Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine ...(5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92-0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.
Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. HIV-1 can replicate in blood monocytes, although only a minor proportion of ...circulating monocytes harbor viral DNA. Resident macrophages in tissues can be infected and function as viral reservoirs. However, their susceptibility to infection, and their capacity to actively replicate the virus, varies greatly depending on the tissue localization and cytokine environment. The susceptibility of monocytes to HIV-1 infection in vitro depends on their differentiation status. Monocytes are refractory to infection and become permissive upon differentiation into macrophages. In addition, the capacity of monocyte-derived macrophages to sustain viral replication varies between individuals. Host determinants regulate HIV-1 replication in monocytes and macrophages, limiting several steps of the viral life-cycle, from viral entry to virus release. Some host factors responsible for HIV-1 restriction are shared with T lymphocytes, but several anti-viral mechanisms are specific to either monocytes or macrophages. Whilst a number of these mechanisms have been identified in monocytes or in monocyte-derived macrophages in vitro, some of them have also been implicated in the regulation of HIV-1 infection in vivo, in particular in the brain and the lung where macrophages are the main cell type infected by HIV-1. This review focuses on cellular factors that have been reported to interfere with HIV-1 infection in monocytes and macrophages, and examines the evidences supporting their role in vivo, highlighting unique aspects of HIV-1 restriction in these two cell types.
Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in ...clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.
DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian ...development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.
The MYTHEN single‐photon‐counting silicon microstrip detector has been developed at the Swiss Light Source for time‐resolved powder diffraction experiments. An upgraded version of the detector has ...been installed at the SLS powder diffraction station allowing the acquisition of diffraction patterns over 120° in 2θ in fractions of seconds. Thanks to the outstanding performance of the detector and to the calibration procedures developed, the quality of the data obtained is now comparable with that of traditional high‐resolution point detectors in terms of FWHM resolution and peak profile shape, with the additional advantage of fast and simultaneous acquisition of the full diffraction pattern. MYTHEN is therefore optimal for time‐resolved or dose‐critical measurements. The characteristics of the MYTHEN detector together with the calibration procedures implemented for the optimization of the data are described in detail. The refinements of two known standard powders are discussed together with a remarkable application of MYTHEN to organic compounds in relation to the problem of radiation damage.
The forkhead transcription factor FOXM1 coordinates expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We previously showed that FOXM1 acts ...downstream of 14-3-3ζ signaling, the elevation of which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high has not been clearly defined yet.
We analyzed FOXM1 protein expression by immunohistochemistry in 501 ER-positive breast cancers. We also mapped genome-wide FOXM1, extracellular signal-regulated kinase 2 and ERα binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. These binding profiles were integrated with gene expression data derived from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. We also modulated the levels of FOXM1 and newly discovered FOXM1-regulated genes and examined their impact on the cancer stem-like cell population and on cell invasiveness and resistance to endocrine treatments.
FOXM1 protein expression was high in 20% of the tumors, which correlated with significantly reduced survival in these patients (P = 0.003 by logrank Mantel-Cox test). ChIP-seq analyses revealed that FOXM1 binding sites were enriched at the transcription start site of genes involved in cell-cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are associated with a poor prognosis in ERα-positive patients treated with tamoxifen. Integration of binding profiles with gene expression highlighted FOXM1 transcriptional networks controlling cell proliferation, stem cell properties, invasion and metastasis. Increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness.
Collectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance. They highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments.
The detection and treatment of cancer has advanced significantly in the past several decades, with important improvements in our understanding of the fundamental molecular and genetic basis of the ...disease. Despite these advancements, drug-screening methodologies have remained essentially unchanged since the introduction of the in vitro human cell line screen in 1990. Although the existing methods provide information on the overall effects of compounds on cell viability, they are restricted by bulk measurements, large sample sizes, and lack capability to measure proliferation kinetics at the individual cell level. To truly understand the nature of cancer cell proliferation and to develop personalized adjuvant therapies, there is a need for new methodologies that provide quantitative information to monitor the effect of drugs on cell growth as well as morphological and phenotypic changes at the single cell level. Here we show that a quantitative phase imaging modality known as spatial light interference microscopy (SLIM) addresses these needs and provides additional advantages over existing proliferation assays. We demonstrate these capabilities through measurements on the effects of the hormone estradiol and the antiestrogen ICI182,780 (Faslodex) on the growth of MCF-7 breast cancer cells. Along with providing information on changes in the overall growth, SLIM provides additional biologically relevant information. For example, we find that exposure to estradiol results in rapidly growing cells with lower dry mass than the control population. Subsequently blocking the estrogen receptor with ICI results in slower growing cells, with lower dry masses than the control. This ability to measure changes in growth kinetics in response to environmental conditions provides new insight on growth regulation mechanisms. Our results establish the capabilities of SLIM as an advanced drug screening technology that provides information on changes in proliferation kinetics at the cellular level with greater sensitivity than any existing method.
