Clonal Expression Research Articles

The clonal expression genes associated with poor prognosis of liver cancer.

The extensive spatial genomic intratumor heterogeneity (ITH) in liver cancer hindered treatment development and limited biomarker design. Early events that drive tumor malignant transformation in tumor founder cells are clonally present in all tumor cell populations, which provide stable biomarkers for the localization of tumor cells and patients’ prognosis. In the present study, we identified the recurrently clonal somatic mutations and copy number alterations (CNAs) (893 clonal somatic mutations and 6,617 clonal CNAs) in 353 liver cancer patients from The Cancer Genome Atlas (TCGA) and evaluated their prognosis potential. We showed that prognosis-related clonal alterations might play essential roles in tumor evolution. We identified 32 prognosis related clonal alterations differentially expressed between paired normal and tumor samples, that their expression was cross-validated by three independent cohorts (50 paired samples in TCGA, 149 paired samples in GSE76297, and 9 paired samples in SUB6779164). These clonal expression alterations were also significantly correlated with clinical phenotypes. Using stepwise regression, we identified five (UCK2, EFNA4, KPAN2, UBE2T, and KIF14) and six (MCM10, UCK2, IQGAP3, EFNA4, UBE2T, and KPNA2) clonal expression alterations for recurrence and survival model construction, respectively. Furthermore, in 10 random repetitions, we showed strong applicability of the multivariate Cox regression models constructed based on the clonal expression genes, which significantly predicted the outcomes of the patients in all the training and validation sets. Taken together, our work may provide a new avenue to overcome spatial ITH and refine biomarker design across cancer types.

Immune repertoire analysis of normal Chinese donors at different ages.

This study investigated the characteristics of the immune repertoire in normal Chinese individuals of different ages. In this study, all seven receptor chains from both B and T cells in peripheral blood of 16 normal Chinese individuals from two age groups were analyzed using high-throughput sequencing and dimer-avoided multiplex PCR amplification. Normal in this study is defined as no chronic, infectious or autoimmune disease within 6months prior to blood draw. We found that compared with the younger group, the clonal expression of T-cell receptor repertoire increased in the older group, while diversity decreased. In addition, we found that the T-cell receptor repertoire was more significantly affected by age than the B-cell receptor repertoire, including significant differences in the use of the unique TCR-alpha and TCR-beta V-J gene combinations, in the two groups of normal participants. We further analyzed the degree of complementarity determining region 3 sequence sharing between the two groups, and found shared TCR-alpha, TCR-gamma, immunoglobulin-kappa and immunoglobulin-lambda chain complementarity determining region 3 sequences in all subjects. Taken together, our study gives us a better understanding of the immune repertoire of different normal Chinese people, and these results can be applied to the treatment of age-related diseases. Immune repertoire analysis also allows us to observe participant's wellness, aiding in early-stage diagnosis.

Abstract 833: Linking phenotype and genotype: Multimodal analysis of surface proteins, intracellular proteins, and SNV in single cells

