High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

ABSTRACTIn the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination.Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

Unitary Structure of Palindromes in DNA

We investigate the quantum behavior encountered in palindromes within DNA structure. In particular we reveal the unitary structure of usual palindromic sequences found in genomic DNAs of all living organisms using the Schwinger approach. We clearly demonstrate the role played by palindromic configurations with special emphasis on physical symmetries in particular subsymmetries of unitary structure. We unveil the prominence of unitary structure in palindromic sequences in the sense that vitally significant information endowed within DNA could be transformed unchangeably in the process of transcription. We introduce a new symmetry relation namely purine-purine or pyrimidine-pyrimidine symmetries (p-symmetry) in addition to the already known symmetry relation of purine-pyrimidine symmetries (pp symmetry) given by Chargaff rule. Therefore important vital functions of a living organisms are protected by means of these symmetric features. It is understood that higher order palindromic sequences could be generated in terms of the basis of the highest prime numbers that make up the palindrome sequence number. We propose that violation of this unitary structure of palindromic sequences by means of our proposed symmetries leads to a mutation in DNA which could offer a new perspective in the scientific studies on the origin and cause of mutation.

Effect of dietary inulin in the gut microbiota of whiteleg shrimp Penaeus vannamei

The effect of dietary inulin on the intestinal bacterial communities of Penaeus vannamei by 16S metagenomic analysis was assessed. PCR amplified the V3 region of the bacterial 16S rDNA. Sequencing reads were generated using the 2×150 (300 cycles) for the base-read length chemistry of the Illumina MiniSeq platform. The software Shaman and MicrobiomeAnalyst were used to analyze the sequences. The phylum Proteobacteria and the genus Vibrio were among the most abundant taxonomic ranks for control and inulin treatment. The relative abundance of the phylum Bacteroidetes and genus Ruegeria was lower in inulin treatment concerning the control condition. Alpha and beta indices did not show significant differences between inulin treatment and control conditions. For all samples, most of the bacterial organisms showed the presence of carbohydrate and amino acid metabolism-related genes, and to a lesser extent, of energy, lipid, and cofactors and vitamin metabolism-related genes. The principal metabolic functions were glycine, serine, threonine, glyoxylate and dicarboxylate, purine, pyrimidine, pyruvate, and quorum sensing. The interaction network analysis showed fewer interactions in the inulin treatment concerning control condition. Proteobacteria, Bacteroidetes, Vibrio, and Ruegeria predominated in all samples, and inulin did not change the net microbial diversity in the intestine of P. vannamei. Streptomyces, Roseobacter, and Ruegeria showed negative interactions with Vibrio, suggesting their use as probiotics. This study sheds light on the inulin supplement on the essential role of microbiota in the shrimp.

Phospholipid-Gold Nanorods Induce Energy Crisis in MCF-7 Cells: Cytotoxicity Evaluation Using LC-MS-Based Metabolomics Approach

Phospholipid-modified gold nanorods (phospholipid-GNRs) have demonstrated drastic cytotoxicity towards MCF-7 breast cancer cells compared to polyethylene glycol-coated GNRs (PEG-GNRs). In this study, the mechanism of cytotoxicity of phospholipid-GNRs towards MCF-7 cells was investigated using mass spectrometry-based global metabolic profiling and compared to PEGylated counterparts. The results showed that when compared to PEG-GNRs, phospholipid-GNRs induced significant and more pronounced impact on the metabolic profile of MCF-7 cells. Phospholipid-GNRs significantly decreased the levels of metabolic intermediates and end-products associated with cellular energy metabolisms resulting in dysfunction in TCA cycle, a reduction in glycolytic activity, and imbalance of the redox state. Additionally, phospholipid-GNRs disrupted several metabolism pathways essential for the normal growth and proliferation of cancer cells including impairment in purine, pyrimidine, and glutathione metabolisms accompanied by lower amino acid pools. On the other hand, the effects of PEG-GNRs were limited to alteration of glycolysis and pyrimidine metabolism. The current work shed light on the importance of metabolomics as a valuable analytical approach to explore the molecular effects of GNRs with different surface chemistry on cancer cell and highlights metabolic targets that might serve as promising treatment strategy in cancer.

