Integration Site Research Articles

Divergent populations of HIV-infected naïve and memory CD4+ T-cell clones in children on antiretroviral therapy.

Naïve cells comprise 90% of the CD4+ T-cell population in neonates and exhibit distinct age-specific capacities for proliferation and activation. We hypothesized that HIV-infected naïve CD4+ T-cell populations in children on long-term antiretroviral therapy (ART) would thus be distinct from infected memory cells. Peripheral blood naïve and memory CD4+ T cells from 8 children with perinatal HIV on ART initiated at age 1.7-17 months were isolated by FACS. DNA was extracted from sorted cells and HIV proviruses counted, evaluated for intactness, and subjected to integration site analysis. Naïve CD4+ T cells containing HIV proviruses were detected in children with 95% statistical confidence. A median of 4.7% of LTR-containing naïve CD4+ T cells also contained HIV genetic elements consistent with intactness. Full-length proviral sequencing confirmed intactness of one provirus. In the participant with the greatest level of naïve cell infection, ISA revealed infected expanded cell clones in both naïve and memory T cells with no common HIV integration sites detected between subsets. Divergent integration site profiles reflected differential gene expression patterns of naïve and memory T cells. These results demonstrate that HIV persists in both naïve and memory CD4+ T cells that undergo clonal expansion and harbor intact proviruses, suggesting that infected memory T-cell clones do not frequently arise from naïve cell differentiation in children with perinatal HIV on long-term ART. Center for Cancer Research, NCI and Office of AIDS Research funding to MFK, NCI FLEX funding to JWR. Children's and Emory JFF pilot to MM.

HPV integration profiling using nanopore sequencing and association with cervical precancerous lesion

ObjectivesHPV infection and HPV DNA integration can lead to cervical cancer, but the relationship with lesion severity is unclear. This study aimed to investigate the correlation between HPV integration profile and cervical lesion extent.Materials and methodsTwenty patients representing cervicitis, CIN I, CIN II, and CIN III underwent nanopore sequencing for HPV genotype and integration site analysis. HPV integration profiles were correlated with lesion severity. Gene Ontology (GO) and KEGG analysis were used to identify stage-specific genes and pathways.ResultsHPV integration rates were 60, 60, 100, and 100% for cervicitis, CIN I, CIN II, and CIN III, respectively, with varying numbers of integrated genes. Each group had specific stage-related genes, with 83 shared genes linked to neuron development and cell–cell processes. CIN II and CIN III displayed more cancer-related pathway enrichment than earlier stages.ConclusionA positive correlation exists between HPV integration frequency and cervical lesion stage. Late-stage lesions showed heightened enrichment in cancer-related pathways through specific HPV-integrated genes.

Context-Sensitive Processing in a Model Neocortical Pyramidal Cell With Two Sites of Input Integration.

Neocortical layer 5 thick-tufted pyramidal cells are prone to exhibiting burst firing on receipt of coincident basal and apical dendritic inputs. These inputs carry different information, with basal inputs coming from feedforward sensory pathways and apical inputs coming from diverse sources that provide context in the cortical hierarchy. We explore the information processing possibilities of this burst firing using computer simulations of a noisy compartmental cell model. Simulated data on stochastic burst firing due to brief, simultaneously injected basal and apical currents allow estimation of burst firing probability for different stimulus current amplitudes. Information-theory-based partial information decomposition (PID) is used to quantify the contributions of the apical and basal input streams to the information in the cell output bursting probability. Four different operating regimes are apparent, depending on the relative strengths of the input streams, with output burst probability carrying more or less information that is uniquely contributed by either the basal or apical input, or shared and synergistic information due to the combined streams. We derive and fit transfer functions for these different regimes that describe burst probability over the different ranges of basal and apical input amplitudes. The operating regimes can be classified into distinct modes of information processing, depending on the contribution of apical input to output bursting: apical cooperation, in which both basal and apical inputs are required to generate a burst; apical amplification, in which basal input alone can generate a burst but the burst probability is modulated by apical input; apical drive, in which apical input alone can produce a burst; and apical integration, in which strong apical or basal inputs alone, as well as their combination, can generate bursting. In particular, PID and the transfer function clarify that the apical amplification mode has the features required for contextually modulated information processing.

