Identification of the novel HLA-DPB1*1461:01 allele in three individuals from Southern India.

HLA-DPB1*1461:01 differs from DPB1*02:01:02:01 in exon 2, codon 51 CTG > CGG a Leucine to Arginine replacement.

Network diffusion-based approach for survival prediction and identification of biomarkers using multi-omics data of papillary renal cell carcinoma.

Identification of cancer subtypes based on molecular knowledge is crucial for improving the patient diagnosis, prognosis, and treatment. In this work, we integrated copy number variations (CNVs) and transcriptomic data of Kidney Papillary Renal Cell Carcinoma (KIRP) using a network diffusion strategy to stratify cancers into clinically and biologically relevant subtypes. We constructed GeneNet, a KIRP specific gene expression network from RNA-seq data. The copy number variation data was projected onto GeneNet and propagated on the network for clustering. We identified robust subtypes that are biologically informative and significantly associated with patient survival, tumor stage and clinical subtypes of KIRP. We performed a Singular Value Decomposition (SVD) analysis of KIRP subtypes, which revealed the genes/silent players related to poor survival. A differential gene expression analysis between subtypes showed that genes related to immune, extracellular matrix organization, and genomic instability are upregulated in the poor survival group. Overall, the network-based approach revealed the molecular subtypes of KIRP and captured the relationship between gene expression and CNVs. This framework can be further expanded to integrate other omics data.

Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.

Early biological responses in ocular tissue after SMILE and LASIK surgery.

We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.

Automated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy.

High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases.

Clumped-MCEM: Inference for multistep transcriptional processes.

Many biochemical events involve multistep reactions. Among them, an important biological process that involves multistep reaction is the transcriptional process. A widely used approach for simplifying multistep reactions is the delayed reaction method. In this work, we devise a model reduction strategy that represents several OFF states by a single state, accompanied by specifying a time delay for burst frequency. Using this model reduction, we develop Clumped-MCEM which enables simulation and parameter inference. We apply this method to time-series data of endogenous mouse glutaminase promoter, to validate the model assumptions and infer the kinetic parameters. Further, we compare efficiency of Clumped-MCEM with state-of-the-art methods - Bursty MCEM2 and delay Bursty MCEM. Simulation results show that Clumped-MCEM inference is more efficient for time-series data and is able to produce similar numerical accuracy as state-of-the-art methods - Bursty MCEM2 and delay Bursty MCEM in less time. Clumped-MCEM reduces computational cost by 57.58% when compared with Bursty MCEM2 and 32.19% when compared with delay Bursty MCEM.

Transcriptional processes: Models and inference.

Many biochemical events involve multistep reactions. One of the most important biological processes that involve multistep reaction is the transcriptional process. Models for multistep reaction necessarily need multiple states and it is a challenge to compute model parameters that best agree with experimental data. Therefore, the aim of this work is to design a multistep promoter model which accurately characterizes transcriptional bursting and is consistent with observed data. To address this issue, we develop a model for promoters with several OFF states and a single ON state using Erlang distribution. To explore the combined effects of model and data, we combine Monte Carlo extension of Expectation Maximization (MCEM) and delay Stochastic Simulation Algorithm (DSSA) and call the resultant algorithm as delay Bursty MCEM. We apply this algorithm to time-series data of endogenous mouse glutaminase promoter to validate the model assumptions and infer the kinetic parameters. Our results show that with multiple OFF states, we are able to infer and produce a model which is more consistent with experimental data. Our results also show that delay Bursty MCEM inference is more efficient.

Chromosome fusions triggered by noncoding RNA.

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination.

V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the "12/23 rule") is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.

Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2.

The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 µM) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997-1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity.

RNA-Mediated Epigenetic Programming of Genome Rearrangements.

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNA-mediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.