The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a non-invasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be non-toxic to a panel of cancer cell lines grown continuously for up to 72 hours and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation and drug-induced cytotoxicity. The overall responses to anti-cancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multi-parameter and kinetic fingerprint of anti-cancer drug action.
DRAQ7; real-time assays; cell viability; drug; cytotoxicity; DNA damage response; cell cycle; microfluidic; cytometry
The effects of controlled release on immune response to an immunomodulating peptide were evaluated in a murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). The peptide, Ac-PLP-BPI-NH2-2 (Ac-HSLGKWLGHPDKF-(AcpGAcpGAcp)2-ITDGEATDSG-NH2; Ac = acetyl, Acp = aminocaproic acid) was designed to suppress T-cell activation in response to PLP139–151, an antigenic peptide in MS. Poly-lactide-co-glycolide (PLGA) microparticles containing Ac-PLP-BPI-NH2-2 (8±4 μm, 1.4±0.2% (w/w)) were prepared by a powder-in oil-in water emulsion-solvent evaporation method, sterilized and administered subcutaneously (s.c.) to SJL/J (H-2s) mice in which EAE had been induced by immunization with PLP139–151. Treatment groups received Ac-PLP-BPI-NH2-2: (i) in solution by repeated i.v. or s.c. injection, (ii) in solution co-administered with blank PLGA microparticles, (iii) in solution co-administered with Ac-PLP-BPI-NH2-2 loaded microparticles, and (iv) as Ac-PLP-BPI-NH2-2 loaded microparticles. Administration of Ac-PLP-BPI-NH2-2 as an s.c. solution produced clinical scores and maintenance of body weight comparable to i.v. solution, but with reduced overall survival, presumably due to anaphylaxis. Administration as s.c. microparticles provided a somewhat less effective reduction in symptoms but with no toxicity during treatment. Thus, the results suggest that s.c. administration of Ac-PLP-BPI-NH2-2 microparticles can provide pharmacological efficacy and reduction in dosing frequency without increased toxicity.
PLP-BPI peptides; Poly(D,L-lactide); Microparticles; EAE
Purine nucleotides are ubiquitous molecules that play vital roles in all kingdoms of life, not only as components of nucleic acids, but also participating in signaling and energy storage. Cellular pools of purines are maintained by the tight control of several complementary and sometimes competing processes including de novo biosynthesis, salvage and catabolism of nucleotides. While great strides have been made over the past sixty years in understanding the biosynthesis of purines, we are experiencing a renaissance in this field. In this feature article we discuss the most recent discoveries relating to purine biosynthesis, with particular emphasis upon the dynamic multi-protein complex called the purinosome. In particular we highlight advances made towards understanding the assembly, control and function of this protein complex and the attempts made to exploit this knowledge for drug discovery.
The characteristic features of senescent cells such as their “flattened” appearance, enlarged nuclei and low saturation density at the plateau phase of cell growth, can be conveniently measured by image-assisted d cytometry such as provided by the laser scanning cytometry (LSC). The “flattening” of senescent cells is reflected by the decline in local density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2- phenylindole (DAPI)] paralleled by an increase in nuclear size (area). Thus, the ratio of the maximal pixel of DAPI fluorescence per nucleus to the nuclear area provides a very sensitive morphometric biomarker of “depth” of senescence, which progressively declines during induction of senescence. Also recorded is cellular DNA content revealing cell cycle phase, as well as the saturation cell density at plateau phase of growth, which is dramatically decreased in cultures of senescent cells. Concurrent immunocytochemical analysis of expression of p21WAF1, p16INK4a or p27KIP1 cyclin kinase inhibitor provides additional markers of senescence. These biomarker indices can be expressed in quantitative terms (“senescence indices”) as a fraction of the same markers of the exponentially growing cells in control cultures.
