Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Knockdown of Smox protects the integrity of the blood-brain barrier through antioxidant effect and Nrf2 pathway activation in stroke.

Once an ischemic stroke occurs, reactive oxygen species (ROS) and oxidative stress degrade the tight connections between cerebral endothelial cells resulting in their damage. The expression of antioxidant genes may be enhanced, and ROS formation may be reduced following Nrf2 activation, which is associated with protection against ischemic stroke. Overexpression of spermine oxidase (Smox) in the neocortex led to increased H2O2 production. However, how Smox impacts the regulation of the blood-brain barrier (BBB) through antioxidants has not been examined yet. We conducted experiments both in the cell level and in the transient middle cerebral artery occlusion (tMCAO) model to evaluate the effect of Smox siRNA lentivirus (si-Smox) knockdown on BBB protection against ischemic stroke. Mice treated with si-Smox showed remarkably decreased BBB breakdown and reduced endothelial inflammation following stroke. The treatment with si-Smox significantly elevated the Bcl-2 to Bax ratio and decreased the production of cleaved caspase-3 in the tMCAO model. Further investigation revealed that the neuroprotective effect was the result of the antioxidant properties of si-Smox, which reduced oxidative stress and enhanced CD31+ cells in the peri-infarct cortical areas. Of significance, si-Smox activated Nrf2 in both bEnd.3 cells and tMCAO animals, and blocking Nrf2 with brusatol diminished the protective effects of si-Smox. The study findings suggest that si-Smox exerts neuroprotective effects and promotes angiogenesis by activating the Nrf2 pathway, thus decreasing oxidative stress and apoptosis caused by tMCAO. As a result, si-Smox may hold potential as a therapeutic candidate for preserving BBB integrity while treating ischemic stroke.

Identification and validation of major-effect quantitative trait locus QMS-5B associated with male sterility in photo-thermo-sensitive genic male sterile wheat.

KEY MESSAGE: QMS-5B, a major QTL for photo-thermo-sensitive genic male sterility in wheat, was fine mapped in a 2.15 Mb region harboring a serine/threonine protein kinase gene TraesCS5B03G0887500, which was the most likely candidate gene. Genic male sterility is an essential trait in the utilization of heterosis and hybrid seed production for wheat. Currently, genic male sterile genes have been reported in wheat mutants, but the sterile genes controlling photo-thermo-sensitive genic male sterility in wheat have not been studied systematically. Here, 235 doubled haploid lines derived from a cross between photo-thermo-sensitive genic male sterile line BS462 and its restorer line CP279 were used to map male sterile gene by GenoBaits® Wheat 100 K Panel, bulked segregant exome sequencing (BSE-Seq) and wheat 660 K array. As a result, the major stable QTL on chromosome 5B, QMS-5B, was identified in all four environments, accounting for 7.3-36.4% of the phenotypic variances. Ulteriorly, QMS-5B was delimited to an approximate 2.15 Mb physical interval between KASP-5B5 and KASP-5B6 using kompetitive allele-specific PCR (KASP) markers. Within the interval, twenty-nine high-confidence genes were predicted according to Chinese Spring RefSeq v2.1. TraesCS5B03G0887500, encoding a serine/threonine protein kinase, was identified as the most likely candidate gene for QMS-5B based on weighted gene co-expression network analysis. Expression analysis confirmed that TraesCS5B03G0887500 was significantly differentially expressed in anthers of BS462 and CP279 at different stages under fertile and sterile environments. In addition, flanking KASP marker KASP-5B6 can effectively genotype male sterile lines and restorer lines, and can be used for molecular marker-assisted selection. This study provides insights into for exploring the mechanism of photo-thermo-sensitive genic male sterility in wheat.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate­resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double­strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)­resistant HT29 cells [DM­ and homogeneously staining region (HSR)­containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM­containing cells. No corresponding changes were found in HSR­containing cells. The present study further verified the involvement of MSH3­regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3­depleted DM­containing cells. Finally, changes in MSH3 expression could affect DHFR amplification­derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA­based drug resistance in cancer.

Genome-Wide Identification and Expression Profiling Analysis of the Long-Chain Acyl-CoA Synthetases Reveal Their Potential Roles in Wheat Male Fertility.

