Cell-specific Activity Research Articles

Abstract P390: The Adipokine Chemerin Does Not Promote Activation or Cytokine Secretion by T Cells Stimulated With a Strong Polyclonal Activator

High fat diet has long been implicated as a risk factor for obesity and other conditions associated with metabolic syndrome, including hypertension. The mechanism by which high fat diet increases risk of hypertension is likely multifactorial, but corresponding low-grade inflammation may play an important role. Low-grade inflammation associated with high fat diet is often accompanied by altered adipokine levels, including an increase in circulating chemerin. However, our understanding of the effects of chemerin and other adipokines on immune cells is limited. Thus, the purpose of these studies was to test the hypothesis that the adipokine chemerin promotes CD4 and CD8 T cell activation and cytokine secretion. To test this hypothesis, primary mouse splenocytes were treated with chemerin or vehicle 30 min prior to activation with a T cell-specific activator (anti-CD3/anti-CD28) for 24 h. T cell activation causes a rapid upregulation of cell surface proteins that are commonly used as experimental markers of activation. We found that chemerin did not impact the induction of the T cell activation markers, CD25, CD69, CD44, CD134, CD137 and CD154, in either CD4 or CD8 T cells. Likewise, we did not see a difference in the downregulation of CD62L, which is rapidly internalized by T cells following activation. Consistent with this, chemerin caused no difference in cytokine secretion by activated T cells. Specifically, the concentrations of IL-2, IL-17A, IFNγ, and TNFα were similar between groups. Overall, this study shows that the adipokine chemerin does not promote acute changes in activation or cytokine expression in primary mouse splenic T cells fully activated with anti-CD3/anti-CD28.

Dual roles of the MPK3 and MPK6 mitogen-activated protein kinases in regulating Arabidopsis stomatal development.

An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)-MKK4/MKK5-MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4DD or MKK5DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development.

HuMSC-EVs Protect Endothelial Cells Against Hypoxia/Reoxygenation Injury by Inhibiting the Pannexin 1/p38-MAPK Pathway

Vascular endothelial cell dysfunction plays an important role in myocardial ischemia–reperfusion (I/R) injury, and pannexin 1 (Panx1), an ATP-permeable channel, is closely associated with the pathophysiological processes of I/R injury. The purpose of this study was to investigate the protective effects of human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (HuMSC-EVs) and the underlying mechanism in a model of I/R injury. For the cellular model of I/R injury, human umbilical vein endothelial cells (HuVECs) were exposed to hypoxia/reoxygenation (H/R) conditions. The model cells were then treated with HuMSC-EVs, and the effects on cell survival and specific signaling activities were observed. The results showed that after H/R exposure, Panx1 expression and other markers of cellular damage were increased in HuVECs. However, treatment with HuMSC-EVs inhibited the H/R-induced increase in Panx1 expression and improved HuVEC survival. Mechanistically, HuMSC-EVs were found to inhibit the p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis pathway, as evidenced by increased Bcl2 expression and reductions in p38 MAPK phosphorylation, Bax expression, and cleaved-caspase 3 expression. Together our data suggest that HuMSC-EVs alleviate H/R-induced apoptosis among HuVECs by inhibiting activity of the Panx1/p38-MAPK-dependent apoptosis pathway.