Decoding a Cell's Fate: How Notch and receptor tyrosine kinase signals specify the Drosophila R7 photoreceptor.

Decoding a Cell's Fate: How Notch and receptor tyrosine kinase signals specify the Drosophila R7 photoreceptor.

Intra-articular injection of inorganic pyrophosphate improves IL-1β-induced cartilage damage in rat model of knee osteoarthritis in vivo.

Osteoarthritis (OA) is the most common form of chronic joint disease, affecting mainly the elderly population. This disorder is caused by cartilage degeneration with complex changes in the chondrocyte phenotype. Inorganic pyrophosphate (PPi) was shown to counteract the detrimental effect of interleukin (IL)-1β challenging in an in vitro OA model based on rat articular chondrocytes. It also maintained the differentiated articular phenotype, mostly by down regulating wingless-related integration site (Wnt)-5a secretion. These observations suggest a PPi protective role for chondrocyte in vitro. To address this hypothesis in vivo, we investigated the impact on knee joint of three intra-articular injection (IAI) of PPi in a rat model of cartilage damage induced by IAI of IL-1β, where cartilage degradation and synovial inflammation are similar to that observed in OA. Cartilage and synovial membrane were collected after 7 days of challenge by IL-1β. PPi was able to reduce the deleterious effect of IL-1β. This effect was observable on the expression of cartilage extracellular matrix metabolism markers and confirmed by histology with safranin O and hematoxylin-eosin-saffron (HES) staining. Inorganic pyrophosphate also repressed the Wnt5a expression induced by IL-1β. No effect was observed on the inflammatory response of the synovial membrane. These results demonstrate that PPi improves IL-1β-induced cartilage damage in rat but not the associated inflammation of synovial membrane. Thus, PPi could become a molecule of interest to restrict the progression of this disorder.

Aberrant DNA methylation of genes regulating CD4+ T cell HIV-1 reservoir in women with HIV.

The HIV-1 reservoir in CD4+ T cells (HRCD4) pose a major challenge to curing HIV, with many of its mechanisms still unclear. HIV-1 DNA integration and immune responses may alter the host's epigenetic landscape, potentially silencing HIV-1 replication. This study used bisulphitecapture DNA methylation sequencing in CD4+ T cells from the blood of 427 virally suppressed women with HIV to identify differentially methylated sites and regions associated with HRCD4. The average total HRCD4 size was 1409 copies per million cells, with most proviruses defective and only a small proportion intact. The study identified 245 differentially methylated CpG sites and 85 regions linked to HRCD4 size, with 52% of significant sites in intronic regions. Genes associated with HRCD4 were involved in viral replication, HIV-1 latency and cell growth and apoptosis. HRCD4 size was inversely related to DNA methylation of interferon signalling genes and positively associated with methylation at known HIV-1 integration sites. HRCD4-associated genes were enriched on the pathways related to immune defence, transcription repression and host-virus interactions. These findings suggest that HIV-1 reservoir is linked to aberrant DNA methylation in CD4+ T cells, offering new insights into epigenetic mechanisms of HIV-1 latency and potential molecular targets for eradication strategies. Study involved 427 women with HIV. Identified 245 aberrant DNA methylation sites and 85 methylation regions in CD4+ T cells linked to the HIV-1 reservoir. Highlighted genes are involved in viral replication, immune defence, and host genome integration. Findings suggest potential molecular targets for eradication strategies.

Effect of CHO cell line constructed with CMAH gene-directed integration on the recombinant protein expression.

Effect of CHO cell line constructed with CMAH gene-directed integration on the recombinant protein expression.

Protocol for generating NGN2 iPSC lines and large-scale human neuron production.

Protocol for generating NGN2 iPSC lines and large-scale human neuron production.

Characterization of the Genomic Landscape in HPV-positive Cervical and Head and Neck Squamous Cell Carcinomas by Whole Genome Next Generation Sequencing.

