding to its promoter [CCNA2 in our study is consistent with the observation that overexpression of BRD2 upregulated the transcription of CCNA2 which led to aggressive cancer in the mouse [CCNA2 transcriptional activation in NIH3T3 cells [CCND1 and CCND2 are known to play a role in G1/S transition, and they associate with their partners CDK4 and CDK6 during cell cycle regulation [CCND1 [CCND1 and CCND2, and that their expression is reduced in NIH3T3 cells after knockdown of BRD4 [CCND1 [c-MYC is an oncogene that is expressed early in the G1 stage and has a crucial role in cell cycle progression [c-MYC has been shown to be regulated by BRD4 in various cancer cell lines such as Hela cells and leukemia cells, and c-MYC expression was reduced upon BRD4 knockdown or cell treatment with small molecules inhibiting BRD4 [E2F transcription factor family was among the highly downregulated groups in the gene expression profile. We confirmed that E2F2 transcription reduced upon JQ1 treatment of MSCs with qRT-PCR. The E2F family plays a critical role in the regulation of the cell cycle through transcriptional regulation of genes involved in the G1 phase and DNA replication [E2F and transactivates E2F responsive genes in NIH3T3 cells [CCND1, CCNA, CCNE, and dihydrofolate reductase were among the E2F responsive genes [RB1 and stemness genes, has been reported in senescent cells [RB2, CDKN1A, CDKN2A, and CDKN1B, suggesting that JQ1-induced arrest in MSCs is not due to senescence.Microarray analysis of JQ1-treated MSCs pointed to cell cycle arrest in the G1 phase. Microarray data confirmed by qRT-PCR analyses showed that a number of genes involved in cell cycle regulation, including 2 phases , 27. BRDpromoter , 29. Dowhe mouse . Also, aT3 cells , 32. CCNgulation , 34. Cycgulation . Studiesn [CCND1 . A similn [CCND1 . The eff of BRD4 . BRD2 al4 [CCND1 , 29. c-Mgression . It is kgression , 38–40. ing BRD4 , 37. JQ1lication . BRD2 haT3 cells . CCND1, ve genes , 29, 32.nt cells , our resBCL2, BIM, Caspase 8, and Caspase 9, which play critical roles in controlling the apoptotic pathways, were determined by qRT-PCR. Even though JQ1 caused a significant downregulation of BCL2, an antiapoptotic marker, expression of proapoptotic genes Caspase 8 and Caspase 9 were also downregulated, while no significant change in the expression of BIM was detected. Western blot analysis also revealed significant downregulation of Caspase 8 but little change in the expression of Caspase 9, which is consistent with the qRT-PCR analysis as discussed. These results suggested that MSCs did not undergo apoptosis when they were subjected to JQ1 treatment.Since JQ1 resulted in cell cycle arrest in MSCs, we further investigated whether JQ1 induced apoptosis as reported in cancer cells. Flow cytometric analysis also showed no significant increase in Annexin V-positive cells, suggesting that JQ1 did not induce apoptosis in MSCs. To further confirm that JQ1 did not exert an apoptotic effect in MSCs, transcriptional levels of OCT4, Nanog, KLF4, and SOX2 [OCT4, Nanog, KLF4, and SOX2, suggesting that BET proteins play an essential role in the self-renewal of MSCs.Stem cells have been reported to express several self-renewal markers, including and SOX2 –47. Our WNT2, FZD2, and FZD4 and upregulation of FZD6, LRP6, CTNNB1, and AXIN. These observations suggest that the WNT pathway is turned off and differentiation of MSCs is induced by JQ1. This is in partial agreement with a previous report where downregulation of WNT2, FZD2, and FZD4 and upregulation of CTNNB1 was involved in neural differentiation of human adipose-derived stem cells [Since signaling pathways are known to play an important role in differentiation processes, we investigated whether WNT signaling was involved in self-renewal and differentiation of stem cells . Microarem cells . Taken tTUJ1, CCL5, INA, and SMURF2 were upregulated, which are neuronal lineage-specific markers [TNF, CCL5, and ICAM1 is interesting as observed by microarray analysis because these genes have been reported to be important for leukocyte recruitment and cell adhesion [It has been proposed that BET proteins exert a dual function where they act as coactivators for cell cycle genes and corepressors for specific differentiation markers . Therefo markers –52. Althadhesion , 54, sugOur results clearly showed that JQ1 inhibited MSC growth by influencing important cellular processes including cell cycle, signal transduction, and differentiation. In addition, genes involved in self-renewal, DNA replication, and mitosis were downregulated. JQ1 is likely to exert a similar effect on MSCs derived from other adult sources such as bone marrow MSCs. Interestingly, recently published reports showed antioxidant activity of JQ1 and its ability to block memory in mice , 56. TheOverall, this study revealed harmful effects of JQ1 and possibly other members of the thienodiazepine class on growth, self-renewal, and cell cycle progression of MSCs. The use of this class of BET inhibitors as anticancer agents should therefore be more carefully evaluated before their use for cancer therapy. Furthermore, our studies provided a promising model to supplement animal studies to investigate the safety of anticancer agents or drugs. This is a simple and inexpensive system compared with animal models and could provide results and predictions directly applicable to humans. These findings are likely to have implications in stem cell biology, cancer therapy, and regenerative medicine and provide a basis for future studies.


Protective clothing for fire fighters is characterized by high thermal insulation to reduce external heat gain through dry and wet heat ,2. Howevre), heart rate (HR) and sweat water loss (change in body mass) were measured. Mean skin temperature  included two exposures to 40 °C ambient temperature including physical activity (treadmill with 3.3 and 5 Mets) of 20 min duration with a recovery phase of 20 min duration at 25 °C in between. The field trial  consisted of an exposure to a mean (SD) temperature at 1m above ground level of 141(18) °C for 15 min without any kind of activity. As for the lab trial, two exposures were separated by a recovery phase of 15 min duration at 25 °C. In both investigations, rectal temperature (T-1 and +0.17(0.11) °C.10 min-1) and the second exposure (+0.28(0.07) °C.10 min-1 and +0.29(0.13) °C.10 min-1). The maximum values for both trials are shown in table In the lab and field trials respectively, similar changes in rectal temperature were observed for the first (+0.15(0.05) °C.10 minMaximum physiological responses during the fire chamber trial were more pronounced as for the climatic chamber trial. This observation underlines the importance of fire fighter protective clothing in harsh conditions. However, changes in thermo-physiological responses were found to be similar for the lab and the field trial.Our findings highlight the importance of the thermo-physiological impact of protective clothing during activity. Increased physical activity itself can lead to non-compensable heat stress when wearing protective clothing, even though the environmental condition is not expected to induce severe heat stress.