Search

We describe the use of Bayesian analysis methods applied to time-of-flight secondary ion mass spectrometer (TOF-SIMS) spectra. The method is applied to the COmetary Secondary Ion Mass Analyzer (COSIMA) TOF-SIMS mass spectra where the analysis can be broken into subgroups of lines close to integer mass values. The effects of the instrumental dead time are discussed in a new way. The method finds the joint probability density functions of measured line parameters (number of lines, and their widths, peak amplitudes, integrated amplitudes and positions). In the case of two or more lines, these distributions can take complex forms. The derived line parameters can be used to further calibrate the mass scaling of TOF-SIMS and to feed the results into other analysis methods such as multivariate analyses of spectra. We intend to use the method, first as a comprehensive tool to perform quantitative analysis of spectra, and second as a fast tool for studying interesting targets for obtaining additional TOF-SIMS measurements of the sample, a property unique to COSIMA. Finally, we point out that the Bayesian method can be thought of as a means to solve inverse problems but with forward calculations, only with no iterative corrections or other manipulation of the observed data. [ABSTRACT FROM AUTHOR]

Human reproductive success relies on the proper differentiation of the uterine endometrium to facilitate implantation, formation of the placenta, and pregnancy. This process involves two critical types of decidual uterine cells: endometrial/decidual stromal cells (dS) and uterine/decidual natural killer (dNK) cells. To better understand the transcription factors governing the in vivo functions of these cells, we analyzed single-cell transcriptomics data from first-trimester terminations of pregnancy, and for the first time conducted gene regulatory network analysis of dS and dNK cell subpopulations. Our analysis revealed stromal cell populations that corresponded to previously described in vitro decidualized cells and senescent decidual cells. We discovered new decidualization driving transcription factors of stromal cells for early pregnancy, including DDIT3 and BRF2, which regulate oxidative stress protection. For dNK cells, we identified transcription factors involved in the immunotolerant (dNK1) subpopulation, including IRX3 and RELB, which repress the NFKB pathway. In contrast, for the less immunotolerant (dNK3) population we predicted TBX21 (T-bet) and IRF2-mediated upregulation of the interferon pathway. To determine the clinical relevance of our findings, we tested the overrepresentation of the predicted transcription factors target genes among cell type-specific regulated genes from pregnancy disorders, such as recurrent pregnancy loss and preeclampsia. We observed that the predicted decidualized stromal and dNK1-specific transcription factor target genes were enriched with the genes downregulated in pregnancy disorders, whereas the predicted dNK3-specific targets were enriched with genes upregulated in pregnancy disorders. Our findings emphasize the importance of stress tolerance pathways in stromal cell decidualization and immunotolerance promoting regulators in dNK differentiation., (© 2024. The Author(s).)