In this study, we provide a comprehensive characterization of HPV-positive primary cervical cancers (CC) and HPV-positive head and neck squamous cell carcinomas (HNSCC) through whole genome next-generation sequencing. Human papillomavirus (HPV) infection, recognized as a definitive human carcinogen, is increasingly acknowledged for its role in development of human cancers. HPV-driven cervical cancers are among the leading causes of cancer-related deaths worldwide, while HPV-driven head and neck cancers exhibit distinct biological and clinical characteristics. Recent data has provided convincing evidence that HPV-related cervical cancer, like HPV head and neck cancer also predict better outcomes, with viral integration patterns further predicting disease related outcomes. We designed an experimental study that encompasses four pairs of HPV-positive patient samples with controls, utilizing state-of-the-art Next Generation Sequencing (NGS) technology including whole genome sequencing, transcriptome sequencing and virus integration. Multiple mutated genes, including TTN, COL6A3, and FLNA, were identified shared between CC and HNSCC. Additionally, we observed a notable proportion of pathways affected by oncogenic alterations, particularly in the RTK-RAS and NOTCH pathways, in both CC and HNSCC. Furthermore, we discovered a shared down-regulation of the Hedgehog signaling pathway based on transcriptome expression analysis in KEGG. We also identified RUNX2 and TFPI as sites of virus integration, and upstream as well as downstream pathway modulators, and represent potential targets for therapeutic interventions. Overall, this study showed a thorough comparison between CC and HNSCC from multiple aspects, including gene variations, oncogenic pathways, KEGG enrichment and virus integration sites. However, further studies, which involve larger patient cohorts should be undertaken to further support these findings.

Synthetic Lethality of SHP2 and XIAP Suppresses Proliferation and Metastasis in KRAS-mutant Nonsmall Cell Lung Cancer.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are associated with poor prognosis and poor response to standard therapeutic regimens in patients with nonsmall cell lung cancer (NSCLC). Identification of novel synthetic lethal partners in oncogenic KRAS is an alternative therapeutic strategy for KRAS-mutant malignancies. After high-throughput screening against a preclinical/clinical compound library, embelin, a known X-linked inhibitor of apoptosis protein (XIAP) inhibitor, specifically inhibits the catalytic activity and phosphorylation of Src homology domain 2 containing tyrosine phosphatase 2 (SHP2) in KRAS-mutant NSCLC cells. Pharmacological inhibition and genetic knockdown of XIAP and SHP2 induce synthetic lethality in KRAS-mutated NSCLC cells and xenograft animal models. Mechanistically, dual inhibition of XIAP and SHP2 by embelin lessens the proliferation and metastasis, activates senescence and endogenous apoptosis, inhibits cancer-related RAS/mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)/AKT, Janus kinase/signal transducers and activators of transcription (JAK/STAT), wingless-related integration site (Wnt), and nuclear factor kappa B (NF-κB) signaling pathways, and overcomes compensatory feedback in the MAPK signals through the modulation of mitogen-inducible gene-6 (MIG-6) and SPROUTY2 (SPRY2). Collectively, SHP2 and XIAP are potential synthetic lethal partners, and embelin warrants further development as a novel therapeutic option for alleviating KRAS-mutant NSCLC by cotargeting SHP2 and XIAP.

Multiomic profiling of T cell lymphoma after therapy with anti-BCMA CAR T cells and GPRC5D-directed bispecific antibody.

Chimeric antigen receptor (CAR) T cells and bispecific T cell engagers have become integral components in the treatment of relapsed/refractory multiple myeloma. We report a 63-year-old male who received ciltacabtagene autoleucel CAR T cells and the GPRC5D × CD3 bispecific talquetamab for early relapse of his multiple myeloma. Nine months after CAR T therapy, he developed a symptomatic leukemic peripheral T cell lymphoma with cutaneous and intestinal involvement. Longitudinal single-cell RNA and T cell receptor sequencing of peripheral blood and bone marrow revealed two hyperexpanded CAR-carrying T cell clones. These expanded clones exhibited an exhausted effector-memory T cell transcriptional signature, and the neoplasm itself was sensitive to dexamethasone treatment. The immunophenotypic and transcriptional alterations of these abnormal T cells resembled those of T-large granular lymphocytic leukemia. Spatial transcriptomes of skin lesions confirmed the aberrant CAR-expressing T cells. Whole-genome sequencing revealed three distinct integration sites, within the introns of ZGPAT, KPNA4 and polycomb-associated noncoding RNAs. Before and after CAR T whole-genome analyses implicated clonal outgrowth of a TET2-mutated precursor propelled by additional subclone-specific loss of heterozygosity and other secondary mechanisms. This case highlights the evolution of a CAR-carrying peripheral T cell lymphoma following CAR T cell and bispecific T cell engager therapy, offering critical insights into the clonal evolution from a predisposed hematopoietic precursor to a mature neoplasm.