DIPG-34. IDENTIFICATION OF CAR-T CELL ENHANCING COMPOUNDS USING HIGH THROUGHPUT SCREENING

Abstract BACKGROUND The infiltrative growth patterns and restrictiveness of the blood brain barrier greatly limit therapeutic options for children diagnosed with diffuse intrinsic pontine gliomas (DIPGs). However, clinical trials using chimeric antigen receptor (CAR)-T cells have shown promise in DIPG patients. Despite some success, the biological limitations plaguing CAR-T cells hinder their overall efficacy. We propose that leveraging chemical compounds in tandem with CAR-T cells may provide an increased therapeutic benefit against this lethal brain tumor. Our study is aimed at using high throughput screening of >2,000 FDA approved and investigational compounds to identify candidates that enhance CAR-T cell mediated cytotoxicity of DIPGs. METHODS In an initial limited screen (32 compounds) used for optimization, compounds were serially diluted using the Echo 650 acoustic liquid handler. Subsequent co-culture conditions of renilla luciferase transduced DIPG007 tumor cells and clinically relevant firefly luciferase transduced CAR-T cells were plated at a 2:1 effector: target (E: T) ratio. In comparison to DMSO control, drug induced changes on cell viability were measured using cell-specific luciferase activity at 24 and 72 hours. RESULTS The limited screen revealed the BET bromodomain inhibitor, GSK-046, as an initial target based on its ability to increase CAR-T cell viability after 72 hours. Preliminary validation of these findings supports this CAR-T enhancing role of GSK-046 via MTS assays. We also identified nonlethal concentrations of GSK-046 for DIPG007 cells that could be further tested in combination with CAR-T cells to determine therapeutic synergy. CONCLUSIONS Our initial findings warrant further investigation into the combinatorial benefit of GSK-046 and CAR-T cells against DIPGs. Currently, we are optimizing and expanding our drug screen to identify additional compounds that may complement CAR-T cell therapy. Furthermore, we intend to validate the cytotoxic benefit of our prioritized combinations in additional in vitro and in vivo settings.

Preclinical efficacy and pharmacokinetics of an RNA-encoded T cell-engaging bispecific antibody targeting human claudin 6.

We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)-formulated RNA (RNA-LNP) encoding a T cell-engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell-specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-μg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530).

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.

Colonic epithelial cell-specific TFEB activation: a key mechanism promoting anti-bacterial defense in response to Salmonella infection.

Colitis caused by infections, especially Salmonella, has long been a common disease, underscoring the urgency to understand its intricate pathogenicity in colonic tissues for the development of effective anti-bacterial approaches. Of note, colonic epithelial cells, which form the first line of defense against bacteria, have received less attention, and the cross-talk between epithelial cells and bacteria requires further exploration. In this study, we revealed that the critical anti-bacterial effector, TFEB, was primarily located in colonic epithelial cells rather than macrophages. Salmonella-derived LPS significantly promoted the expression and nuclear translocation of TFEB in colonic epithelial cells by inactivating the mTOR signaling pathway in vitro, and this enhanced nuclear translocation of TFEB was also confirmed in a Salmonella-infected mouse model. Further investigation uncovered that the infection-activated TFEB contributed to the augmentation of anti-bacterial peptide expression without affecting the intact structure of the colonic epithelium or inflammatory cytokine expression. Our findings identify the preferential distribution of TFEB in colonic epithelial cells, where TFEB can be activated by infection to enhance anti-bacterial peptide expression, holding promising implications for the advancement of anti-bacterial therapeutics.

Differential stability of Gcn4p controls its cell-specific activity in differentiated yeast colonies.

Gcn4p belongs to conserved AP-1 transcription factors involved in many cellular processes, including cell proliferation, stress response, and nutrient availability in yeast and mammals. AP-1 activities are regulated at different levels, such as translational activation or protein degradation, which increases the variability of regulation under different conditions. Gcn4p activity in unstructured yeast liquid cultures increases upon amino acid deficiency and is rapidly eliminated upon amino acid excess. Gcn2p kinase is the major described regulator of Gcn4p that enables GCN4 mRNA translation via the uORFs mechanism. Here, we show that Gcn4p is specifically active in U cells in the upper regions and inactive in L cells in the lower regions of differentiated colonies. Using in situ microscopy in combination with analysis of mutants and strains with GFP at different positions in the translational regulatory region of Gcn4p, we show that cell-specific Gcn4p activity is independent of Gcn2p or other translational or transcriptional regulation. Genetically, biochemically, and microscopically, we identified cell-specific proteasomal degradation as a key mechanism that diversifies Gcn4p function between U and L cells. The identified regulation leading to active Gcn4p in U cells with amino acids and efficient degradation in starved L cells differs from known regulations of Gcn4p in yeast but shows similarities to the activity of AP-1 ATF4 in mammals during insulin signaling. These findings may open new avenues for understanding the parallel activities of Gcn4p/ATF4 and reveal a novel biological role for cell type-specific regulation of proteasome-dependent degradation.IMPORTANCEIn nature, microbes usually live in spatially structured communities and differentiate into precisely localized, functionally specialized cells. The coordinated interplay of cells and their response to environmental changes, such as starvation, followed by metabolic adaptation, is critical for the survival of the entire community. Transcription factor Gcn4p is responsible for yeast adaptation under amino acid starvation in liquid cultures, and its activity is regulated mainly at the level of translation involving Gcn2p kinase. Whether Gcn4p functions in structured communities was unknown. We show that translational regulation of Gcn4p plays no role in the development of colony subpopulations; the main regulation occurs at the level of stabilization of the Gcn4p molecule in the cells of one subpopulation and its proteasomal degradation in the other. This regulation ensures specific spatiotemporal activity of Gcn4p in the colony. Our work highlights differences in regulatory networks in unorganized populations and organized structures of yeast, which in many respects resemble multicellular organisms.