Long-chain acyl-CoA synthetase (LACS), responsible for the conversion of free FAs into acyl-CoAs, is involved in multiple pathways of lipid metabolism. Although LACS genes in Arabidopsis have been well characterized, no detailed information concerning this family is available for wheat. In the present study, a systematic analysis was carried out for the wheat LACS family. As a result, 30 putative TaLACSs were identified. Expression analysis revealed that 22 Takacs were expressed in wheat anthers. Two orthologs of AtLACS1, TaLACS2 and TaLACS3, were repressed at the vacuolated stage in the cold-treated BS366 (a temperature-sensitive genic male-sterile line). Thus, TaLACS2 and TaLACS3 may function like AtLACS1 in wax biosynthesis in anthers, and the repression of both genes may be correlated with the male sterility of BS366. TaLACS5 is an ortholog of AtLACS5, which was expressed exclusively in anthers. TaLACS5 was repressed in the cold-treated BS366 at the tetrad, uninucleate, and vacuolated stages. The negative correlation between TaLACS5 and TaGAMYB-B, and the MYB domain found in the promoter sequence suggested that TaLACS5 may be negatively regulated by TaGAMYB-B to participate in wheat fertility. These findings will provide a valuable foundation for the understanding of the wheat LACS gene family in male fertility.

A Novel Marine Pyran-Isoindolone Compound Enhances Fibrin Lysis Mediated by Single-Chain Urokinase-Type Plasminogen Activator.

Fungi fibrinolytic compound 1 (FGFC1) is a rare pyran-isoindolone derivative with fibrinolytic activity. The aim of this study was to further determine the effect of FGFC1 on fibrin clots lysis in vitro. We constructed a fibrinolytic system containing single-chain urokinase-type plasminogen activator (scu-PA) and plasminogen to measure the fibrinolytic activity of FGFC1 using the chromogenic substrate method. After FITC-fibrin was incubated with increasing concentrations of FGFC1, the changes in the fluorescence intensity and D-dimer in the lysate were measured using a fluorescence microplate reader. The fibrin clot structure induced by FGFC1 was observed and analyzed using a scanning electron microscope and laser confocal microscope. We found that the chromogenic reaction rate of the mixture system increased from (15.9 ± 1.51) × 10−3 min−1 in the control group to (29.7 ± 1.25) × 10−3 min−1 for 12.8 µM FGFC1(p < 0.01). FGFC1 also significantly increased the fluorescence intensity and d-dimer concentration in FITC fibrin lysate. Image analysis showed that FGFC1 significantly reduced the fiber density and increased the fiber diameter and the distance between protofibrils. These results show that FGFC1 can effectively promote fibrin lysis in vitro and may represent a novel candidate agent for thrombolytic therapy.

Inhibiting homologous recombination decreases extrachromosomal amplification but has no effect on intrachromosomal amplification in methotrexate-resistant colon cancer cells.

Gene amplification, which involves the two major topographical structures double minutes (DMs) and homegeneously stained region (HSR), is a common mechanism of treatment resistance in cancer and is initiated by DNA double-strand breaks. NHEJ, one of DSB repair pathways, is involved in gene amplification as we demonstrated previously. However, the involvement of homologous recombination, another DSB repair pathway, in gene amplification remains to be explored. To better understand the association between HR and gene amplification, we detected HR activity in DM- and HSR-containing MTX-resistant HT-29 colon cancer cells. In DM-containing MTX-resistant cells, we found increased homologous recombination activity compared with that in MTX-sensitive cells. Therefore, we suppressed HR activity by silencing BRCA1, the key player in the HR pathway. The attenuation of HR activity decreased the numbers of DMs and DM-form amplified gene copies and increased the exclusion of micronuclei and nuclear buds that contained DM-form amplification; these changes were accompanied by cell cycle acceleration and increased MTX sensitivity. In contrast, BRCA1 silencing did not influence the number of amplified genes and MTX sensitivity in HSR-containing MTX-resistant cells. In conclusion, our results suggest that the HR pathway plays different roles in extrachromosomal and intrachromosomal gene amplification and may be a new target to improve chemotherapeutic outcome by decreasing extrachromosomal amplification in cancer.

Scaling and predictability in stock markets: a comparative study.

