Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.     

SHIV病毒在猴体内的复制与传代

目的为建立SHIV艾滋病动物模型提供毒力较强的病毒株,将新合成的SHIV XJ02170病毒适应猴体,并增强其毒力。方法实验前采集猴血清并进行血清学检查和PCR检测。选出13只无SIV,STLV1,SRV D和B病毒感染的猴。第一批实验,将SHIV前病毒DNA质粒经肌肉注射到猴体内,每只500μg;SHIV病毒液,经静脉注射到猴体内,每只2ml。病毒质粒和病毒液各接种2只猴。当第一批猴体检出病毒后,10ml感染猴的全血,抗凝后静脉注射到第二批猴体内,当第二批猴检出病毒后再将10ml感染猴的全血静脉注射到第三批猴体内,连续传代4次。每批实验均定期采集血液标本,分别用肝素和EDTA抗凝,进行病毒分离;病毒基因PCR检测;CD4,CD8测定;病毒抗体检测。结果SHIV XJ02170病毒和SHIV XJ02170前病毒DNA质粒在猴体内的传代中均能分离出病毒;从传代猴的血浆和外周血单核细胞(PBMC)中检出了病毒DNA和RNA基因;CD4,CD8测定结果显示有暂时性倒置现象,后变为正常倒置与正常交替出现;在传代的猴血清中检测出特异性HIV病毒抗体。结论SHIV XJ02170病毒与SHIV XJ02170前病毒DNA质粒,均能在恒河猴体内复制。     

Distinct Pathogenic Genes Causing Intellectual Disability and Autism Exhibit a Common Neuronal Network Hyperactivity Phenotype

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Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Engagement of the ATR-Dependent DNA Damage Response at the Human Papillomavirus 18 Replication Centers during the Initial Amplification

We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high- and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G₂ phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of γH2AX, ATR-interacting protein (ATRIP), and topoisomerase IIβ-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification.     

Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.     

大鼠及小鼠微卫星引物在社鼠中的跨种扩增

利用微卫星引物在同一属、科、目不同种之间具有通用性的特点,通过PCR扩增、聚丙烯凝胶电泳和银染技术对社鼠(Niviventer confucianus)近缘物种大鼠(Rattus norvegicus)、小鼠(Mus musculus)中已知的70个微卫星位点引物进行跨种扩增,筛选适合社鼠相关研究的多态微卫星引物.结果发现,40个位点引物出现扩增条带,21个位点引物能够稳定扩增,其中15个位点杂合,13个位点具有多态性;PCR扩增的Mg2+浓度主要集中在1.5及2.0 mmol/L,退火温度在50～60℃之间不等.虽然部分扩增产物有影子带的存在,但并不影响等位基因的判读.总体来看,利用大、小鼠的微卫星引物扩增社鼠的微卫星位点是可行的.     

Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G₁, after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.The RecQ helicases are a family of DNA-unwinding enzymes essential for the maintenance of genome integrity in all kingdoms of life. Five RecQ enzymes have been found in human cells: RECQ1 (also called RECQL or RECQL1), BLM (RECQ2 or RECQL3), WRN (RECQ3 or RECQL2), RECQ4 (RECQL4), and RECQ5 (RECQL5) (3, 7). Here we refer to these helicases as RECQ1, RECQ4, and RECQ5, without the “L” that is present in the official gene names. Mutations in the BLM, WRN, and RECQ4 genes are linked to Bloom syndrome (BS), Werner syndrome (WS), and the subset of Rothmund-Thomson syndrome (RTS) patients at high risk of developing osteosarcomas, respectively (19, 31, 71). RECQ4 mutations have also been associated with RAPADILINO and Baller-Gerold syndrome (56, 61). Although these disorders are all associated with inherent genomic instability and cancer predisposition, they show distinct clinical features, suggesting that BLM, WRN, and RECQ4 are involved in different aspects of DNA metabolism. However, the molecular events underlying the pathogenesis of BS, WS, and RTS remain obscure. Mutations in the remaining two human RecQ helicase genes, RECQ1 and RECQ5, have not as yet been identified as causes of either genomic instability or heritable cancer predisposition disorders.Several lines of evidence suggest that RecQ helicases play an important role in DNA replication control (3, 10). In particular, RecQ helicases are thought to facilitate replication by preserving the integrity of stalled replication forks and by remodeling or repairing damaged or collapsed forks to allow the resumption of replication. Consistent with these ideas, several investigators have shown that primary fibroblasts from BS, WS, and RTS patients and RecQ5-deficient mouse embryonic fibroblasts all show differential hypersensitivity to agents that perturb DNA replication (12, 14, 26, 29). Moreover, BLM and WRN are recruited to DNA replication forks after replicative stress, and DNA fiber track analyses have shown that both BLM and WRN are required for normal fork progression after DNA damage or replication arrest (11-13, 47, 54). In particular, BLM in conjunction with DNA topoisomerase III and two other accessory proteins, RMI-1 and RMI-2, has been shown to catalyze the resolution of double-Holliday-junction recombination intermediates to generate noncrossover products. This dissolution reaction could play an important role in the error-free recombinational repair of damaged or stalled forks during S phase (57, 67). WRN also appears to promote error-free repair by contributing to the resolution of gene conversion events to generate noncrossover products (46). In line with the above observations, WRN and BLM can be found associated with replication foci or other DNA damage response proteins in damaged cells. In contrast, in unperturbed cells, a majority of each protein is found in the nucleolus (WRN) or associated with PML bodies (BLM) (5, 37, 62).RECQ4 has also been implicated in DNA replication. Recent studies have shown that hypomorphic mutants of the Drosophila melanogaster homolog of human RECQ4, DmRECQ4, have reduced DNA replication-dependent chorion gene amplification (65). These findings are thus consistent with a postulated role for Xenopus laevis RECQ4 (XRECQ4) in the initiation of DNA replication (39, 48). The N terminus of XRECQ4 bears homology to the N termini of the yeast proteins Sld2 (Saccharomyces cerevisiae [budding yeast]) and DRC1 (Schizosaccharomyces pombe [fission yeast]), which play a central role, in association with budding yeast Dpb11 and the fission yeast homolog Cut5/Rad4, in the establishment of DNA replication forks (38, 41, 63). Consistently, the N terminus of XRECQ4 has been shown to interact with the X. laevis variant of Cut5, and XRECQ4 depletion severely perturbs DNA replication initiation in X. laevis egg extracts (39, 48). The notion that the function of XRECQ4 is evolutionarily conserved in mammals is supported by the observations that the human protein can complement its Xenopus counterpart in cell-free assays for replication initiation and that depletion of human RECQ4 inhibits cellular proliferation and DNA synthesis (39, 48). Moreover, deletion of the N-terminal region of mouse RECQ4 has been shown to be an embryonic lethal mutation (27). These observations suggest that vertebrate RECQ4 might be a functional homolog of Sld2/DRC11, although its precise function during replication initiation and progression is not known. Recent results, published while this work was in progress, indicate that human RECQ4 interacts with the MCM replicative complex during replication initiation and that this interaction is regulated by CDK phosphorylation of RECQ4 (69). These findings, together with our results below, provide clues to the mechanism regulating RECQ4 interaction with the replication machinery.RECQ1 is the most abundant of the human RecQ helicases and was the first of the human RecQ proteins to be discovered on the basis of its potent ATPase activity (50). Despite this, little is known about the cellular functions of RECQ1, and no human disease associations have been identified to date. Recent studies have shown that RECQ1 is involved in the maintenance of genome integrity and that RECQ1 depletion affects cellular proliferation (51). Moreover, biochemical studies have shown that RECQ1 and BLM display distinct substrate specificities, indicating that these helicases are likely to perform nonoverlapping functions (43). These results suggest an important—though as yet mechanistically ill-defined—role for RECQ1 in cell cycle progression and/or DNA repair (52).In order to better delineate the role of human RecQ helicases in DNA replication, we investigated the in vivo interactions of all five human RecQ enzymes with three well-characterized human DNA replication origins in quantitative chromatin immunoprecipitation (ChIP) assays. We also determined how nascent-origin-dependent DNA synthesis, chromatin binding of replication proteins, origin firing frequency, and replication fork rates were altered by depleting specific human RecQ helicase proteins. We found that only two of the five human RecQ helicases, RECQ1 and RECQ4, specifically interact with origins in unperturbed cells. Our results provide new mechanistic insight into the distinct roles of human RECQ1 and RECQ4 in DNA replication initiation and in replication fork progression.     