From Genome-wide Association Studies to Functional Variants: ARL14 Cis-regulatory Variants Are Associated With Severe Malaria.

Genome-wide association studies have identified several nonfunctional tag single-nucleotide polymorphisms (SNPs) associated with severe malaria. We hypothesized that causal SNPs could play a significant role in severe malaria by altering promoter or enhancer activity. Here, we sought to identify such regulatory SNPs. SNPs in linkage disequilibrium with tagSNPs associated with severe malaria were identified and were further annotated using FUMA. Then, SNPs were prioritized using the integrative weighted scoring method to identify regulatory ones. Gene reporter assays were performed to assess the regulatory effect of a region containing candidates. The association between SNPs and severe malaria was assessed using logistic regression models in a Senegalese cohort. Among 418 SNPs, the best candidates were rs116525449 and rs79644959, which were in full disequilibrium between them, and located within the ARL14 promoter. Our gene reporter assay results revealed that the region containing the SNPs exhibited cell-specific promoter or enhancer activity, while the SNPs influenced promoter activity. We detected an association between severe malaria and those 2 SNPs using the overdominance model and we replicated the association of severe malaria with the tagSNP rs116423146. We suggest that these SNPs regulate ARL14 expression in immune cells and the presentation of antigens to T lymphocytes, thus influencing severe malaria development.

Multiomic profiling of human clonal hematopoiesis reveals genotype and cell-specific inflammatory pathway activation

AbstractClonal hematopoiesis (CH) is an age-associated phenomenon that increases the risk of hematologic malignancy and cardiovascular disease. CH is thought to enhance disease risk through inflammation in the peripheral blood.1 Here, we profile peripheral blood gene expression in 66 968 single cells from a cohort of 17 patients with CH and 7 controls. Using a novel mitochondrial DNA barcoding approach, we were able to identify and separately compare mutant Tet methylcytosine dioxygenase 2 (TET2) and DNA methyltransferase 3A (DNMT3A) cells with nonmutant counterparts. We discovered the vast majority of mutated cells were in the myeloid compartment. Additionally, patients harboring DNMT3A and TET2 CH mutations possessed a proinflammatory profile in CD14+ monocytes through previously unrecognized pathways such as galectin and macrophage inhibitory factor. We also found that T cells from patients with CH, although mostly unmutated, had decreased expression of GTPase of the immunity associated protein genes, which are critical to T-cell development, suggesting that CH impairs T-cell function.

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Activation of GFRAL+ neurons induces hypothermia and glucoregulatory responses associated with nausea and torpor

GFRAL-expressing neurons actuate aversion and nausea, are targets for obesity treatment, and may mediate metformin effects by long-term GDF15-GFRAL agonism. Whether GFRAL+ neurons acutely regulate glucose and energy homeostasis is, however, underexplored. Here, we report that cell-specific activation of GFRAL+ neurons using a variety of techniques causes a torpor-like state, including hypothermia, the release of stress hormones, a shift from glucose to lipid oxidation, and impaired insulin sensitivity, glucose tolerance, and skeletal muscle glucose uptake but augmented glucose uptake in visceral fat. Metabolomic analysis of blood and transcriptomics of muscle and fat indicate alterations in ketogenesis, insulin signaling, adipose tissue differentiation and mitogenesis, and energy fluxes. Our findings indicate that acute GFRAL+ neuron activation induces endocrine and gluco- and thermoregulatory responses associated with nausea and torpor. While chronic activation of GFRAL signaling promotes weight loss in obesity, these results show that acute activation of GFRAL+ neurons causes hypothermia and hyperglycemia.