Lyme borreliosis is a disease caused by Borrelia burgdorferi sensu lato bacteria. Borrelia burgdorferi is known to induce prolonged extrafollicular immune responses and abnormal germinal centre formation. The infection fails to generate a neutralizing type of immunity, eventually establishing a persistent infection. Here, we performed single-cell RNA sequencing to characterize the immune landscape of lymph node lymphocytes during the early Borrelia burgdorferi infection in a murine model. Our results indicate key features of an extrafollicular immune response four days after Borrelia burgdorferi infection, including notable B cell proliferation, immunoglobulin class switching to IgG3 and IgG2b isotypes, plasmablast differentiation, and the presence of extrafollicular B cells identified through immunohistochemistry. Additionally, we found infection-derived upregulation of suppressor of cytokine signalling genes Socs1 and Socs3, along with downregulation of genes associated with MHC II antigen presentation in B cells. Our results support the central role of B cells in the immune response of a Borrelia burgdorferi infection, and provide cues of mechanisms behind the determination between extrafollicular and germinal centre responses during Borrelia burgdorferi infection., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Various single-stranded and hairpin-forming DNA and 2'-O-methyl-RNA oligonucleotides bearing a single (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue esterified from either O1 and O2 or O1 and O3 were synthesized. Incubation of these oligonucleotides with equimolar mixtures of formylmethyl derivatives of the canonical nucleobases and 2-methylbenzimidazole under mildly acidic conditions revealed base-filling of the modified site to be strongly favored by base stacking of a double-helix, especially an A-type one. In 2'-O-methyl-RNA hairpin oligonucleotides, base-filling of the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue with nucleobase aldehydes followed the rules of Watson-Crick base pairing, thymine being the only exception. In single-stranded oligonucleotides or the Hoogsteen strand of triple helices, both the yield and selectivity of base-filling were much more modest., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Base-filling, i. e., post-synthetic furnishing of an oligonucleotide scaffold with base moieties or their analogues, is an interesting alternative to the conventional approach of sequential coupling of building blocks (modified or otherwise). Reversible attachment of the base moieties is particularly attractive as it allows the use of dynamic combinatorial chemistry and usually leads to higher fidelity. This concept article summarizes the various backbones and coupling reactions used for base-filling over the past fifteen years, discusses the impact of base stacking and pairing on efficiency and fidelity and highlights potential and realized applications., (© 2024 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Background: Immune-mediated arthritis is a group of autoinflammatory diseases, where the patient's own immune system attacks and destroys synovial joints. Sustained remission is not always achieved with available immunosuppressive treatments, warranting more detailed studies of T cell responses that perpetuate synovial inflammation in treatment-refractory patients., Methods: In this study, we investigated CD4 + and CD8 + T lymphocytes from the synovial tissue and peripheral blood of patients with treatment-resistant immune-mediated arthritis using paired single-cell RNA and TCR-sequencing. To gain insights into the trafficking of clonal families, we compared the phenotypes of clones with the exact same TCRß amino acid sequence between the two tissues., Results: Our results show that both CD4 + and CD8 + T cells display a more activated and inflamed phenotype in the synovial tissue compared to peripheral blood both at the population level and within individual T cell families. Furthermore, we found that both cell subtypes exhibited clonal expansion in the synovial tissue., Conclusions: Our findings suggest that the local environment in the synovium drives the proliferation of activated cytotoxic T cells, and both CD4 + and CD8 + T cells may contribute to tissue destruction and disease pathogenesis., (© 2024. The Author(s).)

To improve their properties or to introduce entirely new functionalities, the intriguing scaffolds of nucleic acids have been decorated with various modifications, most recently also organometallic ones. While challenging to introduce, organometallic modifications offer the potential of expanding the field of application of metal-dependent functionalities to metal-deficient conditions, notably those of biological media. So far, organometallic moieties have been utilized as probes, labels and catalysts. This Feature Article summarizes recent efforts and predicts likely future developments in each of these lines of research.

The serine/threonine-specific Moloney murine leukemia virus (PIM) kinase family (i.e., PIM1, PIM2, and PIM3) has been extensively studied in tumorigenesis. PIM kinases are downstream of several cytokine signaling pathways that drive immune-mediated diseases. Uncontrolled T helper 17 (Th17) cell activation has been associated with the pathogenesis of autoimmunity. However, the detailed molecular function of PIMs in human Th17 cell regulation has yet to be studied. In the present study, we comprehensively investigated how the three PIMs simultaneously alter transcriptional gene regulation during early human Th17 cell differentiation. By combining PIM triple knockdown with bulk and scRNA-seq approaches, we found that PIM deficiency promotes the early expression of key Th17-related genes while suppressing Th1-lineage genes. Further, PIMs modulate Th cell signaling, potentially via STAT1 and STAT3. Overall, our study highlights the inhibitory role of PIMs in human Th17 cell differentiation, thereby suggesting their association with autoimmune phenotypes., Competing Interests: Declaration of interests A.M. is a co-founder of Arsenal Biosciences, Spotlight Therapeutics, and Survey Genomics; serves on the boards of directors at Spotlight Therapeutics and Survey Genomics; is a board observer (and former member of the board of directors) at Arsenal Biosciences; is a member of the scientific advisory boards of Arsenal Biosciences, Spotlight Therapeutics, Survey Genomics, NewLimit, Amgen, and Tenaya; owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma, and Tenaya; and has received fees from Arsenal Biosciences, Spotlight Therapeutics, NewLimit, 23andMe, PACT Pharma, Juno Therapeutics, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein, and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Two oligonucleotide conjugates sharing the same sequence but incorporating a different 5'-terminal organometallic moiety were synthesized, by either direct mercuration in solution or oximation with an organomercury aldehyde on solid support. The potential of these conjugates to serve as new type of artificial ribonucleases was tested with a complementary 2´-O-methyl-RNA target sequence featuring a single cleavable RNA phosphodiester linkage. Both organomercury oligonucleotides greatly outperformed their metal-free counterparts as well as the previously reported small molecule organomercury RNA cleaving agent in catalytic activity, providing an important proof-of-concept. Compared to state-of-the-art metal-dependent artificial ribonucleases, however, the observed activity was modest., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR + ) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Hairpin oligodeoxynucleotides incorporating a (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue in the middle of the double-helical stem and opposite to either one of the canonical nucleobases or an abasic 2-(hydroxymethyl)tetrahydrofuran-3-ol spacer were synthesized. Under mildly acidic conditions, aromatic aldehydes reacted reversibly with these oligonucleotides, converting the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol unit into a 2-aryl-N-methoxy-1,3-oxazinane nucleoside analogue. The equilibrium of this reaction was found to be dependent on both the aldehyde and the nucleobase opposite to the modified residue. 9-Formyl-9-deazaadenine, combining a large stacking surface with an array of hydrogen bond donors and acceptors, showed the highest affinity as well as selectivity consistent with the rules of Watson-Crick base pairing. 5-Formyluracil or indole-3-carbaldehyde, lacking in either stacking or hydrogen bonding ability, were incorporated with a much lower affinity and selectivity., (© 2023The Authors. Published by Wiley-VCH GmbH.)