Replication Patterns of Repetitive DNA Sequences on the W Chromosome Are Altered during Development of the Chick Embryo

A novel method was developed to study developmental changes in the replication pattern of repetitive DNA sequences on the W chromosome (W-DNA) of the female chick embryo. The amount of total nuclear DNA and W-DNA as well as 5-bromodeoxyuridine (BrdU) incorporation was successively measured on the same cells using multiparametric microfluorometry [1]. With this method we first examined the possibility of changes in replication patterns of W-DNA during development. Measurements were conducted on various heterogeneous cell populations obtained from whole embryo on Day 0.4 and Day 1, and from pectoral muscle, neural tube, liver, and oogonium on Day 9. Parameters of W-DNA replication, duration, and timing were found to vary according to the stage of embryonic development. Developmental features of these changes were further studied on specific cell types during their critical developmental processes. In scutate scale dermis, the W-DNA replication duration showed a characteristic lengthening from around 0.45C during Day 5 through Day 7.4 to 0.9C during Day 7.7 through Day 7.9 and shortening to 0.37C during Day 8.1 through Day 12. Transient lengthening in W-DNA replication duration was also observed in erythrocytes; 0.65C → 1.0C → 0.6C during Day 0.9 through Day 2.17. Timing also shifted earlier in accord with changes in the duration. Replication rate of whole genome DNA was monitored by measuring BrdU incorporation on respective cells and found, to a large extent, comparable to that of W-DNA. The data suggest that a link might be operative between replication patterns of genes and the developmental program.     

Cells of the Upper and Lower Epidermis of Barley (Hordeum vulgare L.) Leaves Exhibit Distinct Patterns of Vacuolar Solutes

Vacuolar saps were extracted from individual, anatomically uniform cells of the upper (adaxial) and lower (abaxial) epidermis of the third leaf of barley (Hordeum vulgare L.) using a modified pressure probe. Saps (volume 80-200 pL) were sampled at various times between 3 d before and 7 d after full-leaf expansion and were analyzed for their osmolality and their concentrations of NO3-, malate, CI-, K+, and Ca2+. The osmolalities of upper and lower epidermis both increased with time but were similar to each other. In young leaves, K+ and Ca2+ were evenly distributed between the two epidermal layers, but as the leaf aged, the upper epidermis accumulated high (40-100 mM) Ca2+, whereas cells of the lower epidermis accumulated K+ instead. Nitrate concentration was 100 to 150 mM higher in the upper than in the lower epidermis, whereas CI- was 50 to 120 mM higher in the lower epidermis. These differences did not depend on the leaf developmental stage. The uneven distribution of epidermal NO3- and CI- was maintainedover a wide range of epidermal sap concentrations of these ions and was not affected by NO3- or CI- starvation or by an increase in the light intensity from 120 to 400 [mu]mol m-2 s-1. However, the latter did cause a decrease in epidermal NO3- and the appearance and accumulation of epidermal malate, particularly in the upper epidermis. The physiological implications of the results for solute storage in leaves and for the pathways of ion distribution to the epidermis are discussed.     

Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks

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Dynamics of DNA Replication during Premeiosis and Early Meiosis in Wheat

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.     

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.