Hazard and risk characterization of 56 structurally diverse PFAS using a targeted battery of broad coverage assays using six human cell types

Per- and poly-fluoroalkyl substances (PFAS) are extensively used in commerce leading to their prevalence in the environment. Due to their chemical stability, PFAS are considered to be persistent and bioaccumulative; they are frequently detected in both the environment and humans. Because of this, PFAS as a class (composed of hundreds to thousands of chemicals) are contaminants of very high concern. Little information is available for the vast majority of PFAS, and regulatory agencies lack safety data to determine whether exposure limits or restrictions are needed. Cell-based assays are a pragmatic approach to inform decision-makers on potential health hazards; therefore, we hypothesized that a targeted battery of human in vitro assays can be used to determine whether there are structure-bioactivity relationships for PFAS, and to characterize potential risks by comparing bioactivity (points of departure) to exposure estimates. We tested 56 PFAS from 8 structure-based subclasses in concentration response (0.1–100 μM) using six human cell types selected from target organs with suggested adverse effects of PFAS – human induced pluripotent stem cell (iPSC)-derived hepatocytes, neurons, and cardiomyocytes, primary human hepatocytes, endothelial and HepG2 cells. While many compounds were without effect; certain PFAS demonstrated cell-specific activity highlighting the necessity of using a compendium of in vitro models to identify potential hazards. No class-specific groupings were evident except for some chain length- and structure-related trends. In addition, margins of exposure (MOE) were derived using empirical and predicted exposure data. Conservative MOE calculations showed that most tested PFAS had a MOE in the 1–100 range; ∼20% of PFAS had MOE<1, providing tiered priorities for further studies. Overall, we show that a compendium of human cell-based models can be used to derive bioactivity estimates for a range of PFAS, enabling comparisons with human biomonitoring data. Furthermore, we emphasize that establishing structure-bioactivity relationships may be challenging for the tested PFAS.

Evaluation of PDL1 positive cancer cell-specific binding activity of recombinant anti-PDL1 scFv.

Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.

Increased Activity of MAPKAPK2 within Mesenchymal Cells as a Target for Inflammation-Associated Fibrosis in Crohn's Disease.

Mesenchymal stromal cells are suggested to play a critical role in Crohn's disease [CD]-associated fibrosis. MAPKAPK2 [MK2] has emerged as a potential therapeutic target to reduce inflammation in CD. However, the cell-specific pattern of phospho-MK2 activation and its role in CD-associated fibrosis are unknown. The objectives of this study were to evaluate cell-specific changes in MK2 activity between predominantly inflammatory CD vs CD with fibrotic complications and define the role of stromal cell-specific MK2 activation in CD-associated fibrosis. CD tissue, CD tissue-derived mesenchymal stromal cells known as myo-/fibroblasts [CD-MFs], and fibroblast-specific MK2 conditional knockout [KO] mice were used. In the inflamed area of predominantly inflammatory CD, high MK2 activity was equally distributed between mesenchymal and haematopoietic cells. By contrast, in CD with fibrotic complications, high MK2 activity was mostly associated with mesenchymal stromal cells. Using ex vivo CD tissue explants and an IL-10KO murine colitis model, we demonstrated that pro-fibrotic responses are significantly reduced by treatment with the MK2 inhibitor PF-3644022. Inhibition of MK2 activity in primary cultures of CD-MFs significantly reduced basal and TGF-β1-induced profibrotic responses. Using fibroblast-specific MK2 knockout mice in chronic dextran saline sulphate colitis, we demonstrated that fibroblast intrinsic MK2 signalling is among the key processes involved in the chronic inflammation-induced profibrotic responses. Our data suggest that activation of MK2 within fibroblasts contributes to the chronic inflammation-induced fibrosis in CD and that targeting MK2 has potential for the development of novel therapeutic approaches for fibrosis in CD.

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

BackgroundDespite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.ResultsWe examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.ConclusionsOur study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

Sertoli Cell-Specific Activation of Transforming Growth Factor Beta Receptor 1 Leads to Testicular Granulosa Cell Tumor Formation.