Abstract Cancers evolve via processes of clonal expansion, selection, and somatic variation, which contribute to tumor heterogeneity. While bulk analysis has improved our understanding of cancer, the heterogeneity of a tumor is masked with the average readout provided by a bulk measurement. This problem could be overcome by employing single-cell technologies. One of the most common single-cell technologies is single-cell RNA sequencing (scRNA-seq). Transcriptomic data collected by scRNA-seq are used to imply activity of gene products based on RNA levels. Signaling pathways related to cancer initiation and progression have been mapped solely based on scRNA-seq results. This approach, however, is not an accurate measurement of proteins that are the functional molecules responsible for key events in cancer. Recent breakthroughs of multimodal approaches such as CITE-seq and REAP-seq partially addressed this problem by allowing simultaneous analysis of transcriptomes and surface protein expression levels. These methods still lack a couple components that are required to fully dissect the biology of cancer. First, the methods are limited to the measurements of cell surface proteins, whereas proteins involved in cancer-related signal transduction and transcriptional pathways are mostly localized in the intracellular compartments. Second, the methods do not allow direct analysis of DNA sequences, hence unable to provide an accurate readout of genotypic information such as single-nucleotide variants and copy number variations. Here we describe a technology that could overcome these hurdles. To achieve this, cells are first treated with oligonucleotide-barcoded antibodies targeting surface proteins. Cells are then fixed and permeabilized, followed by incubation with barcoded antibodies for intracellular proteins. The resulting cells are processed on the Mission Bio TapestriⓇ platform, a device that enables encapsulation of single cells in droplets. Targeted DNA sequencing libraries are generated from the single cells using a multiplex panel of primers targeting regions of interest. Protein sequencing libraries are separately generated from the oligonucleotides off the antibodies. The inclusion of intracellular protein detection enables measurement of pivotal proteins in cancer mechanisms, including apoptosis (BCL2 family proteins), transcription factors (GATA3), tumor suppressors (TP53), as well as phosphorylated proteins involved in cell growth signaling pathways (phosphorylated ERK and STAT proteins). This is the first ever method that provides a solution to effectively link surface and intracellular protein measurement with targeted DNA analysis. With this approach, single-cell readout of genotypic and phenotypic information can be collected together, allowing concurrent complex analyses of cancer clonal evolution and driver protein expression. Citation Format: Prithvi Singh, Saurabh Parikh, Tim Lai, Dalia Dhingra, Aik Ooi. Linking phenotype and genotype: Multimodal analysis of surface proteins, intracellular proteins, and SNV in single cells [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 833.

Abstract 643: Non-classical sources of tumour specific antigen in checkpoint inhibitor response

Abstract A large-scale meta-analysis of over 1000 CPI (immune checkpoint blockade) treated patients across multiple cancer types broadly categorised biomarkers into four categories: sources of antigen leading to T-cell responses, host-derived factors, immune evasion and infiltration mechanisms. Our analyses established tumour mutation burden (TMB) as the strongest predictor of CPI response, alongside clonal TMB and CXCL9 expression. However, over half of the patients in our cohort who responded to CPI treatment did not have high-TMB and showed no evidence of oncogenic viral infections, hence suggesting alternative sources of antigen could be driving the immune response. As well as this, data from the CPI1000 cohort suggested that the biomarkers identified in an extensive systematic review only accounted for ~60% of the variance in CPI response, promoting the search for non-classical sources of antigen. Extending the cohort to encompass 2500+ patients, using available genomic and transcriptomic data from checkpoint inhibitor trials and industry partners, the previously published multivariate predictor will be further validated in a larger patient cohort inclusive of more cancer types. The CPI1000 cohort utilised primarily whole exome-sequencing therefore, had limited ability to discern non-coding sources of tumour antigen and cell-type specific predictors of response. The CPI2500+ cohort will incorporate several additional cohorts as well as single-cell and T-cell reactivity data. The raw data will be harmonised across studies and processed using a robust bioinformatics pipeline. As well as this, alternative (non-classical) sources of tumour specific antigen will be studied. Alternative sources of antigen can originate from a diverse range of biological processes including retained introns, stop codon loss mutations and cancer associated bacterial/viral epitopes. Data from this exploratory analysis will be coupled with peptide reactivity assays from local UCL patient TIL (tumour infiltrating lymphocyte) and PMBC (peripheral blood mononuclear cells) samples to validate immunogenicity. Current analysis has identified a novel subgroup of CPI-responders with low-TMB who have significantly higher rates of microbial diversity, consistent with a diverse tumour microbiome providing a source of immunogenic antigens in the absence of high-TMB. Here we will present insights from CPI2500+ and highlight the importance of emerging non-classical sources of tumour specific antigen. Citation Format: Krupa Thakkar, Danwen Qian, Hongui Cha, Thomas B. Watkins, Charles Swanton, Kevin Litchfield. Non-classical sources of tumour specific antigen in checkpoint inhibitor response [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 643.