Most people who invest in stock markets want to be rich, thus, many technical methods have been created to beat the market. If one knows the predictability of the price series in different markets, it would be easier for him/her to make the technical analysis, at least to some extent. Here we use one of the most basic sold-and-bought trading strategies to establish the profit landscape, and then calculate the parameters to characterize the strength of predictability. According to the analysis of scaling of the profit landscape, we find that the Chinese individual stocks are harder to predict than US ones, and the individual stocks are harder to predict than indexes in both Chinese stock market and US stock market. Since the Chinese (US) stock market is a representative of emerging (developed) markets, our comparative study on the markets of these two countries is of potential value not only for conducting technical analysis, but also for understanding physical mechanisms of different kinds of markets in terms of scaling.

A statistical physics view of pitch fluctuations in the classical music from Bach to Chopin: evidence for scaling.

Because classical music has greatly affected our life and culture in its long history, it has attracted extensive attention from researchers to understand laws behind it. Based on statistical physics, here we use a different method to investigate classical music, namely, by analyzing cumulative distribution functions (CDFs) and autocorrelation functions of pitch fluctuations in compositions. We analyze 1,876 compositions of five representative classical music composers across 164 years from Bach, to Mozart, to Beethoven, to Mendelsohn, and to Chopin. We report that the biggest pitch fluctuations of a composer gradually increase as time evolves from Bach time to Mendelsohn/Chopin time. In particular, for the compositions of a composer, the positive and negative tails of a CDF of pitch fluctuations are distributed not only in power laws (with the scale-free property), but also in symmetry (namely, the probability of a treble following a bass and that of a bass following a treble are basically the same for each composer). The power-law exponent decreases as time elapses. Further, we also calculate the autocorrelation function of the pitch fluctuation. The autocorrelation function shows a power-law distribution for each composer. Especially, the power-law exponents vary with the composers, indicating their different levels of long-range correlation of notes. This work not only suggests a way to understand and develop music from a viewpoint of statistical physics, but also enriches the realm of traditional statistical physics by analyzing music.

[Preparation and characterization of visible-light response activated carbon with antibacterial behavior].

A visible-light response activated carbon with antibacterial activity was prepared by calcinations of the mixture of TiO2 precursor obtained by acid catalyzed hydrolysis method and commercial granular activated carbon (GAC) in NH3/N2 atmosphere. The antibacterial activity of the prepared activated carbon towards E. coil was investigated under the visible-light irradiation. X-ray diffraction (XRD), Scanning electron spectroscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and N2 adsorption analyser were used to characterize the crystal phase structure, surface morphology, spectral characteristics and pore properties. The results show there is no influence for crystal phase structure of TiO2 supported AC. The crystal size of TiO2 is 9.8 nm. Non-crystal phase layer and strong adsorption force of carrier can inhibit the grain growth of TiO2, and AC combines with TiO2 in Ti-O-C so that TiO2 formestight film on AC. The sample calcinated at 500 degrees C for 5 h exhibits the highest bactericidal performance, and the bactericidal rate reached up to 67% after 4 h irradiation, which was better than that of nature light (39%) in the same condition.

High glucose condition upregulated Txnip expression level in rat mesangial cells through ROS/MEK/MAPK pathway.

Thioredoxin interacting protein (Txnip) is one of the most abundantly up-regulated genes in response to hyperglycemia. The increased renal expression of Txnip was associated with type IV collagen accumulation in streptozotocin-induced diabetic mice. As the mechanism of action of high glucose is unknown, we undertook the investigation of the signaling pathway on the upregulation of Txnip expression induced by high glucose in rat mesangial cells. Rat mesangial cells were exposed to normal (5.5 mM) or high (25 mM) glucose at different time points. Txnip expression was determined using real-time RT-PCR and western-blotting at transcription and translation level, respectively. Intracellular reactive oxygen species (ROS) was detected by FACS Calibur flow cytometer using fluorescent probe (DCFH-DA).The treatment with high glucose resulted in an increase of Txnip mRNA from 4 h to 12 h and Txnip protein from 12 to 24 h in comparison with normal glucose condition. In addition, N-acetyl-cysteine (NAC) was found to decrease Txnip protein expression under high glucose condition. Furthermore, p38MAPK inhibitor SB203580 suppressed Txnip expression at transcription and protein level significantly to high glucose exposure. These results suggest that high glucose exposure improves Txnip mRNA and protein expression level by ROS/MEK/MAPK signaling pathway.