Coronary artery disease (CAD) is a pandemic disease where up to half of the risk is explained by genetic factors. Advanced insights into the genetic basis of CAD require deeper understanding of the contributions of different cell types, molecular pathways, and genes to disease heritability. Here, we investigate the biological diversity of atherosclerosis-associated cell states and interrogate their contribution to the genetic risk of CAD by using single-cell and bulk RNA sequencing (RNA-seq) of mouse and human lesions. We identified 12 disease-associated cell states that we characterized further by gene set functional profiling, ligand-receptor prediction, and transcription factor inference. Importantly, Vcam1+ smooth muscle cell state genes contributed most to SNP-based heritability of CAD. In line with this, genetic variants near smooth muscle cell state genes and regulatory elements explained the largest fraction of CAD-risk variance between individuals. Using this information for variant prioritization, we derived a hybrid polygenic risk score (PRS) that demonstrated improved performance over a classical PRS. Our results provide insights into the biological mechanisms associated with CAD risk, which could make a promising contribution to precision medicine and tailored therapeutic interventions in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

BackgroundRelatlimab plus nivolumab (anti-lymphocyte-activation gene 3 plus anti-programmed death 1 [anti-LAG-3+anti-PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown.MethodsWe evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy-refractory patients with metastatic melanoma treated with anti-LAG-3+anti-PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-Seq) combined with other multiomics profiling.ResultsThe highest LAG3 expression was noted in NK cells, Tregs, and CD8+ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3+ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8+ T cell clones gained a more cytotoxic and NK-like phenotype.ConclusionAnti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells.Trial registrationClinicalTrials.gov (NCT01968109)FundingCancer Foundation Finland, Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Relander Foundation, State funding for university-level health research in Finland, a Helsinki Institute of Life Sciences Fellow grant, Academy of Finland (grant numbers 314442, 311081, 335432, and 335436), and an investigator-initiated research grant from BMS.

Short oligonucleotides incorporating either 1-mercuri-6-phenylcarbazole, 8-mercuri-6-phenylcarbazole, or 1,8-dimercuri-6-phenylcarbazole C-nucleoside in the middle of the chain have been synthesized and studied for their potential as hybridization probes for sequences containing thiopyrimidine nucleobases. All of these oligonucleotides formed very stable duplexes with complementary sequences pairing the organometallic moiety with either 2- or 4-thiothymine. The isomeric monomercurated oligonucleotides were also able to discriminate between 2- and 4-thiothymine based on the different melting temperatures of the respective duplexes. DFT-optimized structures of the most stable mononuclear Hg II -mediated base pairs featured a coordinated covalent bond between Hg II and either S2 or S4 and a hydrogen bond between the carbazole nitrogen and N3. The dinuclear Hg II -mediated base pairs, in turn, were geometrically very similar to the one previously reported to form between 1,8-dimercuri-6-phenylcarbazole and thymine and had one Hg II ion coordinated to a thio and the other one to an oxo substituent., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

In Brief: Preeclampsia is a common serious disorder that can occur during pregnancy. This study uses integrative analysis of preeclampsia transcriptomes and single-cell transcriptomes to predict cell type-specific contributions to preeclampsia., Abstract: Preeclampsia is a devastating pregnancy disorder and a major cause of maternal and perinatal mortality. By combining previous transcriptomic results on preeclampsia with single-cell sequencing data, we here predict distinct and partly unanticipated contributions of decidual stromal cells and uterine natural killer cells in early- and late-onset preeclampsia.

Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCRαβ-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders., (© 2022. The Author(s).)

(2 R ,3 S )-4-(Methoxyamino)butane-1,2,3-triol was converted into a protected phosphoramidite building block and incorporated into the middle of a short DNA oligonucleotide. O1 and O3 of the (2 R ,3 S )-4-(methoxyamino)butane-1,2,3-triol were engaged in phosphodiester linkages, leaving O2 and the methoxyamino function available to form an N -methoxy-1,3-oxazinane ring through reaction with an aldehyde. In modified oligonucleotides thus obtained, the oxazinane ring formally replaces the furanose ring and the aldehyde, the base moiety of natural nucleosides. The feasibility of synthesizing base-modified oligonucleotides by this approach was demonstrated with several aromatic and aliphatic aldehydes featuring various functional groups.

Background: Allergen-specific type 2 CD4 + T H 2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the T H 2 response has only recently been appreciated., Objective: We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4 + T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy., Methods: Dog allergen-specific memory CD4 + T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing., Results: Dog allergen-specific T-cell responses in allergic subjects were dominantly of T H 2 type. T H 2 cells could be phenotypically further divided into 3 subsets, which consisted of T H 2-like (CCR6 - CXCR3 - CRTH2 - ), T H 2 (CCR6 - CXCR3 - CRTH2 + CD161 - ), and T H 2A (CCR6 - CXCR3 - CRTH2 + CD161 + CD27 - ) cells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the T H 2-biased signature in allergen-specific T cells from allergic subjects and revealed a T H 1/T H 17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between T H 2-like, T H 2, and T H 2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype., Conclusions: Our study demonstrates heterogeneity within allergen-specific T H 2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

T cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of mature, clonally expanded T cells, where somatic-activating STAT3 mutations are common. Although T-LGLL has been described as a chronic T cell response to an antigen, the function of the non-leukemic immune system in this response is largely uncharacterized. Here, by utilizing single-cell RNA and T cell receptor profiling (scRNA+TCRαβ-seq), we show that irrespective of STAT3 mutation status, T-LGLL clonotypes are more cytotoxic and exhausted than healthy reactive clonotypes. In addition, T-LGLL clonotypes show more active cell communication than reactive clones with non-leukemic immune cells via costimulatory cell-cell interactions, monocyte-secreted proinflammatory cytokines, and T-LGLL-clone-secreted IFNγ. Besides the leukemic repertoire, the non-leukemic T cell repertoire in T-LGLL is also more mature, cytotoxic, and clonally restricted than in other cancers and autoimmune disorders. Finally, 72% of the leukemic T-LGLL clonotypes share T cell receptor similarities with their non-leukemic repertoire, linking the leukemic and non-leukemic repertoires together via possible common target antigens. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clonotype., (© 2022. The Author(s).)

A C-nucleoside derivative of phenylpyridine or the respective palladacycle was incorporated at either 3'- or 5'-terminus of a short oligodeoxynucleotide. Hybridization properties of these modified oligonucleotides were studied in a fluorescence-based competition assay in addition to conventional UV melting temperature analysis and compared with those of a previously prepared analogue featuring the modified nucleoside in the middle of the sequence. With the unpalladated phenylpyridine oligonucleotides, UV melting temperature qualitatively correlated with the ability to displace a strand from a double helix in the competition assay, decreasing in the order 5' > 3' > middle. Corresponding results on the palladacyclic oligonucleotides were more difficult to interpret but both UV melting and competition experiments revealed a decrease in the duplex stability upon palladation in most cases. On the other hand, dependence of the UV melting temperature on the identity of the canonical nucleobase opposite to the modified nucleobase analogue was much more pronounced with the palladacyclic duplexes than with their unpalladated counterparts. Furthermore, UV melting profiles of the palladacyclic duplexes featured an additional transition at a temperature exceeding the melting temperature of the unmodified part of the duplex. Taken together, these results lend support to the idea of Pd(II)-mediated base pairs that are highly stable but incompatible with the geometry of a double helix., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell-targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8 + T-cell-mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1-targeting antibodies for cancer treatment., Patients and Methods: In this study, we analyzed the mode of action of a humanized IgG4 anti-Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer ( n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305-induced systemic immune activation in patients with cancer., Results: Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase-mediated endosomal acidification and improved the ability of macrophages to activate CD8 + T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients., Conclusions: Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral proinflammatory responses in patients with metastatic cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

[Figure: see text].

