Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain.

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

BACKGROUND: Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. METHODS: We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. RESULTS: Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. CONCLUSIONS: Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.

Sox2-Evf2 lncRNA-mediated mechanisms of chromosome topological control in developing forebrain.

The Evf2 long non-coding RNA directs Dlx5/6 ultraconserved enhancer(UCE)-intrachromosomal interactions, regulating genes across a 27 Mb region on chromosome 6 in mouse developing forebrain. Here, we show that Evf2 long-range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2. Evf2 directly interacts with Sox2, antagonizing Sox2 activation of Dlx5/6UCE, and recruits Sox2 to the Dlx5/6eii shadow enhancer and key Dlx5/6UCE interaction sites. Sox2 directly interacts with Dlx1 and Smarca4, as part of the Evf2 ribonucleoprotein complex, forming spherical subnuclear domains (protein pools, PPs). Evf2 targets Sox2 PPs to one long-range repressed target gene (Rbm28), at the expense of another (Akr1b8). Evf2 and Sox2 shift Dlx5/6UCE interactions towards Rbm28, linking Evf2/Sox2 co-regulated topological control and gene repression. We propose a model that distinguishes Evf2 gene repression mechanisms at Rbm28 (Dlx5/6UCE position) and Akr1b8 (limited Sox2 availability). Genome-wide control of RNPs (Sox2, Dlx and Smarca4) shows that co-recruitment influences Sox2 DNA binding. Together, these data suggest that Evf2 organizes a Sox2 PP subnuclear domain and, through Sox2-RNP sequestration and recruitment, regulates chromosome 6 long-range UCE targeting and activity with genome-wide consequences.

Aortic Stress Activates an Adaptive Program in Thoracic Aortic Smooth Muscle Cells That Maintains Aortic Strength and Protects Against Aneurysm and Dissection in Mice.

BACKGROUND: When aortic cells are under stress, such as increased hemodynamic pressure, they adapt to the environment by modifying their functions, allowing the aorta to maintain its strength. To understand the regulation of this adaptive response, we examined transcriptomic and epigenomic programs in aortic smooth muscle cells (SMCs) during the adaptive response to AngII (angiotensin II) infusion and determined its importance in protecting against aortic aneurysm and dissection (AAD). METHODS: We performed single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) analyses in a mouse model of sporadic AAD induced by AngII infusion. We also examined the direct effects of YAP (yes-associated protein) on the SMC adaptive response in vitro. The role of YAP in AAD development was further evaluated in AngII-infused mice with SMC-specific Yap deletion. RESULTS: In wild-type mice, AngII infusion increased medial thickness in the thoracic aorta. Single-cell RNA sequencing analysis revealed an adaptive response in thoracic SMCs characterized by upregulated genes with roles in wound healing, elastin and collagen production, proliferation, migration, cytoskeleton organization, cell-matrix focal adhesion, and PI3K-PKB/Akt (phosphoinositide-3-kinase-protein kinase B/Akt) and TGF-ß (transforming growth factor beta) signaling. ScATAC-seq analysis showed increased chromatin accessibility at regulatory regions of adaptive genes and revealed the mechanical sensor YAP/transcriptional enhanced associate domains as a top candidate transcription complex driving the expression of these genes (eg, Lox, Col5a2, Tgfb2). In cultured human aortic SMCs, cyclic stretch activated YAP, which directly bound to adaptive gene regulatory regions (eg, Lox) and increased their transcript abundance. SMC-specific Yap deletion in mice compromised this adaptive response in SMCs, leading to an increased AAD incidence. CONCLUSIONS: Aortic stress triggers the systemic epigenetic induction of an adaptive response (eg, wound healing, proliferation, matrix organization) in thoracic aortic SMCs that depends on functional biomechanical signal transduction (eg, YAP signaling). Our study highlights the importance of the adaptive response in maintaining aortic homeostasis and preventing AAD in mice.

The cap-proximal RNA secondary structure inhibits preinitiation complex formation on HAC1 mRNA.