Cell aging; chromatin structure; cell cycle; apoptosis; nuclear size; laser scanning cytometry; p21WAF1; p16INK4a; p27KIP1; premature cell senescence
We sought to compare the safety and accuracy of a new free-hand pedicle screw placement technique to that of the conventional technique. One hundred fifty-three consecutive adult patients with simple fracture in the thoracic or/and lumbar spine were alternately assigned to either the new free-hand or the conventional group. In the new free-hand technique group, preoperative computerized tomography (CT) images were used to calculate the targeted medial-lateral angle of each pedicle trajectory and the pedicle screw was inserted perpendicular to the correspond-ing supraspinal ligament. In the conventional technique group, the medial-lateral and cranial-caudal angle of each pedicle trajectory was determined by intraoperatively under fluoroscopic guidance. The accuracy rate of pedicle screw placement, the time of intraoperative fluoroscopy, the operating time and the amount of blood loss during operation were respectively compared. All screws were analyzed by using intraoperative radiographs, intraoperative triggered electromyography (EMG) monitoring data, postoperative CT data and clinical outcomes. The accuracy rate of pedicle screw placement in the new free-hand technique group and the conventional technique group was 96.3% and 94.2% (P < 0.05), respectively. The intraoperative fluoroscopy time of the new technique group was less than that of the conventional technique group (5.37 seconds vs. 8.79 seconds, P < 0.05). However, there was no statistical difference in the operating time and the amount of blood loss during operation (P > 0.05). Pedicle screw placement with the free-hand technique which keeps the screw perpendicular to the supraspinal ligament is an accurate, reliable and safe technique to treat simple fracture in the thoracic or lumbar spine.
spine fracture; pedicle screw placement; radiation exposure; supraspinal ligament; anatomy reference
Both hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure can cause liver damage as well as increase the probability of hepatocellular carcinoma (HCC). To investigate the underlying genetic changes that may influence development of HCC associated with HBV infection and AFB1 exposure, HCC patients were subdivided into 4 groups depending upon HBV and AFB1 exposure status: (HBV(+)/AFB1(+), HBV(+)/AFB1(-), HBV(-)/AFB1(+), HBV(-)/AFB1(-)). Genetic abnormalities and protein expression profiles were analyzed by array-based comparative genomic hybridization and isobaric tagging for quantitation. A total of 573 chromosomal aberrations (CNAs) including 184 increased and 389 decreased were detected in our study population. Twenty-five recurrently altered regions (RARs; chromosomal alterations observed in ≥10 patients) in chromosomes were identified. Loss of 4q13.3-q35.2, 13q12.1-q21.2 and gain of 7q11.2-q35 were observed with a higher frequency in the HBV(+)/AFB1(+), HBV(+)/AFB1(-) and HBV(-)/AFB1(+) groups compared to the HBV(-)/AFB(-) group. Loss of 8p12-p23.2 was associated with high TNM stage tumors (P = 0.038) and was an unfavorable prognostic factor for tumor-free survival (P =0.045). A total of 133 differentially expressed proteins were identified in iTRAQ proteomics analysis, 69 (51.8%) of which mapped within identified RARs. The most common biological processes affected by HBV and AFB1 status in HCC tumorigenesis were detoxification and drug metabolism pathways, antigen processing and anti-apoptosis pathways. Expression of AKR1B10 was increased significantly in the HBV(+)/AFB1(+) and HBV(-)/AFB1(+) groups. A significant correlation between the expression of AKR1B10 mRNA and protein levels as well as AKR1B10 copy number was observered, which suggest that AKR1B10 may play a role in AFB1-related hepatocarcinogenesis. In summary, a number of genetic and gene expression alterations were found to be associated with HBV and AFB1- related HCC. The possible synergistic effects of HBV and AFB1 in hepatocarcinogenesis warrant further investigations.
To investigate the feasibility of contouring the planning risk organ volume (PRV) for the heart, and to determine the probability of evaluating radiation dose to the heart using kilovoltage-cone beam computed tomography (kV-CBCT) in early-stage non-small-cell lung cancer (NSCLC) patients, who received stereotactic body radiotherapy (SBRT).