Predicting patient treatment response and resistance via single-cell transcriptomics of their tumors.

e20540 Background: Tailoring the best treatments to cancer patients is an important open challenge. Here, we build a precision oncology data science and software framework for PERsonalized single-Cell Expression-based Planning for Treatments In Oncology (PERCEPTION). Methods: Our approach capitalizes on recently published matched bulk and single-cell transcriptome profiles of large-scale cell-line drug screens to build treatment response models from patients’ single-cell (SC) tumor transcriptomics. Our approach is a two-step process: first, the prediction models are trained on large-scale bulk-expression profiles of cancer cell lines and then, in a second step, the models’ performance is further optimized by training on SC-expression profiles of cancer cell lines. Results: First, we show that PERCEPTION successfully predicts the response to monotherapy and combination treatments in screens performed in cancer and patient-tumor-derived primary cells based on SC-expression profiles. Second, it successfully stratifies responders to combination therapy based on the patients’ tumor’s SC-expression in two very recent multiple myeloma and breast cancer clinical trials. Thirdly, it captures the development of clinical resistance to five standard tyrosine kinase inhibitors using tumor SC-expression profiles obtained during treatment in a lung cancer patients’ cohort. Notably, PERCEPTION outperforms state-of-the-art bulk expression-based predictors in all three clinical cohorts. Conclusions: In sum, this study provides a first-of-its-kind conceptual and computational method that is predictive of response to therapy in patients, based on the clonal SC gene expression of their tumors.

Reliable Detection of T-Cell Clonality by Flow Cytometry in Mature T-Cell Neoplasms Using TRBC1: Implementation as a Reflex Test and Comparison with PCR-Based Clonality Testing.

The T-cell receptor β constant region 1 (TRBC1) antibody can identify T-cell clonality and distinguish pathological from normal T cells. This study aims to establish optimal cutpoints for establishing monotypia and validate the diagnostic abilities of the TRBC1 antibody when used as a reflex test in conjunction with an existing T-cell antibody panel. We used 46 normal peripheral blood specimens and examined 8 patients with reactive lymphoproliferations to determine the normal biological range of TRBC1 on CD4+ and CD8+ T cells. We also evaluated 43 patient specimens that were submitted for investigation of a lymphoproliferative disorder for CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD26/CD45/CD56/TCR αβ/TCR γδ, along with TRBC1 expression. The results were compared to TCR gene rearrangement patterns using polymerase chain reaction (PCR) analysis. Statistical analysis established differing cutoff points for establishing monotypia dependent on restricted TRBC1 or TRBC2 usage. Direct comparison with molecular analysis indicated that no specimen identified with the restricted expression of TRBC1 was reported as polyclonal by PCR with a concordance rate of 97% between a clonal PCR result and monotypic TRBC1 expression. Incorporation of the TRBC1 antibody using statistically derived cutoff points in a reflex setting for the evaluation of a suspected T-cell neoplasm improves the identification of clonal T-cell populations by flow cytometry and correlates well with molecular methods.

Mitochondrial-Linked De Novo Pyrimidine Biosynthesis Dictates Human T-Cell Proliferation but Not Expression of Effector Molecules.

T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells.

In Situ Vaccination Induces Changes in Follicular Lymphoma Tumor Cells That Correlate with Abscopal Clinical Regressions

In Situ Vaccination Induces Changes in Follicular Lymphoma Tumor Cells That Correlate with Abscopal Clinical Regressions