When first introduced, single-photon counting detectors reshaped crystallography at synchrotrons. Their fast readout speed enabled, for example, shutter-less data collection and fine slicing of the ...rotation angle and boosted the development of new experimental techniques like ptychography. Under optimal conditions, single-photon counting detectors provide an unlimited dynamic range with image noise only limited by the Poisson statistics of the incoming photons. Counting the pulses from individual photons, essentially what made the detectors so successful, also causes the main drawback, which is the loss of efficiency at high photon fluxes due to pulse pileup in the analog front end. To fully take advantage of diffraction-limited light sources, the next-generation single-photon counters need to improve their count rate capabilities in the same order of magnitude as the increased flux. Moreover, fast frame rates (a few kHz) are required to cope with the shorter dwell time achievable, thanks to the higher flux. Detector architecture with multiple comparators and counters can open new possibilities for energy-resolved imaging, while interpixel communication can overcome the issues arising from charge sharing and reduce the loss of efficiency at the pixel corners. Coupling single-photon counting detectors to high-Z sensors for hard X-ray detection (
>
20 keV) and to low-gain avalanche diodes (LGADs) for soft X-rays is also necessary to make use of the increased coherence of the new light sources over the full radiation spectrum. In this paper, we present possible strategies to improve the performance of single-photon counting detectors at the fourth-generation synchrotron sources and compare them to charge integrating detectors.
Tripartite motif (TRIM) proteins constitute a family of proteins that share a conserved tripartite architecture. The recent discovery of the anti-HIV activity of TRIM5alpha in primate cells has ...stimulated much interest in the potential role of TRIM proteins in antiviral activities and innate immunity.
To test if TRIM genes are up-regulated during antiviral immune responses, we performed a systematic analysis of TRIM gene expression in human primary lymphocytes and monocyte-derived macrophages in response to interferons (IFNs, type I and II) or following FcgammaR-mediated activation of macrophages. We found that 27 of the 72 human TRIM genes are sensitive to IFN. Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity. Also, we found 2 TRIM proteins, TRIM9 and 54, to be specifically up-regulated in FcgammaR-activated macrophages.
Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.
EIGER is an advanced family of single photon counting hybrid pixel detectors, primarily aimed at diffraction experiments at synchrotrons. Optimization of maximal functionality and minimal pixel size ...(using a
0.25
μ
m
process and conserving the radiation tolerant design) has resulted in
75
×
75
μ
m
2
pixels. Every pixel comprises a preamplifier, shaper, discriminator (with a 6 bit DAC for threshold trimming), a configurable 4/8/12 bit counter with double buffering, as well as readout, control and test circuitry. A novel feature of this chip is its double buffered counter, meaning a next frame can be acquired while the previous one is being readout. An array of 256×256 pixels fits on a
∼
2
×
2
cm
2
chip and a sensor of
∼
8
×
4
cm
2
will be equipped with eight readout chips to form a
module containing 0.5
Mpixel. Several modules can then be tiled to form larger area detectors. Detectors up to 4×8 modules (16
Mpixel) are planned. To achieve frame rates of up to 24
kHz the readout architecture is highly parallel, and the chip readout happens in parallel on 32 readout lines with a 100
MHz Double Data Rate clock.
Several chips and
singles (i.e. a single chip bump-bonded to a single chip silicon sensor) were tested both with a lab X-ray source and at Swiss Light Source (SLS) beamlines. These tests demonstrate the full functionality of the chip and provide a first assessment of its performance. High resolution X-ray images and “high speed movies” were produced, even without threshold trimming, at the target system frame rates (up to
∼
24
kHz
in 4 bit mode). In parallel, dedicated hardware, firmware and software had to be developed to comply with the enormous data rate the chip is capable of delivering. Details of the chip design and tests will be given, as well as highlights of both test and final readout systems.
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