Translation of HAC1 mRNA in the budding yeast Saccharomyces cerevisiae is derepressed when RNase Ire1 removes its intron via nonconventional cytosolic splicing in response to accumulation of unfolded proteins inside the endoplasmic reticulum. The spliced HAC1 mRNA is translated into a transcription factor that changes the cellular gene expression patterns to increase the protein folding capacity of cells. Previously, we showed that a segment of the intronic sequence interacts with the 5'-UTR of the unspliced mRNA, resulting in repression of HAC1 translation at the initiation stage. However, the exact mechanism of translational derepression is not clear. Here, we show that at least 11-base-pairing interactions between the 5'-UTR and intron (UI) are sufficient to repress HAC1 translation. We also show that overexpression of the helicase eukaryotic initiation factor 4A derepressed translation of an unspliced HAC1 mRNA containing only 11-bp interactions between the 5'-UTR and intronic sequences. In addition, our genetic screen identifies that single mutations in the UI interaction site could derepress translation of the unspliced HAC1 mRNA. Furthermore, we show that the addition of 24 RNA bases between the mRNA 5'-cap and the UI interaction site derepressed translation of the unspliced HAC1 mRNA. Together, our data provide a mechanistic explanation for why the cap-proximal UI-RNA duplex inhibits the recruitment of translating ribosomes to HAC1 mRNA, thus keeping mRNA translationally repressed.

Programmed cell death in aortic aneurysm and dissection: A potential therapeutic target.

Rupture of aortic aneurysm and dissection (AAD) remains a leading cause of death. Progressive smooth muscle cell (SMC) loss is a crucial feature of AAD that contributes to aortic dysfunction and degeneration, leading to aortic aneurysm, dissection, and, ultimately, rupture. Understanding the molecular mechanisms of SMC loss and identifying pathways that promote SMC death in AAD are critical for developing an effective pharmacologic therapy to prevent aortic destruction and disease progression. Cell death is controlled by programmed cell death pathways, including apoptosis, necroptosis, pyroptosis, and ferroptosis. Although these pathways share common stimuli and triggers, each type of programmed cell death has unique features and activation pathways. A growing body of evidence supports a critical role for programmed cell death in the pathogenesis of AAD, and inhibitors of various types of programmed cell death represent a promising therapeutic strategy. This review discusses the different types of programmed cell death pathways and their features, induction, contributions to AAD development, and therapeutic potential. We also highlight the clinical significance of programmed cell death for further studies.

Gastric tumorigenesis induced by combining Helicobacter pylori infection and chronic alcohol through IL-10 inhibition.

Helicobacter pylori infection and alcohol intake are independent risk factors in gastric carcinogenesis; however, until now, the combined effect of H. pylori infection and alcohol consumption and the specific mechanism is still problematic. Here, we developed a series of mouse models that progress from chronic gastritis to gastric cancer, induced by infecting H. pylori combined with chronic alcohol consumption and then determining the molecular mechanism of the progression by flow cytometry, western blotting, qPCR, Mito Traker assay in the gastric cancer and T-cell lines. Interleukin-10 (IL-10) knockout mice was used to determine whether IL-10 deficiency directly contributes to H. pylori and alcohol induced gastric tumorigenesis. Alcohol consumption, together with H. pylori infection, causes gastric cancer; IL-10 downregulation and mitochondrial metabolic dysfunction in CD8+ cells are also involved. IL-10 knockout accelerates tumor development in mice with either H. pylori infection or alcohol induced gastric cancer or both. IL-10 inhibits glucose uptake and glycolysis and promotes oxidative phosphorylation with lactate inhibition. Consequently, in the absence of IL-10 signaling, CD8+ cells accumulate damaged mitochondria in a mouse model of gastric cancer induced with the combination of alcohol plus H. pylori infection, and this results in mitochondrial dysfunction and production of IL-1ß. IL-1ß promotes H. pylori infection and reduces NKX6.3 gene expression, resulting in increased cancer cell survival and proliferation. Gastric cancer can be induced by the combination of H. pylori infection and chronic alcohol consumption through IL-10 inhibition induced CD8+ cells dysfunction and NKX6.3 suppression.

Replication timing networks reveal a link between transcription regulatory circuits and replication timing control.