USE OF TCR Vβ2 REPERTOIRES TO DIFFERENTIATE CD8 T-CELL LYMPHOMAS FROM INFECTIOUS DISEASE REACTIVE LYMPHOCYTOSIS

Reactive lymphocytosis can be difficult to differentiate from clonal lymphocytosis, especially when it involves CD8 positive T-lymphocytes. Leukemias and lymphomas cause an increase in this population at the expense of replication of clonal cells with genetic and/or phenotypic aberrations. Infections, mainly of viral etiology, often cause an exacerbated response of this population. The expansion resulting from a viral infection is not selective, so multiple cell clones are involved. Defining the cause of lymphocytosis is essential for determining therapeutic measures. To define T-cell clonality in patients with peripheral blood lymphocytosis using TCR Vβ2 repertoire by flow cytometry multiparametric analysis (FCM). We included five patients with suspected T-cell lymphocytosis, one patient with immunodeficiency, and one control. Analysis of TCR Vβ2 rearrangements was performed on whole blood sample using the IOTest Beta Mark TCR Vβ Repertoire Kit (IOTest, Beckman Coulter, Indianapolis, USA) antibody set. BD FACSCanto TM II Cytometer and Infinicyt TM 2.0 analysis software were used. Patient 1 was considered as negative control (lymphocytes 3.41x10 3 /μL) showed expression of various Vβ2 clones, ranging from 0.05 to 1.40%. Patients 2 and 3, both increased CD8 T-lymphocytes associated with a history of infectious disease, had a polyclonal profile of TCR Vβ2 expression, Patient 4 had lymphocytosis (4.25x10 3 /μl) with majority expression of Vβ5.1 clones on CD4 (10.84%) and CD8 (10.56%) lymphocytes and of Vβ 13.2 clone on CD4 lymphocytes (11.53%). This patient was considered with reactive oligoclonal proliferation. Patient 5 had lymphocytosis with an abnormal CD8 T cell phenotype suggestive of Large Granular T-Cell Leukemia. TCR rearrangement analysis confirmed the presence of the Vβ 13.1 clone in CD8 T-cells. Patient 6 had a large lymphocytosis (462.92x10 3 /μL) with a predominance of CD8 positive T-cells and a phenotype suggestive of Prolymphocytic T Cell Leukemia. TCR rearrangement indicated the majority presence of the Vβ8 clone on CD8 T-cell. And finally, patient 7 had mild lymphocytosis (6.87x103/μL) with a predominance of CD8 T-cells with expression of clone Vβ 4 (8.90%) and Vβ17 (2.01%) and of clone Vβ3 on CD4 cells (6.51%) and was considered as oligoclonal expansion. The confirmation of monoclonal expansion of CD8 T lymphocytes could help in the diagnosis of T-cell neoplasms, confirming the diagnosis of large granular T-cell leukemia and CD8 T-cell prolymphocytic leukemia in two patients. Among the patients with polyclonal or oligoclonal expression, all patients had a history of viral infectious diseases, such as CMV or EBV infection. Patient 7 had a diagnosis of immunodeficiency with oligoclonal T expansion that can be discussed separately. Patients presenting with CD8 positive T lymphocytosis and often nonspecific clinical symptoms may benefit from combined evaluation of phenotype and clonal expression by the Vβ2 repertoire.

Locus specific epigenetic modalities of random allelic expression imbalance

Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives.

Role of KIR Receptor in NK Regulation during Viral Infections

Natural Killer (NK) cells are key effectors of the innate immune system which represent the first line of defense against viral infections. NK cell activation depends on the engagement of a complex receptor repertoire expressed on their surface, consisting of both activating and inhibitory receptors. Among the known NK cell receptors, the family of killer Ig-like receptors (KIRs) consists in activating/inhibitory receptors that interact with specific human leukocyte antigen (HLA) molecules expressed on target cells. In particular, the expression of peculiar KIRs have been reported to be associated to viral infection susceptibility. Interestingly, a significant association between the development and onset of different human pathologies, such as tumors, neurodegeneration and infertility, and a clonal KIRs expression on NK cells has been described in presence of viral infections, supporting the crucial role of KIRs in defining the effect of viral infections in different tissues and organs. This review aims to report the state of art about the role of KIRs receptors in NK cell activation and viral infection control.

Streamlined Human Cell-Based Recombinase-Mediated Cassette Exchange Platform Enables Multigene Expression for the Production of Therapeutic Proteins.