Materials and methods
Seventeen NSCLC patients who received SBRT (5Gy/f × 10f dose) were enrolled and subjected to CBCT and CT imaging analyses to plan treatment. Sequential planning CBCT images of individual patient’s hearts were analyzed for reproducibility of heart contouring and volume. Comparative analyses were made between the planning CT- and CBCT-detected heart margins and dose-volume indices for treatment.
The heart volume from planning CT images was significantly smaller than that from CBCT scans (p < 0.05), and the volumes based on the different series of CBCT images were similar (p > 0.05).The overlap of the heart region on the same anatomical section between the first series of CBCT scans and other scans reached 0.985 ± 0.020 without statistically significant differences (p > 0.05). The mean margins of the heart from planning CT and CBCT scans were 10.5 ± 2.8 mm in the left direction, 5.9 ± 2.8 mm in the right direction, 2.2 ± 1.6 mm in the direction of the head, 3.3 ± 2.2 mm in the direction of the foot, 6.7 ± 1.1 mm in the anterior direction, and 4.5 mm ± 2.5 mm in the posterior direction. All relative and absolute dose-volume indices obtained from CBCT images were significantly larger than those from planning CT scans (p < 0.05), with the exception of the volume in the 5Gy region.
The PRV of heart contouring based on kV-CBCT is feasible with good reproducibility. More accurate and objective dose-volume indices may be obtained for NSCLC patients by using kV-CBCT, instead of CT, to plan SBRT.
Non-small-cell lung cancer; Stereotactic body radiotherapy; Kilovoltage-cone beam computed tomography; Heart
Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, itwould be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenicDNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previouslywe showed that phosphorylation of H2AX (defined as γH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that γH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring γH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that γH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that γH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.
Tobacco smoke; H2AX; Double strand breaks; DNA damage
The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty- to 60-min exposure of cells to 100 or 200 μM H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24–48 h under hypoxic conditions (3% O2) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20–30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 μM) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.
H2AX phosphorylation; ATM activation; reactive oxygen species; hypoxia; hydrogen peroxide; 3-bromopyruvate; dichloroacetate; celecoxib; aging; caloric restriction; mitochondria
D-isoascorbic acid is a food antioxidant additive and used in accordance with Good Manufacturing Practice (GMP). High solubility in water (about 150 g/L at 25°C) reduces its effectiveness in stabilizing fats and oils. Our research group had successfully synthesized D-isoascorbyl palmitate using immobilized lipase Novozym 435 as a biocatalyst. Low production efficiency of D-isoascorbyl palmitate is still a problem for industrial production due to the long reaction time of over 24 h. In the present work, ultrasonic treatment was applied for accelerating the reaction process. The operation parameters were optimized to obtain the maximum D-isoascorbyl palmitate conversion rate by using a 5-level-4-factor Central Composite Design (CCD) and Response Surface Methdology (RSM). The reaction apparent kinetic parameters under the ultrasound treatment and mechanical shaking conditions were also determined and compared.
Results showed that ultrasound treatment decreased the reaction time by over 50%. D-isoascorbyl palmitate yielded to 94.32 ± 0.17% and the productivity reached to 8.67 g L-1 h-1 under the optimized conditions as: 9% of enzyme load (w/w), 61°C of reaction temperature, 1:5 of D- isoascorbic-to-palmitic acid molar ratio, and 137 W of the ultrasound power. The immobilized lipase Novozym 435 could be reused for 7 times with 65% of the remained D-isoascorbyl palmitate conversion rate. The reaction kinetics showed that the maximum apparent reaction rate (vmax) of the ultrasound-assisted reaction was 2.85 times higher than that of the mechanical shaking, which proved that ultrasound treatment significantly enhanced the reaction efficiency.
A systematic study on ultrasound-assisted enzymatic esterification for D-isoascorbyl palmitate production is reported. The results show a promising perspective of the ultrasound technique to reduce the reaction time and improve the production efficiency. The commercial D-isoascorbyl palmitate synthesis will be potentially realized due to this ultrasound-promoted esters synthesis method.
