We evaluated how variations in sequencing depth and other parameters influence interpretation of chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. Using Drosophila melanogaster S2 cells, we generated ChIP-seq data sets for a site-specific transcription factor (Suppressor of Hairy-wing) and a histone modification (H3K36me3). We detected a chromatin-state bias: open chromatin regions yielded higher coverage, which led to false positives if not corrected. This bias had a greater effect on detection specificity than any base-composition bias. Paired-end sequencing revealed that single-end data underestimated ChIP-library complexity at high coverage. Removal of reads originating at the same base reduced false-positives but had little effect on detection sensitivity. Even at mappable-genome coverage depth of ∼1 read per base pair, ∼1% of the narrow peaks detected on a tiling array were missed by ChIP-seq. Evaluation of widely used ChIP-seq analysis tools suggests that adjustments or algorithm improvements are required to handle data sets with deep coverage.

RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.

To describe and interpret lung cancer incidence and mortality trends in Croatia between 1988 and 2008.

Alzheimer's disease (AD) is the most common form of dementia affecting millions of people worldwide and will continue to affect millions more with population aging on the rise. AD causality is multifactorial. Known causal factors include genetic predisposition, age, and sex. Environmental toxins such as air pollution (AP) have also been implicated in AD causation. Exposure to AP can lead to chronic oxidative stress (OS), which is involved in the pathogenesis of AD. Whereas AP plays a role in AD pathology, the epidemiological evidence for this association is limited. Given the significant prevalence of AP exposure combined with increased population aging, epidemiological evidence for this link is important to consider. In this paper, we examine the existing evidence supporting the relationship between AP, OS, and AD and provide recommendations for future research on the population level, which will provide evidence in support of public health interventions.

To determine if caloric restriction (CR) would cause changes in plasma metabolic intermediates in response to a mixed meal, suggestive of changes in the capacity to adapt fuel oxidation to fuel availability or metabolic flexibility, and to determine how any such changes relate to insulin sensitivity (S(I)).

In this study, we have demonstrated that Korean Panax ginseng (KG) significantly enhances myelopoiesis in vitro and reconstitutes bone marrow after 5-flurouracil-induced (5FU) myelosuppression in mice. KG promoted total white blood cell, lymphocyte, neutrophil and platelet counts and improved body weight, spleen weight, and thymus weight. The number of CFU-GM in bone marrow cells of mice and serum levels of IL-3 and GM-CSF were significantly improved after KG treatment. KG induced significant c-Kit, SCF and IL-1 mRNA expression in spleen. Moreover, treatment with KG led to marked improvements in 5FU-induced histopathological changes in bone marrow and spleen, and partial suppression of thymus damage. The levels of IL-3 and GM-CSF in cultured bone marrow cells after 24 h stimulation with KG were considerably increased. The mechanism underlying promotion of myelopoiesis by KG was assessed by monitoring gene expression at two time-points of 4 and 8 h. Treatment with Rg1 (0.5, 1 and 1.5 µmol) specifically enhanced c-Kit, IL-6 and TNF-α mRNA expression in cultured bone marrow cells. Our results collectively suggest that the anti-myelotoxicity activity and promotion of myelopoiesis by KG are mediated through cytokines. Moreover, the ginsenoside, Rg1, supports the role of KG in myelopoiesis to some extent.

Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists.

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) have been associated with neurodevelopmental and neuropsychiatric disorders including Rett Syndrome, X-linked mental retardation syndrome, severe neonatal encephalopathy, and Angelman syndrome. Although alterations in the performance of MeCP2-deficient mice in specific behavioral tasks have been documented, it remains unclear whether or not MeCP2 dysfunction affects patterns of periodic behavioral and electroencephalographic (EEG) activity. The aim of the current study was therefore to determine whether a deficiency in MeCP2 is sufficient to alter the normal daily rhythmic patterns of core body temperature, gross motor activity and cortical delta power. To address this, we monitored individual wild-type and MeCP2-deficient mice in their home cage environment via telemetric recording over 24 hour cycles. Our results show that the normal daily rhythmic behavioral patterning of cortical delta wave activity, core body temperature and mobility are disrupted in one-year old female MeCP2-deficient mice. Moreover, female MeCP2-deficient mice display diminished overall motor activity, lower average core body temperature, and significantly greater body temperature fluctuation than wild-type mice in their home-cage environment. Finally, we show that the epileptiform discharge activity in female MeCP2-deficient mice is more predominant during times of behavioral activity compared to inactivity. Collectively, these results indicate that MeCP2 deficiency is sufficient to disrupt the normal patterning of daily biological rhythmic activities.

