Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC

The Botanical Record of Archaeobotany Italian Network - BRAIN: a cooperative network, database and website

Con autorización de la revista para autores CSIC[EN] The BRAIN (Botanical Records of Archaeobotany Italian Network) database and network was developed by the cooperation of archaeobotanists working on Italian archaeological sites. Examples of recent research including pollen or other plant remains in analytical and synthetic papers are reported as an exemplar reference list. This paper retraces the main steps of the creation of BRAIN, from the scientific need for the first research cooperation to the website which has a free online access since 2015.Peer reviewe

Rubin-Euclid Derived Data Products:Initial Recommendations

This report is the result of a joint discussion between the Rubin and Euclid scientific communities. The work presented in this report was focused on designing and recommending an initial set of Derived Data products (DDPs) that could realize the science goals enabled by joint processing. All interested Rubin and Euclid data rights holders were invited to contribute via an online discussion forum and a series of virtual meetings. Strong interest in enhancing science with joint DDPs emerged from across a wide range of astrophysical domains: Solar System, the Galaxy, the Local Volume, from the nearby to the primaeval Universe, and cosmology

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs

<div><p>The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a “non-specific” fashion <i>in vitro</i> and <i>in vivo</i>. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput <i>in vitro</i> protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci <i>in vivo</i>. We identified all hexameric motifs preferentially bound by MYC/MAX <i>in vitro</i>, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX <i>in vitro</i>. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT <i>in vitro</i> and that elevated MYC levels <i>in vivo</i> more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.</p></div

The NE motif AACGTT and several E-box variants have a similar affinity for MAX:MAX.

<p><b>(A)</b> Sequences of the oligo competitors. <b>(B)</b> EMSA competition assays with MAX:MAX and the labeled CME probe and the indicated amounts (pmol) of competitor oligos. Three independent experiments were quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180147#pone.0180147.g003" target="_blank">Fig 3C</a>. No statistically significant difference was observed between NE and the different competitors (p>0.05 by two-tailed t-test).</p

Summary of relative affinities of MYC:MAX and MAX:MAX complexes for different E-box and non-E-box sequences.

<p>Affinities were normalized relative to the affinity of the NE sequence, which was arbitrarily set to 1 (*). Relative affinities from the PBM scores are indicated between brackets. Sequences not analyzed by EMSA are indicated by “n.a.” and non-detectable binding is indicated by “-“.</p

Sequence requirements for MYC/MAX binding to the NE motif AACGTT.

<p><b>(A)</b> Sequences of the oligo competitors. <b>(B, C)</b> EMSA competition assays with MAX:MAX and the labeled CME probe and the indicated amounts (pmol) of competitor oligos. Three independent experiments were quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180147#pone.0180147.g003" target="_blank">Fig 3C</a>. Significant differences were observed between NE and NE-V1/-V2/V3/V4 (p<0.005 by two-tailed t-test).</p

PBM analysis of the DNA-binding specificity of MYC/MAX complexes.

<p><b>(A)</b> The custom PBM design contained immobilized DNA probes in triplicates with the indicated sequences (N and n indicate the varying positions having any of the 4 nucleotides). The total number of different sequences is shown in brackets. <b>(B)</b> Coomassie-stained SDS-PAGE showing the purified recombinant MYC/MAX complexes. <b>(C)</b> Images of fluorescent spot signals on PBM slides bound by MYC:MAX and MAX:MAX complexes and probed with anti-MAX antibody. <b>(D)</b> Scatter plot illustrating the correlation between MYC:MAX and MAX:MAX binding scores (normalized fluorescent intensities) for each individual probe spotted on the PBM (R² = 0.764). Dashed lines indicate the threshold values for binding scores above 80% of random probes. <b>(E)</b> Ranked order list of top-scoring motifs in PBM probes bound by MYC:MAX and MAX:MAX (with binding scores above 95% of random probes). Double arrows indicate identical motifs. Itallics are the low complexity G/C-rich motifs. Asterisks indicate non-E-box motifs, including the AACGTT motif (underlined). Motifs highlighted in bold were bound by both MYC:MAX and MAX:MAX above the 80% random threshold.</p

Characterization of MYC-occupied CME (CACGTG) and NE (AACGTT) genomic loci and influence of MYC expression levels in P493-6 cells.

<p><b>(A)</b> Venn diagrams show the number of MYC ChIP-seq peaks and summits that contain the CME, the NE or both motifs in high MYC-expressing P493-6 cells. <b>(B, C)</b> Frequency distribution plots show the fraction (%) of all MYC ChIP-seq peaks and summits and those specifically containing the CME or NE motifs that are located within promoters (± 2 kb from a TSS), intergenic or intragenic regions. <b>(D)</b> Frequency distribution of the CME and NE motifs in the human genome. <b>(E)</b> Effect of MYC overexpression on the number and average (mean) height of MYC ChIP-seq summits is shown as fold change (high MYC vs. low MYC) for the summits containing the CME or NE motifs. <b>(F)</b> Effect of MYC overexpression on the number of MYC ChIP-seq summits at promoters, intergenic and intragenic regions is shown as fold change (high MYC vs. low MYC) for CME- or NE-containing summits. <b>(G)</b> Examples of NE and CME ChIP-seq peaks from human genome browser in P493-6 cells expressing low and high MYC levels.</p

Normalized Frequencies of E-boxes and of the NE motif AACGTT at MYC-occupied genomic loci in human P493-6 B-cells.

<p>MYC-bound sequences were obtained from ChIP-seq data of P493-6 cells overexpressing MYC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180147#pone.0180147.ref022" target="_blank">22</a>]. The frequencies of all ten possible E-boxes (CANNTG), the NE motif AACGTT, and two control (Ctrl) sequences under MYC ChIP-seq peaks <b>(A)</b> and summits <b>(B)</b> were normalized to the occurrence of each motif in the human genome. The summits are the ±100 bp region from the apex of ChIP-seq peaks. The normalized frequencies are shown for all MYC peaks/summits (left) and for the most significant MYC peaks/summits (top 33% based on p-value; right).</p