D-isoascorbyl palmitate; Ultrasound treatment; Process optimization; Kinetic evaluation
In China, there is increasing concern because of the rapid increase in HIV infection recorded over recent years. Migrant workers are recognized as one of the groups most affected. In this study, HIV/AIDS-related knowledge, attitudes, and behavior among unmarried migrant workers in Shanghai are investigated, with the aim of providing critical information for policy makers and sex educators to reinforce sexual health services and sex health education targeting the behavior and sexual health of unmarried male migrants.
A cross-sectional survey was conducted among unmarried male migrant workers in Shanghai, China’s largest city and housing the most migrants. A self-administered, anonymous questionnaire was used to collect information on knowledge, attitudes, and behavior associated with increased risk of HIV/AIDS.
A total of 2254 subjects were questioned, with a response rate of 91.3%. Among those interviewed, 63.5% reported sexual activities. Misconceptions regarding HIV transmission, poor perception of HIV infection, and low use of condoms were not uncommon. Among those who had sexual intercourse, 73.7% had not used condoms in their last sexual intercourse, and 28.6% reported having engaged in sexual risk behavior (defined as having at least one non-regular partner). Multivariate logistic regression analyses identified several indicators of sexual risk behavior, including younger age at first sexual intercourse (OR: 0.67, 95% CI: 0.31–0.91 for older age at first sexual intercourse), more cities of migration (OR: 2.91, 95% CI: 2.17–3.81 for high level; OR: 1.15, 95% CI: 1.06–1.29 for medium level), poor perception of acquiring HIV/AIDS (OR: 1.52, 95% CI: 1.33–1.96 for unlikely; OR: 2.38, 95% CI: 1.61–3.70 for impossible), frequent exposure to pornography (OR: 0.33, 95% CI: 0.11–0.43 for never; OR: 0.69, 95% CI: 0.60–1.81 for less frequently), not knowing someone who had or had died of HIV/AIDS and related diseases (OR: 2.13, 95% CI: 1.70–2.53 for no), and having peers who engaged in sex with a non-regular sex partner (OR: 4.40, 95% CI: 3.37–5.56 for yes).
Today, it is necessary to reinforce sex health education among unmarried migrants and sexual health services should target vulnerable migrant young people.
One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H2O2. Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon as 10 min after subjecting cells to UV or H2O2. While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H2O2 exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.
UV light; oxidative DNA damage; H2AX phosphorylation; cell cycle; DNA denaturation; acridine orange; metachromasia; ssDNA; lymphocytes
This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis.
ionizing radiation; DNA topoisomerase inhibitors; DNA double-strand breaks; carcinogens; tobacco smoke; replication stress; genotoxins; DNA photolysis
The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor κB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor κB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.
activation; differentiation; osteoclast; osteoprotegerin; serum-free
Infectious mononucleosis (IM) is the clinical presentation of primary infection with Epstein-Barr virus. Although the literature contains a massive amount of information on IM, most of this is related specifically to only children or adults separately. In order to distinguish any differences between preschool children and youth patients, we retrospectively analyzed their demographic and clinical features.
Records of patients hospitalized from December 2001 to September 2011 with a diagnosis of IM were retrieved from Peking University First Hospital, which is a tertiary teaching hospital in Beijing. The demographic data and clinical characteristics were collected.
IM was diagnosed in 287 patients during this 10-year period, with incidence peaks among preschool children (≤7 years old, 130/287, 45.3%) and youth patients (>15 and <24 years old, 101/287, 35.2%). Although the complaints at admission did not differ between these two patient groups, the incidence of clinical signs (tonsillopharyngitis, lymphadenopathy, hepatomegaly, and edema of the eyelids) was much higher in preschool children. The incidence of liver lesion and percentage of atypical lymphocytes were significantly higher in the youth group (P<0.001), and the average hospital stay was longer in this group. Pneumonia was the most common complication, and there was no case of mortality.
The incidence of IM peaks among preschool children and youth patients in Beijing, China. The levels of liver enzymes and atypical lymphocytes increase with age.