Turning "trashed" genomic loci into treasurable sites for integrating chimeric antigen receptors in T and NK cells.

Turning "trashed" genomic loci into treasurable sites for integrating chimeric antigen receptors in T and NK cells.

Longitudinal analysis of lentiviral and retroviral chimeric antigen receptors' integration sites reveals distinct clonal evolutionary patterns.

T-cell malignancies following chimeric antigen receptor (CAR) therapies are partly related to insertional mutagenesis, but the longitudinal evolution of CAR-integration sites (IS) remains understudied. We performed an IS analysis in blood from three tisagenlecleucel (lentiviral), one axicabtagene-ciloleucel and one brexucabtagene-autoleucel (gammaretrovirals) patient at peak expansion and 1-year follow-up. All were complete responders. Peak expansion IS patterns were vector dependent: lentiviral CAR integrated mostly in introns and gammaretrovirals in intergenic regions, closer to transcription start sites. At 1-year post-infusion, lentiviral CAR showed no major clonal proliferation. Gammaretroviral CARs had divergent outcomes: no detectable CAR (axicabtagene-ciloleucel) or low-level oligoclonal persistence (brexucabtagene-autoleucel). Whether this latter evolution is at risk of further CAR malignancies needs further investigations.

Virusplot: a web server for viral integration analysis and visualization.

The integration of viral DNA into the human genome is a critical event in the pathogenesis of various cancers. This process leads to genomic instability, disrupts cellular regulatory mechanisms, and activates oncogenes or inactivates tumor suppressor genes. Despite significant advancements in genome sequencing technologies, there remains a notable lack of computational tools, particularly web-based applications, specifically designed for viral integration analysis and visualization. To address this gap, we present virusPlot, a web server with the following functional modules: (i) automatic retrieval of virus genome sequences and their annotation; (ii) visualization of virus integration locations and read counts through a graphical representation that links viral and host genome integration sites, facilitating the interpretation of integration patterns; (iii) analysis of virus integration hotspots using Fisher's exact test; and (iv) integration of various functions into an interactive web platform via shinyapp. VirusPlot efficiently processes and visualizes integration data from viruses and host genomes, providing researchers with an intuitive and user-friendly analytical tool that simplifies the complexity of virus integration analysis.

Multistrategy Optimization for High-Yield 3-Fucosyllactose Production in Escherichia coli BL21 Star (DE3).

3-Fucosyllactose (3-FL), an essential component of human milk oligosaccharides, is crucial for infant health. However, the extraction of 3-FL from milk presents a significant challenge. In this study, E. coli BL21 Star (DE3) was engineered for 3-FL biosynthesis, and the gene combinations and metabolic pathways were optimized to obtain a high-yield 3-FL production strain. First, the 3-FL production module was integrated into E. coli. Second, an efficient alpha-1,3-fucosyltransferase was screened. Then, different integration sites were used to optimize the precursor synthesis gene cluster and the transferase genes and to screen for transporter proteins and glycerol utilization genes that could enhance 3-FL production. The 3FL05-1 strain yielded a 3-FL titer of 11.26 g/L in shake flasks. Further amplification in a 5 L bioreactor resulted in a 3-FL titer of 60.24 g/L, which represents the highest reported titer to date.

Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides.

Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides.

Barcoded HIV-1 reveals viral persistence driven by clonal proliferation and distinct epigenetic patterns

The HIV reservoir consists of infected cells in which the HIV-1 genome persists as provirus despite effective antiretroviral therapy (ART). Studies exploring HIV cure therapies often measure intact proviral DNA levels, time to rebound after ART interruption, or ex vivo stimulation assays of latently infected cells. This study utilizes barcoded HIV to analyze the reservoir in humanized mice. Using bulk PCR and deep sequencing methodologies, we retrieve 890 viral RNA barcodes and 504 proviral barcodes linked to 15,305 integration sites at the single RNA or DNA molecule in vivo. We track viral genetic diversity throughout early infection, ART, and rebound. The proviral reservoir retains genetic diversity despite cellular clonal proliferation and viral seeding by rebounding virus. Non-proliferated cell clones are likely the result of elimination of proviruses associated with transcriptional activation and viremia. Elimination of proviruses associated with viremia is less prominent among proliferated cell clones. Proliferated, but not massively expanded, cell clones contribute to proviral expansion and viremia, suggesting they fuel viral persistence. This approach enables comprehensive assessment of viral levels, lineages, integration sites, clonal proliferation and proviral epigenetic patterns in vivo. These findings highlight complex reservoir dynamics and the role of proliferated cell clones in viral persistence.

