We perform a systematic study of models involving leptoquarks and diquarks with masses well below the grand unification scale and demonstrate that a large class of them is excluded due to rapid ...proton decay. After singling out the few phenomenologically viable color triplet and sextet scenarios, we show that there exist only two leptoquark models which do not suffer from tree-level proton decay and which have the potential for explaining the recently discovered anomalies in B meson decays. Both of those models, however, contain dimension five operators contributing to proton decay and require a new symmetry forbidding them to emerge at a higher scale. This has a particularly nice realization for the model with the vector leptoquark (3,1)2/3, which points to a specific extension of the Standard Model, namely the Pati–Salam unification model, where this leptoquark naturally arises as the new gauge boson. We explore this possibility in light of recent B physics measurements. Finally, we analyze also a vector diquark model, discussing its LHC phenomenology and showing that it has nontrivial predictions for neutron–antineutron oscillation experiments.
Zta, the Epstein–Barr virus bZIP transcription factor (TF), binds both unmethylated and methylated double-stranded DNA (dsDNA) in a sequence-specific manner. We studied the contribution of a ...conserved asparagine (N182) to sequence-specific dsDNA binding to four types of dsDNA: (i) dsDNA with cytosine in both strands ((DNA(C|C)), (ii, iii) dsDNA with 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand ((DNA(5mC|C) and DNA(5hmC|C)), and (iv) dsDNA with methylated cytosine in both strands in all CG dinucleotides ((DNA(5mCG)). We replaced asparagine with five similarly sized amino acids (glutamine (Q), serine (S), threonine (T), isoleucine (I), or valine (V)) and used protein binding microarrays to evaluate sequence-specific dsDNA binding. Zta preferentially binds the pseudo-palindrome TRE (AP1) motif (T–4G–3A‑2G/C 0T2C3A4 ). Zta (N182Q) changes binding to A3 in only one half-site. Zta(N182S) changes binding to G3 in one or both halves of the motif. Zta(N182S) and Zta(N182Q) have 34- and 17-fold weaker median dsDNA binding, respectively. Zta(N182V) and Zta(N182I) have increased binding to dsDNA(5mC|C). Molecular dynamics simulations rationalize some of these results, identifying hydrogen bonds between glutamine and A3 , but do not reveal why serine preferentially binds G3 , suggesting that entropic interactions may mediate this new binding specificity.
Previously, cooperative binding of the bZIP domain of CREB1 and the ETS domain of GABPα was observed for the composite DNA ETS ⇔ CRE motif (A 0 C 1 C 2 G 3 G 4 A 5 A 6 G 7 T 8 G 9 A 10 C 11 G 12 T 13 ...C 14 A 15 ). Single nucleotide polymorphisms (SNPs) at the beginning and end of the ETS motif ( ACCGGAAGT ) increased cooperative binding. Here, we use an Agilent microarray of 60-mers containing all double nucleotide polymorphisms (DNPs) of the ETS ⇔ CRE motif to explore GABPα and CREB1 binding to their individual motifs and their cooperative binding. For GABPα, all DNPs were bound as if each SNP acted independently. In contrast, CREB1 binding to some DNPs was stronger or weaker than expected, depending on the locations of each SNP. CREB1 binding to DNPs where both SNPs were in the same half site, T 8 G 9 A 10 or T 13 C 14 A 15 , was greater than expected, indicating that an additional SNP cannot destroy binding as much as expected, suggesting that an individual SNP is enough to abolish sequence-specific DNA binding of a single bZIP monomer. If a DNP contains SNPs in each half site, binding is weaker than expected. Similar results were observed for additional ETS and bZIP family members. Cooperative binding between GABPα and CREB1 to the ETS ⇔ CRE motif was weaker than expected except for DNPs containing A 7 and SNPs at the beginning of the ETS motif.
In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally ...to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.
Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess ...their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.
The bZIP homodimers CEBPB and CREB1 bind DNA containing methylated cytosines differently. CREB1 binds stronger to the C/EBP half-site GCAA when the cytosine is methylated. For CEBPB, methylation of ...the same cytosine does not affect DNA binding. The X-ray structure of CREB1 binding the half site GTCA identifies an alanine in the DNA binding region interacting with the methyl group of T, structurally analogous to the methyl group of methylated C. This alanine is replaced with a valine in CEBPB. To explore the contribution of this amino acid to binding with methylated cytosine of the GCAA half-site, we made the reciprocal mutants CEBPB(V285A) and CREB1(A297V) and used protein binding microarrays (PBM) to examine binding to four types of double-stranded DNA (dsDNA): 1) DNA with cytosine in both strands (DNA(C|C)), 2) DNA with 5-methylcytosine (M) in one strand and cytosine in the second strand (DNA(M|C)), 3) DNA with 5-hydroxymethylcytosine (H) in one strand and cytosine in the second strand (DNA(H|C)), and 4) DNA with both cytosines in all CG dinucleotides containing 5-methylcytosine (DNA(5mCG)). When binding to DNA(C|C), CEBPB (V285A) preferentially binds the CRE consensus motif (TGACGTCA), similar to CREB1. The reciprocal mutant, CREB1(A297V) binds DNA with some similarity to CEBPB, with strongest binding to the methylated PAR site 8-mer TTACGTAA. These data demonstrate that V285 residue inhibits CEBPB binding to methylated cytosine of the GCAA half-site.