A platform, based on targeted integration of transgenes using recombinase-mediated cassette exchange (RMCE) coupled with CRISPR/Cas9, is increasingly being used for the development of mammalian cell lines that produce therapeutic proteins, because of reduced clonal variation and predictable transgene expression. However, low efficiency of the RMCE process has hampered its application in multicopy or multisite integration of transgenes. To improve RMCE efficiency, nuclear transport of RMCE components such as site-specific recombinase and donor plasmid was accelerated by incorporation of nuclear localization signal and DNA nuclear-targeting sequence, respectively. Consequently, the efficiency of RMCE in dual-landing pad human embryonic kidney 293 (HEK293) cell lines harboring identical or orthogonal pairs of recombination sites at two well-known human safe harbors (AAVS1 and ROSA26 loci), increased 6.7- and 8.1-fold, respectively. This platform with enhanced RMCE efficiency enabled simultaneous integration of transgenes at the two sites using a single transfection without performing selection and enrichment processes. The use of a homotypic dual-landing pad HEK293 cell line capable of incorporating the same transgenes at two sites resulted in a 2-fold increase in the transgene expression level compared to a single-landing pad HEK293 cell line. In addition, the use of a heterotypic dual-landing pad HEK293 cell line, which can incorporate transgenes for a recombinant protein at one site and an effector transgene for cell engineering at another site, increased recombinant protein production. Overall, a streamlined RMCE platform can be a versatile tool for mammalian cell line development by facilitating multigene expression at genomic safe harbors.

IRG-4mC: Neural Network Based Tool for Identification of DNA 4mC Sites in Rosaceae Genome

DNA N4-Methylcytosine is a genetic modification process which has an essential role in changing different biological processes such as DNA conformation, DNA replication, DNA stability, cell development and structural alteration in DNA. Due to its negative effects, it is important to identify the modified 4mC sites. Further, methylcytosine may develop anywhere at cytosine residue, however, clonal gene expression patterns are most likely transmitted just for cytosine residues in strand-symmetrical sequences. For this reason many different experiments are introduced but they proved not to be viable choice due to time limitation and high expenses. Therefore, to date there is still need for an efficient computational method to deal with 4mC sites identification. Keeping it in mind, in this research we have proposed an efficient model for Fragaria vesca (F. vesca) and Rosa chinensis (R. chinensis) genome. The proposed iRG-4mC tool is developed based on neural network architecture with two encoding schemes to identify the 4mC sites. The iRG-4mC predictor outperformed the existing state-of-the-art computational model by an accuracy difference of 9.95% on F. vesca (training dataset), 8.7% on R. chinesis (training dataset), 6.2% on F. vesca (independent dataset) and 10.6% on R. chinesis (independent dataset). We have also established a webserver which is freely accessible for the research community.

Continuous MYD88 Activation Is Associated With Expansion and Then Transformation of IgM Differentiating Plasma Cells.

Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.

Visualization of leukemic B cell clonal dynamics during therapy using tSNE.

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+ CD5+ clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Many patients with CLL never receive treatment for the leukemia and therapy is typically only initiated upon progression of disease-related clinical complications. Treatment options for progressing patients range from chemoimmunotherapy (CIT) to small molecule inhibitors that target pro-survival signaling pathways in leukemic B cells, such as the BTK inhibitor ibrutinib. Although therapy regimens have improved, there is still no cure for CLL, in part due to clonal evolution of the leukemic B cell. The goal of this study is to document the clonal dynamics of CLL B cells at baseline and at response evaluation(s) for alterations in clonal architecture and expression of cell surface markers associated with therapy resistance using the single cell flow cytometry analysis tool, t-distributed stochastic neighbor embedding (t-SNE). Biobanked peripheral blood mononuclear cells were obtained from patients that underwent frontline CIT or ibrutinib, or secondary ibrutinib therapy. Fourteen parameter flow cytometry was performed and subjected to tSNE analysis to document CLL B cell clonal architecture and evolution in patients undergoing the various therapies. Profound differences in treatment responses and leukemic cell clonal dynamics were observed which may inform the discovery of novel biomarkers associated with therapy resistance.

Modulation of Immune Response to Chlamydia muridarum by Host miR-135a.