Epstein-Barr virus; Infectious mononucleosis; Preschool children; Youth
Reviewed are the phosphorylation events reporting activation of protein kinases and the key substrates critical for the DNA damage signaling (DDS). These DDS events are detected immunocytochemically using phospho-specific Abs; flow cytometry or image-assisted cytometry provide the means to quantitatively assess them on a cell by cell basis. The multiparameter analysis of the data is used to correlate these events with each other and relate to the cell cycle phase, DNA replication and induction of apoptosis. Expression of γH2AX as a possible marker of induction of DNA double strand breaks is the most widely studied event of DDS. Reviewed are applications of this multiparameter approach to investigate constitutive DDS reporting DNA damage by endogenous oxidants byproducts of oxidative phosphorylation. Also reviewed are its applications to detect and explore mechanisms of DDS induced by variety of exogenous agents targeting DNA such as exogenous oxidants, ionizing radiation, radiomimetic drugs, UV light, DNA topoisomerase I and II inhibitors, DNA crosslinking drugs and variety of environmental genotoxins. Analysis of DDS induced by these agents provides often a wealth of information about mechanism of induction and the type of DNA damage (lesion) and is reviewed in the context of cell cycle phase specificity, DNA replication, and induction of apoptosis or cell senescence. Critically assessed is interpretation of the data as to whether the observed DDS events report induction of a particular type of DNA lesion.
ATM; γH2AX; Chk2; DNA double stand breaks; chromatin relaxation; DNA replication; DNA topoisomerase inhibitors; UV; cytometry; confocal microscopy
Abiotic stress is a major environmental factor that limits cotton growth and yield, moreover, this problem has become more and more serious recently, as multiple stresses often occur simultaneously due to the global climate change and environmental pollution. In this study, we sought to identify genes involved in diverse stresses including abscisic acid (ABA), cold, drought, salinity and alkalinity by comparative microarray analysis. Our result showed that 5790, 3067, 5608, 778 and 6148 transcripts, were differentially expressed in cotton seedlings under treatment of ABA (1μM ABA), cold (4°C), drought (200mM mannitol), salinity (200mM NaCl) and alkalinity (pH=11) respectively. Among the induced or suppressed genes, 126 transcripts were shared by all of the five kinds of abiotic stresses, with 64 up-regulated and 62 down-regulated. These common members are grouped as stress signal transduction, transcription factors (TFs), stress response/defense proteins, metabolism, transport facilitation, as well as cell wall/structure, according to the function annotation. We also noticed that large proportion of significant differentially expressed genes specifically regulated in response to different stress. Nine of the common transcripts of multiple stresses were selected for further validation with quantitative real time RT-PCR (qRT-PCR). Furthermore, several well characterized TF families, for example, WRKY, MYB, NAC, AP2/ERF and zinc finger were shown to be involved in different stresses. As an original report using comparative microarray to analyze transcriptome of cotton under five abiotic stresses, valuable information about functional genes and related pathways of anti-stress, and/or stress tolerance in cotton seedlings was unveiled in our result. Besides this, some important common factors were focused for detailed identification and characterization. According to our analysis, it suggested that there was crosstalk of responsive genes or pathways to multiple abiotic or even biotic stresses, in cotton. These candidate genes will be worthy of functional study under diverse stresses.
The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61–vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.
pseudorabies virus; virus variant; virulent; immune evasion; viruses; pigs; Bartha-K61 vaccine strain; China
Connexin 26 (Cx26, GJB2) mutations can induce congenital deafness and are responsible for ~50 % of nonsyndromic hearing loss in children. Mouse models show that Cx26 deficiency induces cochlear development disorder, hair cell loss, and spiral ganglion (SG) neuron degeneration. Hair cell loss and cell degeneration have been considered as a primary causer responsible for Cx26 deficiency associated hearing loss. In this study, by coincidental examination of cochlear postnatal development with recording of auditory brainstem response (ABR) and hair cell function, we found that occurrence of hearing loss in Cx26 knockout (KO) mice was ahead of hair cell loss and cochlear cell degeneration. ABR was absent at any frequencies (8 – 40 kHz) after birth. However, cochlear cells including SG neurons had no significant degeneration throughout postnatal development. Severe cochlear hair cell loss and SG neuron degeneration were only visible in middle and basal turns, i.e., in middle and high frequency regions, in the adult Cx26 KO mouse cochlea. Functional tests show that hair cells in Cx26 KO mice functioned normally; outer hair cells retained electromotility. These data suggest that cell degeneration is not a primary causer of Cx26 deficiency associated hearing loss. Some mechanisms other than cell degeneration, such as cochlear development disorders, may play an essential role in this common hereditary deafness.