Cranial repair-promoting effect of oxidised sodium alginate/amino gelatine injectable hydrogel loaded with deer antler blood peptides.

Cranial repair-promoting effect of oxidised sodium alginate/amino gelatine injectable hydrogel loaded with deer antler blood peptides.

Discovery of Human PIM Kinase Inhibitors as a Class of Anthelmintic Drugs to Treat Intestinal Nematode Infections.

Soil-transmitted helminth (STH) infections affect one-fourth of the global population and pose a significant threat to human and animal health, with limited treatment options and emerging drug resistance. Trichuris trichiura (whipworm) stands out as a neglected disease, necessitating new drugs to address this unmet medical need. We discovered that several different chemical series of related human Provirus Integration sites for Moloney murine leukemia virus (PIM) family kinase inhibitors possess potent anthelmintic activity by using whole-worm motility assays. Systematic structure-activity relationship (SAR) studies based on the pan-PIM kinase inhibitor CX-6258 were conducted to identify compounds displaying improved in vitro motility inhibition of both adult hookworm (Ancylostoma ceylanicum) and adult whipworm (Trichuris muris) nematodes. A broad kinase selectivity screen of >450 human kinases confirms PIM1 kinase and others as potential targets for CX-6258 and analogues thereof. In addition, we demonstrated that CX-6258 significantly reduced worm burden and egg counts in the T. muris infection model of mice, establishing it as a new oral small molecule anthelmintic therapeutic.

A rare deleterious missense mutation in the AXIN2 gene in Chinese women with polycystic ovary syndrome

Background Polycystic ovary syndrome (PCOS) is an endocrine disorders and characterized by polycystic ovary morphology and oligomenorrhea, affecting fertility and health condition of female around the world. The causative factors of PCOS are complex, and genetic structure remains a long-standing medical challenge in genetics. Previous genome-wide association study (GWAS) showed that Wing-less-related integration site (Wnt) signaling is the most affected pathway among PCOS-related risk genes, and genetic mutations in the Wnt/β-catenin signaling may lead to abnormal development of PCOS. Objective To explore the possibility of axis inhibitor-2 (AXIN2) variants in Chinese women with PCOS and assess their pathogenicities. Methods A total of 365 Chinese women with PCOS and 905 women without PCOS as control were recruited from Jiangxi Provincial Maternal and Child Health Hospital, All of the 11 exons and flanking regions of the AXIN2 gene were amplified by polymerase chain reaction (PCR), the potential variants were analyzed by Sanger sequencing. The evolutionary conservation analysis of the identified Axin-2 mutant was analyzed among 15 vertebrates from human to zebrafish. The protein structure change was analyzed between the wild-type and mutation-type. The pathogenicity of AXIN2 variant was further analyzed in silico. Results We totally identified 7 genetic variants of AXIN2 in this study, including 4 synonymous and 3 missense. Among them, we find a rare deleterious missense variant [p.R714W (c.2140C > T)]. The allele frequencies of this variant were 0.82% and 0.17% in PCOS cases and matched controls, respectively. And it was ranging from 7.89e-5 to 1.47e-4 in public databases. Fisher’s exact test indicated that the allele frequencies in PCOS were p < 0.05 compared to both the controls and the databases. Especially, the mutant amino acid site is highly conserved in vertebrates, while the mutation changed the 714th arginine to tryptophan resulting in significant change in the protein structural of Axin-2. Conclusion In this study, we identified a rare deleterious missense AXIN2 mutation [p.R714W (c.2140C > T)] in Chinese women with PCOS, and this mutant is probably pathogenic. This study may provide a new perspective on revealing the genetic variation of PCOS.