A water-soluble arylmercury complex has been synthesized, and its ability to catalyze the cleavage of the phosphodiester linkage of the RNA model compound adenylyl-3',5'-(2',3'-O-methyleneadenosine) has been assessed over a pH range of 3-8.5 and a catalyst concentration range of 0-7 mM. In the presence of 1 mM catalyst, the observed pH-rate profile featured a new pH-independent region between pH 6 and 7, the catalyzed reaction being as much as eight times faster than the background reaction. At pH 7, the acceleration increased linearly from three- to 17-fold upon increasing the catalyst concentration from 1 to 7 mM. The linear dependence indicates a relatively low affinity of the catalyst for the substrate and, hence, the potential for considerable improvement on tethering to an appropriate targeting group, such as an oligonucleotide., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Synthetic efforts towards nucleosides, nucleotides, oligonucleotides and nucleic acids covalently mercurated at one or more of their base moieties are summarized, followed by a discussion of the proposed, realized and abandoned applications of this unique class of compounds. Special emphasis is given to fields in which active research is ongoing, notably the use of Hg II -mediated base pairing to improve the hybridization properties of oligonucleotide probes. Finally, this minireview attempts to anticipate potential future applications of organomercury nucleic acids., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Objective: Adipose tissue-derived stem cells (ASCs) might play an important role in adipose microenvironment remodelling during tissue expansion through their response to hypoxia. We examined the cytokine profiles of hypoxic visceral ASCs (hypox-visASCs) from subjects with different metabolic risk, the interactions between cytokines as well as the impact of TNFα-induced death in the behavior of surviving hypoxic subcutaneous ASCs (hypox-subASCs) both at bulk population and single-cell level., Materials/methods: Visceral adipose tissue was processed to isolate the ASCs from 33 subjects grouped into normal weight, obese with and without metabolic syndrome. Multiplex assay was used to simultaneously measure multiple inflammatory, anti-inflammatory and angiogenic cytokines in hypox-visASCs from these patients and to elucidate cytokine profiles of hypox-subASCs upon stimulation with IL1β or TNFα and after TNFα-induced death. qPCR and single-cell RNA-sequencing were also performed to elucidate transcriptional impact in surviving hypox-subASCs after TNFα-induced apoptosis., Results: Hypox-visASCs from subjects without metabolic syndrome showed greater secretion levels of inflammatory, anti-inflammatory and angiogenic cytokines compared with those from patients with metabolic syndrome. While IL-1β stimulation was sufficient to increase the secretion levels of these cytokines in hypox-subASCs, TNFα-induced apoptosis also increased their levels and impacted on the expression levels of extracellular matrix proteins, acetyl-CoA producing enzymes and redox-balance proteins in surviving hypox-subASCs. TNFα-induced apoptosis under different glucose concentrations caused selective impoverishment of cell clusters and differentially influenced gene expression profiles of surviving hypox-subASCs., Conclusions: Immunoregulatory and angiogenic functions of hypox-visASCs from patients with metabolic syndrome could be insufficient to promote healthy adipose tissue expansion. TNFα-induced apoptosis may impact on functionality of hypox-subASC populations, whose differential metabolic sensitivity to death could serve to manipulate individual populations selectively in order to elucidate their role in shaping adipose heterogeneity and treating metabolic disorders., Competing Interests: Declaration of competing interest No potential conflicts of interest relevant to this article were reported., (Copyright © 2020. Published by Elsevier Inc.)

A highly nucleobase-discriminating metalated nucleoside analogue, 3-fluoro-2-mercuri-6-methylaniline, was incorporated into an oligonucleotide molecular beacon. Fluorescence emission spectra were measured after the addition of four different complementary strands, in which the nucleobase opposite the metalated analogue varies. The fluorescence results showed a clear binding selectivity at room temperature, in the order G>T>C>A. The selectivity is based on the different affinities between the metalated nucleoside analogue and the canonical nucleobases. The synthesized probe is capable of robust discrimination between the two purine as well as the two pyrimidine bases by fluorescence at room temperature, and more sophisticated temperature analysis allows clear separation of every canonical nucleobase. The probe would, hence, be a suitable method for the detection of single nucleotide polymorphisms., (© 2020 Wiley-VCH GmbH.)