•Generated reciprocal mutants of CEBPB and CREB1 to explore DNA binding differences with modified cytosine.•Used protein binding microarray to study the DNA binding to 4 types of dsDNA.•When binding to DNA(C|C), CEBPB(V285A) binds the CRE consensus motif like CREB1.•CEBPB(V285) inhibits binding to methylated C2 of C/EBP half-site GC2AA.
Total elbow arthroplasty rates continue to rise year after year with up to 7.6% annual growth, yet complication and failure rates have been reported as high as 90%. Proper identification of implants ...prior to revision is crucial to optimize clinical resources and postoperative outcomes. This study aims to evaluate a machine learning algorithm for quick and accurate identification of elbow implants from radiographic images.
A total of 142 elbow implant radiographs representing 5 unique prostheses were obtained from deidentified patients within the Mount Sinai Health System in New York. Images were split into training (n = 97) and test (n = 45) sets. A modified version of the convolutional neural network DenseNet-121 was used to train and evaluate the data. The model was then assessed for accuracy, sensitivity, positive predictive value, and f-1 scores.
Overall model accuracy was 97.8% and top-3 accuracy was 100% for each of the 5 prostheses. Sensitivity, positive predictive value, and f-1 scores for the model were 0.978, 0.980, and 0.96, respectively. The algorithm took 14.10 seconds to classify the 45 images in the test set, averaging 0.31 seconds per image.
The model demonstrated strong performance in the identification of elbow implants among the sample of 142 images and 5 implant types. While these results suggest promise in providing future clinical utility within surgical workflows, the method requires external validation and further testing with a greater number and wider variety of implants.
The Epstein-Barr virus (EBV) B-ZIP transcription factor Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the sequence ...specific DNA binding of Zta to four kinds of double-stranded DNA (dsDNA): (1) DNA containing cytosine in both strands, (2) DNA with 5-methylcytosine (5mC) in one strand and cytosine in the second strand, (3) DNA with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand, and (4) DNA in which both cytosines in all CG dinucleotides contain 5mC. We compared these data to PBM data for three additional B-ZIP proteins (CREB1 and CEBPB homodimers and cJun|cFos heterodimers). With cytosine, Zta binds the TRE motif TGA C / G TCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M = 5mC), were preferentially bound. 5mC inhibits binding of Zta to both TRE motif half-sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer more strongly bind the C/EBP half-site tetranucleotide GCAA when it contains 5mC. Zta also binds dsDNA sequences containing 5hmC in one strand, although the effect is less dramatic than that observed for 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, uncovering the potential for discovery of new viral and host regulatory programs controlled by EBV.
Abstract
Therapeutic immunity against tumors is mainly determined by effector T cells. However, T cells are educated by antigen presenting cells, particularly dendritic cells (DC), that control their ...differentiation into effector T cells able to eliminate target tumor cells. While most studies focused on understanding T cell features leading to antitumor immunity, little is known about DC determinants required to promote effective antitumor T cell function. Yet several studies are revealing that high DC content in tumor lesions correlate with improved tumor outcome. To identify molecular program that modulate DC functionality in tissues we have profiled tumor-associated DC in mice and tumor lesions. We identified a DC molecular program that was shared between mice and human tumors that included maturation genes associated with T cell simulation and migration to the LN (Cd40, Cd80, Fscn, Ccr7), but also genes associated with immunoregulation (Socs1, Socs2, Socs3, Cd200, Pd-l1, Pd-l2, Fasl), we named mregDC (Maier et al., Nature 2020). We also found that mregDC molecular state is induced in DC1 and DC2 upon capture of tumor debris leading to hypothesize that the mregDC state identifies antigen-charged DC that migrate to lymphoid structures, including tertiary lymphoid structures and tumor draining lymph nodes to modulate tumor specific T cell immune responses. While the role of DC in the draining LN has been established the exact role played by DC in the tumor microenvironment (TME) remains unclear. To explore this question, we used multiplex imaging and spatial transcriptomics platforms to identify the distribution and spatial interactions of DC1, DC2 and mregDC within the TME of human NSCLC and HCC lesions during treatment with PD-1 blockade. We found that mregDC accumulate within the tertiary lymphoid structures together with activated T cell and B cell programs. We are continuing to generate additional data on mregDC interactions in human tumors tissues that respond or resist PD-1 blockade and should be able to provide additional information at the time of the meeting.
Citation Format: Raphael Mattiuz, Nima Assad, Pauline Hamon, Assaf Magen, Jessica Le Berichel, Miriam Merad. Determining the regulation and role of mreg DC in tumor immunity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3611.
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