GJB2; gap junction; connexin; hair cell loss; deafness; inner ear
ATP is an important extracellular signaling molecule and can activate both ionotropic (P2X) and metabotropic purinergic (P2Y) receptors to influence cellular function in many aspects. Gap junction is an intercellular channel and plays a critical role in hearing. Here, we report that stimulation of ATP reduced gap junctional coupling between cochlear supporting cells. This uncoupling effect could be evoked by nanomolar physiological levels of ATP. A P2X receptor agonist benzoylbenzoyl-ATP (BzATP) but not a P2Y receptor agonist UTP stimulated this uncoupling effect. Application of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or oxidized ATP (oATP, 0.1 mM) eliminated this uncoupling effect. We further found that ATP activated P2X receptors in the cochlear supporting cells allowing Ca++ influxing, thereby increasing intracellular Ca++ concentration to mediate gap junctions. These data suggest that ATP can mediate cochlear gap junctions at the physiological level by the activation of P2X receptors rather than P2Y receptors. This P2X receptor-mediated purinergic control on the cochlear gap junctions may play an important role in the regulation of K+-recycling for ionic homeostasis in the cochlea and the reduction of hearing sensitivity under noise stress for protection.
ATP; gap junction; purinergic receptor; potassium; inner ear
Burkitt lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. We used high throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways that cooperate with MYC, the defining oncogene of this cancer. In 38% of sporadic BL (sBL) cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. In 70% of sBL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival PI(3) kinase pathway in BL, in part by augmenting constitutive B cell receptor signaling. These findings suggest opportunities to improve therapy for patients with BL.
454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
extracorporeal shock wave lithotripsy; pancreatic stones; hematoma
To analyze the clinicopathologic characteristics and prognostic factors of small gastrointestinal stromal tumor (GIST) of the stomach.
A total of 31 small gastric GIST patients, including 10 males and 21 females, with a median age of 58 years (37-81 years), who underwent surgery at any time from 1999 to 2012 were included in this study. The clinical records of the patients were analyzed retrospectively.
Abdominal discomfort and pain (10 cases, 32.3%, respectively) were the two most common complaints among the patients. All patients received surgery, 11 received gastric wedge resection, 11 received subtotal gastrectomy, 5 received laparoscopic gastric wedge resection, and 4 received endoscopic submucosal dissection. No severe adverse complication was observed. A total of 29 patients (93.5%) were followed up. During the follow-up, 2 patients were found to exhibit tumor recurrence, and 1 patient had liver metastases. One patient died of tumor progression, while another died of another malignant tumor. Median progression free survival (PFS) time was 120.3 months, and median overall survival (OS) time was 130.4 months.
Small gastric GIST has better prognosis. Surgery is the best choice for therapy. Micro-invasive procedures are safe and effective for elective patients. Tumor necrosis, tumor bleeding, and muscle invasion are potential prognostic factors of small gastric GIST.
Gastrointestinal stromal tumors; endoscopy; prognosis
DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ) and asthenozoospermia (AZ) usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control), with only complete methylation and mild hypomethylation(<50% unmethylated CpGs) identified, and there was no significant difference in the occurrence of these two methylation patterns between AZ and NZ (P>0.05). However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs ) and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05%) and AZ (0.58±1.77%) (P<0.001).Additional, our data indicated the occurrence of >20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05). In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.