We conducted a pilot study to evaluate the effects of pelvic radiotherapy on biologic markers of oxidative stress and plasma endotoxin levels, and to assess the relationship between the changes of such factors and radiotherapy-related complications.

CD8 T-cell responses were examined in subjects with incident (new following negative visits) or prevalent (lasting ≥ 4 months) human papillomavirus type 16 (HPV16) or human papillomavirus (HPV18) infection. The groups were chosen from a cohort of women being followed every 4 months with cervical cytology and HPV-DNA testing. Enzyme-linked immunospot (ELISPOT) assay was performed at enrollment (time zero) and one year later. At time zero, 1 (6%) of 17 subjects with incident HPV 16/18 infections had positive ELISPOT results which increased to 6 (35%) at one year. For the subjects with prevalent HPV 16/18 infections, the ELISPOT results were similar at time zero (2 (15%) of 15 subjects positive) and at one year (3 (20%)). While all of the 11 women with prevalent HPV16 infection showed clearance one year later, unexpectedly only 1 (25%) of 4 women with prevalent HPV18 infection demonstrated clearance one year later (P = .009).

The β-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, β-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli.

Acupuncture has been used for millennia to treat pain, although its efficacy and duration of action is limited. Acupuncture also has brief (1-2 h) antinociceptive effects in mice and these effects are dependent on localized adenosine A₁ receptor (A₁R) activation. Intriguingly, adenosine 5'-monophosphate (AMP) is basally elevated near acupuncture points. This finding suggested that it might be possible to inhibit nociception for a longer period of time by injecting prostatic acid phosphatase (PAP, ACPP) into acupuncture points. PAP is an ectonucleotidase that dephosphorylates extracellular AMP to adenosine, has a long half-life in vivo and is endogenously found in muscle tissue surrounding acupuncture points. Here, we found that injection of PAP into the popliteal fossa--a space behind the knee that encompasses the Weizhong acupuncture point--had dose- and A₁R-dependent antinociceptive effects in mouse models of acute and chronic pain. These inhibitory effects lasted up to six days following a single injection, much longer than the hour-long inhibition provided by acupuncture. Antinociception could be transiently boosted with additional substrate (AMP) or transiently blocked with an A₁R antagonist or an inhibitor of phospholipase C. This novel therapeutic approach--which we term "PAPupuncture"--locally inhibits pain for an extended period of time (100x acupuncture), exploits a molecular mechanism that is common to acupuncture, yet does not require acupuncture needle stimulation.

Hypoxia, acidosis and programmed cell death are each hallmarks of acute myocardial infarction (AMI). We previously described a death pathway of cardiac myocytes mediated by hypoxia-acidosis that was characterized by activation of the Bcl2-family protein Bnip3 and programmed necrosis. The pathway included extensive DNA fragmentation that was sensitive to inhibition of the mitochondrial permeability transition pore (mPTP) and calpain inhibitors, but not caspase inhibitors. We did not identify the DNases responsible for DNA cleavage.