The dynamics of CD4 + T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4 + T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (T H 1) and follicular helper T (T FH ) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T H 1 and T FH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T FH and central memory were revealed, with antimalarials modulating these responses and boosting T H 1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4 + T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).

Background: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention., Methods: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion., Results: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq., Conclusions: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients' repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients., (Copyright © 2020 Kelkka, Savola, Bhattacharya, Huuhtanen, Lönnberg, Kankainen, Paalanen, Tyster, Lepistö, Ellonen, Smolander, Eldfors, Yadav, Khan, Koivuniemi, Sjöwall, Elo, Lähdesmäki, Maeda, Nishikawa, Leirisalo-Repo, Sokka-Isler and Mustjoki.)

Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed., Objective: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis., Methods and Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 + and CD8 + T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells., Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

Oligonucleotides incorporating a central C-nucleoside with either a rigid or flexible benzaldoxime base moiety have been synthesized, and the hybridization properties of their metallacyclic derivatives have been studied by UV melting experiments. In all cases, the metallated duplexes were less stable than their unmetallated counterparts, and the metallacyclic nucleobases did not show a clear preference for any of the canonical nucleobases as a base-pairing partner. With palladated oligonucleotides, increased flexibility translated to less severe destabilization, whereas the opposite was true for the mercurated oligonucleotides; this reflects the greater difficulties in accommodating a rigid Pd II -mediated base pair than a rigid Hg II -mediated base pair within the base stack of a double helix., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4 + T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4 + T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.

A C-nucleoside with 6-phenyl-1H-carbazole as the base moiety has been synthesized and incorporated in the middle of an oligonucleotide. Mercuration of this modified residue at positions 1 and 8 gave the first example of an oligonucleotide featuring a monofacial dinuclear organometallic nucleobase. The dimercurated oligonucleotide formed stable duplexes with unmodified oligonucleotides placing either cytosine, guanine, or thymine opposite to the organometallic nucleobase. A highly stabilizing (ΔT m =7.3 °C) Hg II -mediated base pair was formed with thymine. According to DFT calculations performed at the PBE0DH level of theory, this base pair is most likely dinuclear, with the two Hg II ions coordinated to O2 and O4 of the thymine base., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transcriptome analysis at a single-cell level with single-cell RNA sequencing (scRNA-seq) is a powerful method for detailed characterization of heterogeneous cell populations. Recent developments have enabled parallel analysis of both transcript and protein levels by using antibodies conjugated to barcoded oligonucleotides. These antibodies enable protein levels to be converted into nucleotide format, allowing the sequencing-based detection of both modalities at single-cell level. Here we present a simple and reliable method for conjugation of oligonucleotides with antibodies and a protocol for their use in single-cell transcriptome sequencing.

The appearance of type 1 diabetes (T1D)-associated autoantibodies is the first and only measurable parameter to predict progression toward T1D in genetically susceptible individuals. However, autoantibodies indicate an active autoimmune reaction, wherein the immune tolerance is already broken. Therefore, there is a clear and urgent need for new biomarkers that predict the onset of the autoimmune reaction preceding autoantibody positivity or reflect progressive β-cell destruction. Here we report the mRNA sequencing-based analysis of 306 samples including fractionated samples of CD4 + and CD8 + T cells as well as CD4 - CD8 - cell fractions and unfractionated peripheral blood mononuclear cell samples longitudinally collected from seven children who developed β-cell autoimmunity (case subjects) at a young age and matched control subjects. We identified transcripts, including interleukin 32 ( IL32 ), that were upregulated before T1D-associated autoantibodies appeared. Single-cell RNA sequencing studies revealed that high IL32 in case samples was contributed mainly by activated T cells and NK cells. Further, we showed that IL32 expression can be induced by a virus and cytokines in pancreatic islets and β-cells, respectively. The results provide a basis for early detection of aberrations in the immune system function before T1D and suggest a potential role for IL32 in the pathogenesis of T1D., (© 2019 by the American Diabetes Association.)