Previously, our laboratory established the role of small, noncoding RNA species, i.e., microRNA (miRNA) including miR-135a in anti-chlamydial immunity in infected hosts. We report here chlamydial infection results in decreased miR-135a expression in mouse genital tissue and a fibroblast cell line. Several chemokine and chemokine receptor genes (including CXCL10, CCR5) associated with chlamydial pathogenesis were identified in silico to contain putative miR-135a binding sequence(s) in the 3’ untranslated region. The role of miR-135a in the host immune response was investigated using exogenous miR-135a mimic to restore the immune phenotype associated with decreased miR-135a following Chlamydia muridarum (Cm) infection. We observed miR-135a regulation of Cm-primed bone marrow derived dendritic cells (BMDC) via activation of Cm-immune CD4+ T cells for clonal expansion and CCR5 expression. Using a transwell cell migration assay, we explore the role of miR-135a in regulation of genital tract CXCL10 expression and recruitment of CXCR3+ CD4+ T cells via the CXCL10/CXCR3 axis. Collectively, data reported here support miR-135a affecting multiple cellular processes in response to chlamydial infection.

Imaging cell lineage with a synthetic digital recording system.

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.

Single-Antibody Evaluation of T-Cell Receptor β Constant Chain Monotypia by Flow Cytometry Facilitates the Diagnosis of T-Cell Large Granular Lymphocytic Leukemia.

The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL. We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed. Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression. Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

Genetic diversity and characteristics of high-level tigecycline resistance Tet(X) in Acinetobacter species

BackgroundThe recent emergence and dissemination of high-level mobile tigecycline resistance Tet(X) challenge the clinical effectiveness of tigecycline, one of the last-resort therapeutic options for complicated infections caused by multidrug-resistant Gram-negative and Gram-positive pathogens. Although tet(X) has been found in various bacterial species, less is known about phylogeographic distribution and phenotypic variance of different genetic variants.MethodsHerein, we conducted a multiregional whole-genome sequencing study of tet(X)-positive Acinetobacter isolates from human, animal, and their surrounding environmental sources in China. The molecular and enzymatic features of tet(X) variants were characterized by clonal expression, microbial degradation, reverse transcription, and gene transfer experiments, while the tet(X) genetic diversity and molecular evolution were explored by comparative genomic and Bayesian evolutionary analyses.ResultsWe identified 193 tet(X)-positive isolates from 3846 samples, with the prevalence ranging from 2.3 to 25.3% in nine provinces in China. The tet(X) was broadly distributed in 12 Acinetobacter species, including six novel species firstly described here. Besides tet(X3) (n = 188) and tet(X4) (n = 5), two tet(X5) variants, tet(X5.2) (n = 36) and tet(X5.3) (n = 4), were also found together with tet(X3) or tet(X4) but without additive effects on tetracyclines. These tet(X)-positive Acinetobacter spp. isolates exhibited 100% resistance rates to tigecycline and tetracycline, as well as high minimum inhibitory concentrations to eravacycline (2–8 μg/mL) and omadacycline (8–16 μg/mL). Genetic analysis revealed that different tet(X) variants shared an analogous ISCR2-mediated transposon structure. The molecular evolutionary analysis indicated that Tet(X) variants likely shared the same common ancestor with the chromosomal monooxygenases that are found in environmental Flavobacteriaceae bacteria, but sequence divergence suggested separation ~ 9900 years ago (7887 BC), presumably associated with the mobilization of tet(X)-like genes through horizontal transfer.ConclusionsFour tet(X) variants were identified in this study, and they were widely distributed in multiple Acinetobacter spp. strains from various ecological niches across China. Our research also highlighted the crucial role of ISCR2 in mobilizing tet(X)-like genes between different Acinetobacter species and explored the evolutionary history of Tet(X)-like monooxygenases. Further studies are needed to evaluate the clinical impact of these mobile tigecycline resistance genes.

Autosomal Clonal Monoallelic Expression: Natural or Artifactual?

The prevalence of mitotically heritable clonal random monoallelic expression of autosomal genes (aRME) remains controversial. Specifically, presence of clonal aRME is well supported in vitro but remains elusive in vivo. Here, we provide critical insights into this matter and discuss whether prevalent clonal aRME is natural or artifactual.