DNA replication occurs in a defined temporal order known as the replication timing (RT) program and is regulated during development, coordinated with 3D genome organization and transcriptional activity. However, transcription and RT are not sufficiently coordinated to predict each other, suggesting an indirect relationship. Here, we exploit genome-wide RT profiles from 15 human cell types and intermediate differentiation stages derived from human embryonic stem cells to construct different types of RT regulatory networks. First, we constructed networks based on the coordinated RT changes during cell fate commitment to create highly complex RT networks composed of thousands of interactions that form specific functional subnetwork communities. We also constructed directional regulatory networks based on the order of RT changes within cell lineages, and identified master regulators of differentiation pathways. Finally, we explored relationships between RT networks and transcriptional regulatory networks (TRNs) by combining them into more complex circuitries of composite and bipartite networks. Results identified novel trans interactions linking transcription factors that are core to the regulatory circuitry of each cell type to RT changes occurring in those cell types. These core transcription factors were found to bind cooperatively to sites in the affected replication domains, providing provocative evidence that they constitute biologically significant directional interactions. Our findings suggest a regulatory link between the establishment of cell-type-specific TRNs and RT control during lineage specification.

ExTraMapper: exon- and transcript-level mappings for orthologous gene pairs.

MOTIVATION: Access to large-scale genomics and transcriptomics data from various tissues and cell lines allowed the discovery of wide-spread alternative splicing events and alternative promoter usage in mammalians. Between human and mouse, gene-level orthology is currently present for nearly 16k protein-coding genes spanning a diverse repertoire of over 200k total transcript isoforms. RESULTS: Here, we describe a novel method, ExTraMapper, which leverages sequence conservation between exons of a pair of organisms and identifies a fine-scale orthology mapping at the exon and then transcript level. ExTraMapper identifies more than 350k exon mappings, as well as 30k transcript mappings between human and mouse using only sequence and gene annotation information. We demonstrate that ExTraMapper identifies a larger number of exon and transcript mappings compared to previous methods. Further, it identifies exon fusions, splits and losses due to splice site mutations, and finds mappings between microexons that are previously missed. By reanalysis of RNA-seq data from 13 matched human and mouse tissues, we show that ExTraMapper improves the correlation of transcript-specific expression levels suggesting a more accurate mapping of human and mouse transcripts. We also applied the method to detect conserved exon and transcript pairs between human and rhesus macaque genomes to highlight the point that ExTraMapper is applicable to any pair of organisms that have orthologous gene pairs. AVAILABILITY AND IMPLEMENTATION: The source code and the results are available at https://github.com/ay-lab/ExTraMapper and http://ay-lab-tools.lji.org/extramapper. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ubiquitination of ADRα1d/SerpinA1 complex stimulates hypoxia to induce gastric tumorigenesis with a combination of Helicobacter pylori and chronic stress through IL-1α.

BACKGROUND: Helicobacter pylori (H. pylori) has been recognized as the class I carcinogen of gastric cancer and several studies have demonstrated that chronic stress may accelerate gastric cancer progression. However, the evidence is not sufficient. METHODS: Here, we developed a mouse model that combined H. pylori infection with chronic stress. Gastric inflammation promotes gastric tumor development progression. To evaluate the number of pro-inflammatory cells through observing the numbers of activated macrophages and neutrophils in mice gastric tumors compared with untreated mice or only treated with one factor. ADRα1d /SerpinA1 expression and localization were assessed under stress conditions and H. pylori infection, and evaluated by analyzing IL-1α, CD8, platelet, and RBC status using α- or ß- blockers against gastritis to prevent gastric cancer. RESULTS: Further mechanism study showed that stress hormones increase the number of CD8+ lymphocytes by activating ADRß2 receptors, leading to IL-1α secretion and tumorigenicity. Gastric carcinogenesis also involves gastric muscle contraction mediated through ADRα1d/Serpina1 interaction. Specifically, we showed that the ADRα1d/SerpinA1 complex increases glucose uptake and the development of hypoxia conditions. These responses promote platelet aggregation and muscle contraction. In turn, gastric cancer cells increase lactate production and promote gastric cell proliferation through Muc-13 and IL-1α stimulation. CONCLUSION: H. pylori infection in combination with chronic stress can lead to gastric cancer, and the synergistic effects of cytokine production (i.e. IL-1α), T lymphocyte dysfunction contributes to gastric carcinogenesis which will offer treatment opportunities for stress-associated gastric cancer and provide new strategies for the prevention and treatment of gastric cancer in clinics.

The role of 3D genome organization in disease: From compartments to single nucleotides.