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking that is crucial for parietal cell function and epithelial cell homeostasis in the stomach. Gastric parietal cells in Hip1r-deficient mice are lost by apoptotic cell death, which leads to a progressive epithelial cell derangement, including glandular hypertrophy, zymogenic cell loss and expansion of a metaplastic mucous cell lineage known as spasmolytic polypeptide-expressing metaplasia (SPEM). The epithelial cell changes are associated with infiltration of inflammatory cells. As inflammatory mediators, such as IFNγ, have been shown to contribute to the development of the gastric epithelial cell metaplasia after Helicobacter infection, we tested whether IFNγ played a role in the spontaneous progressive epithelial metaplasia observed in Hip1r-deficient mice. Hip1r-deficient mice were crossed with IFNγ-deficient mice and single- and double-mutant mice were analyzed at 3 and 12 months of age. Histopathology scoring showed that loss of IFNγ tempered the spontaneous development of metaplastic lesions in Hip1r-deficient mice. Loss of IFNγ was observed to abrogate the glandular hypertrophy evident in Hip1r mutant stomach, although increased epithelial cell proliferation and elevated gastrin levels were not affected by the presence or absence of this pro-inflammatory cytokine. An analysis of cell lineage markers in the double-mutant mice demonstrated that IFNγ specifically affected the development of metaplastic mucous cells in the neck region, whereas the parietal cell, surface mucous cell and zymogenic cell alterations remained similar to the histopathology in the Hip1r mutant. Morphometric analysis showed that IFNγ was required for the mucous cell hypertrophy and hyperplasia observed in Hip1r-deficient mice. Together, these findings demonstrate that IFNγ is critical for the development of the gastric epithelial cell metaplasia that results from parietal cell atrophy in the Hip1r-deficient mice.

A facile, convenient and high yielding synthesis of novel S-glycosides and N-glycosides incorporating 1,2,3,4-tetrahydronaphthalene and or 1,2-dihydropyridines moieties has been described. The aglycons 2, 4, and 7 were coupled with different activated halosugars in the presence of basic and acidic medium. The preliminary in-vitro cytotoxic evaluation revealed that compounds 3c, 3f, 5c and 7b show promising activity. A molecular docking study was performed against tyrosine kinase (TK) (PDB code: 1t46) by Autodock Vina. The docking output was analyzed and some compounds have shown hydrogen bond (H-B) formation with reasonable distances ranged from 2.06 A° to 3.06 A° with Thr 670 and Cys 673 residues found in the specified pocket. No hydrogen bond was observed with either Glu 640 nor Asp 810 residues, as was expected from pdbsum.

One of the most extensively investigated and well characterized models of non-genomic steroid actions initiated at the cell surface is the induction of oocyte maturation (OM) in fish and amphibians by progestin. Gonadotropin induces the final phase of oocyte maturation indirectly by inducing the synthesis of maturation inducing steroids (MIS) by the ovarian follicles via its membrane receptor, membrane progestin receptor (mPR). Three mPR subtypes (α, β and γ) have been identified by cDNA cloning or by in silico analysis of genome sequence databases. Previously, we described the cloning of the mPRα cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIS induction of OM in goldfish. Then we cloned one β and two γ subtypes (hereafter referred to as γ-1 and γ-2) from a goldfish ovarian cDNA library. RT-PCR showed different tissue expression patterns of the mRNAs for these mPR subtypes. However, in addition to mPRα, the β, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRβ blocked the induction of oocyte maturational competence, whereas injection of antisense oligonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that goldfish mPRβ protein acts as an intermediary during MIS induction of OM in goldfish, in a manner similar to mPRα. We are establishing mutant strains of Medaka fish to investigate the roles of mPR proteins in vivo produced by Targeting Induced Local Lesions in Genomes (Tilling) strategy. By the screening, we have selected three strains in which a point mutation was induced in each strain at the coding sequence of mPRα. In near future results of phenotypic analysis of mPRα defective fish will be introduced.

Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25-85% of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape.

Osteoclasts are specialized multinucleated cells with the unique capacity to resorb bone. Despite insight into the various steps of the interaction of osteoclast precursors leading to osteoclast formation, surprisingly little is known about what happens with the multinucleated cell itself after it has been formed. Is fusion limited to the short period of its formation, or do osteoclasts have the capacity to change their size and number of nuclei at a later stage? To visualize these processes we analyzed osteoclasts generated in vitro with M-CSF and RANKL from mouse bone marrow and native osteoclasts isolated from rabbit bones by live cell microscopy. We show that osteoclasts fuse not only with mononuclear cells but also with other multinucleated cells. The most intriguing finding was fission of the osteoclasts. Osteoclasts were shown to have the capacity to generate functional multinucleated compartments as well as compartments that contained apoptotic nuclei. These compartments were separated from each other, each giving rise to a novel functional osteoclast or to a compartment that contained apoptotic nuclei. Our findings suggest that osteoclasts have the capacity to regulate their own population in number and function, probably to adapt quickly to changing situations.

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.