A Quantitative Comparisons of $\beta$-Coronavirus Genomes and Their Associated Genes

A precise understanding of the genes and associated genomes of SARS-CoV2 is important for various reasons such as discovering origin of the virus and virulence and so on. A thorough descriptive understanding of the SARS-CoV2 genomes and other coronavirus of the beta-coronavirus genus is primarily important. In this article, a set of ten genomes of four CoVs and their associated genes are considered for this present study. A spatial representations of nucleotide bases including purine-pyrimidine representations of the different genes of the corresponding genomes are quantified using Hurst exponent, Shannon entropy and density estimation of different nucleotides including GC content, in order to draw a comparison and contrast among the ten genomes of different types of CoVs which include MERS, SARS-CoV, HKU1 (Human Coronavirus) and associated their genes.

A Quantitative Genomic View of the Coronaviruses: SARS-COV2

In 2020, the pandemic caused by the Coronaviruses (CoV) that are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV2). The Coronavirus disease (COVID-19) is a new strain that was discovered in 2019 and has not been previously identified in humans. It is the high time to investigate the quantitative and/or qualitative genomic informations of the virus SARS-CoV2 in order to strengthen the healthcare facility to fight against this viral disease. In this article, a through quantitative understanding of the purine and pyrimidine spatial distribution/organization of all 89 complete sequences of SARS-CoV (available as on date in the NCBI virus database, is made using different parameters such as fractal dimension, Hurst exponent, Shannon entropy and GC content of the nucleotide sequences of the genome of SARS-CoV2. Also a cluster among all the the SARS-CoV sequences of nucleotide have been made based on their phylogeny made through their closeness (Hamming distance) based on respective purine-pyrimidine distribution.

DNA/TNA Mesoscopic Modeling of Melting Temperatures Suggest Weaker Hydrogen Bonding of CG than in DNA/RNA

TNA/DNA hybrids share several similarities to RNA/DNA, such as the tendency to form A-type helices and a strong dependency of their thermodynamic properties on purine/pyrimidine ratio. However, unlike RNA/DNA, not much is known about the base-pair properties of TNA. Here, we use a mesoscopic analysis of measured melting temperatures to obtain an estimate of hydrogen bonds and stacking interactions. Our results reveal that the AT base pairs in TNA/DNA have nearly identical hydrogen bond strengths than their counterparts in RNA/DNA, but surprisingly CG turned out to be much weaker despite similar stability.

DNA/TNA Mesoscopic Modeling of Melting Temperatures Suggest Weaker Hydrogen Bonding of CG than in DNA/RNA

TNA/DNA hybrids share several similarities to RNA/DNA, such as the tendency to form A-type helices and a strong dependency of their thermodynamic properties on purine/pyrimidine ratio. However, unlike RNA/DNA, not much is known about the base-pair properties of TNA. Here, we use a mesoscopic analysis of measured melting temperatures to obtain an estimate of hydrogen bonds and stacking interactions. Our results reveal that the AT base pairs in TNA/DNA have nearly identical hydrogen bond strengths than their counterparts in RNA/DNA, but surprisingly CG turned out to be much weaker despite similar stability.

Tannin supplementation modulates the composition and function of ruminal microbiome in lambs infected with gastrointestinal nematodes

ABSTRACT This study was carried out to evaluate the effects of tannin supplementation on ruminal microbiota of sixteen lambs infected and non-infected with Haemonchus contortus and Trichostrongylus colubriformis. Animals were fed with hay, concentrate and supplemented with Acacia mearnsii (A. mearnsii). The animals were divided into four treatments: two control groups without infection, either receiving A. mearnsii (C+) or not (C-), and two infected groups, one with A. mearnsii (I+) and another without A. mearnsii (I-). Ruminal short-chain fatty acids (SCFA) and metagenome sequencing of ruminal microbiota were used to evaluate the effect of tannin and infection on ruminal microbiome. For SCFA, differences were observed only with A. mearnsii. Total SCFA and acetate molar percentage were decreased in C+ and I+ (P<0.05). Butyrate, valerate and isovalerate were higher in lambs that received A. mearnsii in the diet (P<0.05). The infection changed the microbiome structure and decreased the abundance of butyrate-producing microorganisms. In addition, A. mearnsii supplementation also affected the structure the microbial community, increasing the diversity and abundance of the butyrate-producing and probiotics bacteria, amino acid metabolic pathways, purine, pyrimidine and sphingolipid metabolism. Together, our findings indicate that A. mearnsii supplementation modulates important groups related to nitrogen, amino acid, purine and pyrimidine metabolism, in rumen microbiome, affected by gastrointestinal nematodes infection in lambs.