Since the advent of the chromosome conformation capture technology, our understanding of the human genome 3D organization has grown rapidly and we now know that human interphase chromosomes are folded into multiple layers of hierarchical structures and each layer can play a critical role in transcriptional regulation. Alterations in any one of these finely-tuned layers can lead to unwanted cascade of molecular events and ultimately drive the manifestation of diseases and phenotypes. Here we discuss, starting from chromosome level organization going down to single nucleotide changes, recent studies linking diseases or phenotypes to changes in the 3D genome architecture.

PresRAT: a server for identification of bacterial small-RNA sequences and their targets with probable binding region.

Bacterial small-RNA (sRNA) sequences are functional RNAs, which play an important role in regulating the expression of a diverse class of genes. It is thus critical to identify such sRNA sequences and their probable mRNA targets. Here, we discuss new procedures to identify and characterize sRNA and their targets via the introduction of an integrated online platform 'PresRAT'. PresRAT uses the primary and secondary structural attributes of sRNA sequences to predict sRNA from a given sequence or bacterial genome. PresRAT also finds probable target mRNAs of sRNA sequences from a given bacterial chromosome and further concentrates on the identification of the probable sRNA-mRNA binding regions. Using PresRAT, we have identified a total of 66,209 potential sRNA sequences from 292 bacterial genomes and 2247 potential targets from 13 bacterial genomes. We have also implemented a protocol to build and refine 3D models of sRNA and sRNA-mRNA duplex regions and generated 3D models of 50 known sRNAs and 81 sRNA-mRNA duplexes using this platform. Along with the server part, PresRAT also contains a database section, which enlists the predicted sRNA sequences, sRNA targets, and their corresponding 3D models with structural dynamics information.

Periodontal Disease in Type 1 Diabetes Mellitus: Influence of Pubertal Stage and Glycemic Control.

OBJECTIVE: Though gingivitis is common in children with type 1 diabetes mellitus (T1DM), the overall periodontal health in T1DM during the pubertal stage is less well-characterized. The study was undertaken to explore the possible influence of puberty and metabolic derangement on periodontal health in T1DM. METHODS: In this cross-sectional study, 110 subjects between 10-18 years with T1DM and 52 healthy siblings of similar age were evaluated for pubertal stage, glycosylated hemoglobin (HbA1c), and periodontal health. Simplified oral hygiene index (OHIS), gingival index (GI), plaque index (PI), bleeding on probing (BOP), and probing depth (PPD) were evaluated at 4 sites per tooth as per 6 Ramfjord index teeth used to assess periodontal disease (PD). RESULTS: PD not merely gingivitis was significantly higher in T1DM (84/110, 76.36%) than the control group (28/52, 53.8%) (P = .004). Irrespective of pubertal status, children with T1DM had worse GI, PI, BOP, and PPD than nondiabetic subjects, although OHIS was better in diabetes. In both T1DM and nondiabetic subjects, pubertal subjects showed significantly worse OHIS, PPD, BOP, and GI than prepubertal subjects. PD was correlated with pubertal stage, age, and HbA1c, although less strongly with the duration of diabetes. In logistic regression, pubertal stage was a stronger predictor of PD (OR = 14.26) than age (OR = 2.22), and HbA1c (OR = 1.5) rather than the presence of diabetes and its duration. CONCLUSIONS: Though pubertal status, age, and poor glycemic control rather than the presence of diabetes and its duration are associated with gingivitis and other forms of PD, puberty had a more profound effect in the pathogenesis of PD in T1DM.

Molecular mechanisms and epidemiology of COVID-19 from an allergist's perspective.

The global pandemic caused by the newly described severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused worldwide suffering and death of unimaginable magnitude from coronavirus disease 2019 (COVID-19). The virus is transmitted through aerosol droplets, and causes severe acute respiratory syndrome. SARS-CoV-2 uses the receptor-binding domain of its spike protein S1 to attach to the host angiotensin-converting enzyme 2 receptor in lung and airway cells. Binding requires the help of another host protein, transmembrane protease serine S1 member 2. Several factors likely contribute to the efficient transmission of SARS-CoV-2. The receptor-binding domain of SARS-CoV-2 has a 10- to 20-fold higher receptor-binding capacity compared with previous pandemic coronaviruses. In addition, because asymptomatic persons infected with SARS-CoV-2 have high viral loads in their nasal secretions, they can silently and efficiently spread the disease. PCR-based tests have emerged as the criterion standard for the diagnosis of infection. Caution must be exercised in interpreting antibody-based tests because they have not yet been validated, and may give a false sense of security of being "immune" to SARS-CoV-2. We discuss how the development of some symptoms in allergic rhinitis can serve as clues for new-onset COVID-19. There are mixed reports that asthma is a risk factor for severe COVID-19, possibly due to differences in asthma endotypes. The rapid spread of COVID-19 has focused the efforts of scientists on repurposing existing Food and Drug Administration-approved drugs that inhibit viral entry, endocytosis, genome assembly, translation, and replication. Numerous clinical trials have been launched to identify effective treatments for COVID-19. Initial data from a placebo-controlled study suggest faster time to recovery in patients on remdesivir; it is now being evaluated in additional controlled studies. As discussed in this review, till effective vaccines and treatments emerge, it is important to understand the scientific rationale of pandemic-mitigation strategies such as wearing facemasks and social distancing, and implement them.

Identification of copy number variations and translocations in cancer cells from Hi-C data.

MOTIVATION: Eukaryotic chromosomes adapt a complex and highly dynamic three-dimensional (3D) structure, which profoundly affects different cellular functions and outcomes including changes in epigenetic landscape and in gene expression. Making the scenario even more complex, cancer cells harbor chromosomal abnormalities [e.g. copy number variations (CNVs) and translocations] altering their genomes both at the sequence level and at the level of 3D organization. High-throughput chromosome conformation capture techniques (e.g. Hi-C), which are originally developed for decoding the 3D structure of the chromatin, provide a great opportunity to simultaneously identify the locations of genomic rearrangements and to investigate the 3D genome organization in cancer cells. Even though Hi-C data has been used for validating known rearrangements, computational methods that can distinguish rearrangement signals from the inherent biases of Hi-C data and from the actual 3D conformation of chromatin, and can precisely detect rearrangement locations de novo have been missing. RESULTS: In this work, we characterize how intra and inter-chromosomal Hi-C contacts are distributed for normal and rearranged chromosomes to devise a new set of algorithms (i) to identify genomic segments that correspond to CNV regions such as amplifications and deletions (HiCnv), (ii) to call inter-chromosomal translocations and their boundaries (HiCtrans) from Hi-C experiments and (iii) to simulate Hi-C data from genomes with desired rearrangements and abnormalities (AveSim) in order to select optimal parameters for and to benchmark the accuracy of our methods. Our results on 10 different cancer cell lines with Hi-C data show that we identify a total number of 105 amplifications and 45 deletions together with 90 translocations, whereas we identify virtually no such events for two karyotypically normal cell lines. Our CNV predictions correlate very well with whole genome sequencing data among chromosomes with CNV events for a breast cancer cell line (r = 0.89) and capture most of the CNVs we simulate using Avesim. For HiCtrans predictions, we report evidence from the literature for 30 out of 90 translocations for eight of our cancer cell lines. Furthermore, we show that our tools identify and correctly classify relatively understudied rearrangements such as double minutes and homogeneously staining regions. Considering the inherent limitations of existing techniques for karyotyping (i.e. missing balanced rearrangements and those near repetitive regions), the accurate identification of CNVs and translocations in a cost-effective and high-throughput setting is still a challenge. Our results show that the set of tools we develop effectively utilize moderately sequenced Hi-C libraries (100-300 million reads) to identify known and de novo chromosomal rearrangements/abnormalities in well-established cancer cell lines. With the decrease in required number of cells and the increase in attainable resolution, we believe that our framework will pave the way towards comprehensive mapping of genomic rearrangements in primary cells from cancer patients using Hi-C. AVAILABILITY AND IMPLEMENTATION: CNV calling: https://github.com/ay-lab/HiCnv, Translocation calling: https://github.com/ay-lab/HiCtrans and Hi-C simulation: https://github.com/ay-lab/AveSim. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia.

Tisagenlecleucel (CTL019) is an investigational immunotherapy that involves reprogramming a patient's own T cells with a transgene encoding a chimeric antigen receptor to identify and eliminate CD19-expressing cells. We previously reported that CTL019 achieved impressive clinical efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), including the expansion and persistence of CTL019 cells, which correlates with response to therapy. Here, we performed formal cellular kinetic analyses of CTL019 in a larger cohort of 103 patients treated with CTL019 in 2 different diseases (ALL and CLL). CTL019 was measured in peripheral blood and bone marrow, using quantitative polymerase chain reaction and flow cytometry. CTL019 levels in peripheral blood typically peaked at 10 to 14 days postinfusion and then declined slowly over time. Patients with complete response (CR)/CR with incomplete count recovery had higher levels of CTL019 in peripheral blood, with greater maximal concentration and area under the curve values compared with nonresponding patients (P < .0001 for each). CTL019 transgene levels were measurable up to 780 days in peripheral blood. CTL019 trafficking and persistence were observed in bone marrow and cerebrospinal fluid. CTL019 expansion correlated with severity of cytokine release syndrome (CRS) and preinfusion tumor burden in pediatric ALL. The results described here are the first detailed formal presentation of cellular kinetics across 2 diseases and highlight the importance of the application of in vivo cellular kinetic analyses to characterize clinical efficacy and CRS severity associated with CTL019 therapy.

Butyrate-Induced In Vitro Colonocyte Differentiation Network Model Identifies ITGB1, SYK, CDKN2A, CHAF1A, and LRP1 as the Prognostic Markers for Colorectal Cancer Recurrence.

Numerous mechanisms are believed to contribute to the role of dietary fiber-derived butyrate in the protection against the development of colorectal cancers (CRCs). To identify the most crucial butyrate-regulated genes, we exploited whole genome microarray of HT-29 cells differentiated in vitro by butyrate treatment. Butyrate differentiates HT-29 cells by relaxing the perturbation, caused by mutations of Adenomatous polyposis coli (APC) and TP53 genes, the most frequent mutations observed in CRC. We constructed protein-protein interaction network (PPIN) with the differentially expressed genes after butyrate treatment and extracted the hub genes from the PPIN, which also participated in the APC-TP53 network. The idea behind this approach was that the expression of these hub genes also regulated cell differentiation, and subsequently CRC prognosis by evading the APC-TP53 mutational effect. We used mRNA expression profile of these critical hub genes from seven large CRC cohorts. Logistic Regression showed strong evidence for association of these common hubs with CRC recurrence. In this study, we exploited PPIN to reduce the dimension of microarray biologically and identified five prognostic markers for the CRC recurrence, which were validated across different datasets. Moreover, these five biomarkers we identified increase the predictive value of the TNM staging for CRC recurrence.

Enhanced basepair dynamics pre-disposes protein-assisted flips of key bases in DNA strand separation during transcription initiation.

Localized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes, including transcription and replication. Little is known about how these strand separations occur. The strand-separated E.coli RNA polymerase-promoter open-complex structure showed four bases of the non-template strand, the master base -11A, -7, -6 and +2, in a flipped state and inserted into protein pockets. To explore whether any property of these bases in the duplex state pre-disposes them to flipping, NMR studies were performed on a wild-type promoter in the duplex state. Measurement of relaxation times indicates that a limited number of base pairs, including the flipped ones, have faster opening rates than the rest. Molecular dynamics studies also show an inherently high dynamic character of the -11A:T base pair in the wild-type strand-paired state. In order to explore the role of the RNA polymerase in the flipping process, we have used 2-aminopurine as a fluorescent probe. Slower kinetics of the increase of 2-aminopurine fluorescence was observed with RNA polymerases containing several mutant σ70s. This may be interpreted as the protein playing an important role in enhancing the flipping rate. These results suggest that flipping of -11A, and perhaps other flipped bases observed in the open-complex, is facilitated by its inherent proclivity to open-up with further assistance from the protein, thus leading to a strand-open state. Other DNA-based processes that require strand-separation may use similar pathways for strand separation. We conclude that not only basepair stability, but also dynamics may play an important role in the strand-separation.

A survey on prediction of specificity-determining sites in proteins.

Specificity-determining sites (SDS) are the key positions of a protein family that show a specific conservation of amino acids, related to the subfamily members of that family. SDS play crucial role in developing functional variation within the protein family during the course of evolution. Thus, it is important to identify SDS to understand the evolutionary process of diversification of biological functions within a protein family. A wide range of computational tools have been designed to detect such SDS. In this review, we intend to examine the concept of SDS in more details along with the advancements and drawbacks of different computational approaches designed towards successful prediction of SDS. Further, we discussed the algorithms behind the computational approaches developed till date and provide an exhaustive comparison of performance of each method. We also introduce a new ensemble approach, SubSite as another tool to predict SDS through a user-friendly webserver available at www.hpppi.iicb.res.in/subsite.