The transforming growth factor β (TGFβ) superfamily, consisting of protein ligands, receptors, and intracellular SMAD transducers, regulates fundamental biological processes and cancer development. Our previous study has shown that sustained activation of TGFβ receptor 1 (TGFBR1) driven by anti-Mullerian hormone receptor type 2 (Amhr2)-Cre in the mouse testis induces the formation of testicular granulosa cell tumors (TGCTs). As Amhr2-Cre is expressed in both Sertoli cells and Leydig cells, it remains unclear whether the activation of TGFBR1 in Sertoli cells alone is sufficient to induce TGCT formation. Therefore, the objective of this study was to determine whether Sertoli cell-activation of TGFBR1 drives oncogenesis in the testis. Our hypothesis was that overactivation of TGFBR1 in Sertoli cells would promote their transdifferentiation into granulosa-like cells and the formation of TGCTs. To test this hypothesis, we generated mice harboring constitutive activation of TGFBR1 in Sertoli cells using anti-Mullerian hormone (Amh)-Cre. Disorganized seminiferous tubules and tumor nodules were found in TGFBR1CA; Amh-Cre mice. A histological analysis showed that Sertoli cell-specific activation of TGFBR1 led to the development of neoplasms resembling granulosa cell tumors, which derailed spermatogenesis. Moreover, TGCTs expressed granulosa cell markers including FOXL2, FOXO1, and INHA. Using a dual fluorescence reporter line, the membrane-targeted tdTomato (mT)/membrane-targeted EGFP (mG) mouse, we provided evidence that Sertoli cells transdifferentiated toward a granulosa cell fate during tumorigenesis. Thus, our findings indicate that Sertoli cell-specific activation of TGFBR1 leads to the formation of TGCTs, supporting a key contribution of Sertoli cell reprogramming to the development of this testicular malignancy in our model.

Nutrient-dependent interactions between a marine copiotroph Alteromonas and a diatom Thalassiosira pseudonana.

As the major producers and consumers, phytoplankton and bacteria play central roles in marine ecosystems and their interactions show great ecological significance. Whether mutualistic or antagonistic, the interaction between certain phytoplankton and bacterial species is usually seen as a derivative of intrinsic physiological properties and rarely changes. This study demonstrated that the interactions between the ubiquitously co-occurring bacteria and diatom, Alteromonas and Thalassiosira pseudonana, varied with nutrient conditions. They overcame hardship together in oligotrophic seawater but showed antagonistic effects against each other under nutrient amendment. The contact-dependent algicidal behavior of Alteromonas based on protease activity solved the paradox among bacterial proliferation, nutrient viability, and algal demise haunting other known non-contact-dependent algicidal processes and might actually trigger the collapse of algal blooms in situ. The chemotactic and swarming movement of Alteromonas might also contribute greatly to the breakdown of "marine snow," which could redirect the carbon sequestration pathway in the ocean.

Single cell dynamics and nitrogen transformations in the chain forming diatom Chaetoceros affinis

Colony formation in phytoplankton is often considered a disadvantage during nutrient limitation in aquatic systems. Using stable isotopic tracers combined with secondary ion mass spectrometry (SIMS), we unravel cell-specific activities of a chain-forming diatom and interactions with attached bacteria. The uptake of 13C-bicarbonate and15N-nitrate or 15N-ammonium was studied in Chaetoceros affinis during the stationary growth phase. Low cell-to-cell variance of 13C-bicarbonate and 15N-nitrate assimilation within diatom chains prevailed during the early stationary phase. Up to 5% of freshly assimilated 13C and 15N was detected in attached bacteria within 12 h and supported bacterial C- and N-growth rates up to 0.026 h−1. During the mid-stationary phase, diatom chain-length decreased and 13C and 15N-nitrate assimilation was significantly higher in solitary cells as compared to that in chain cells. During the late stationary phase, nitrate assimilation ceased and ammonium assimilation balanced C fixation. At this stage, we observed highly active cells neighboring inactive cells within the same chain. In N-limited regimes, bacterial remineralization of N and the short diffusion distance between neighbors in chains may support surviving cells. This combination of “microbial gardening” and nutrient transfer within diatom chains represents a strategy which challenges current paradigms of nutrient fluxes in plankton communities.

Mapping the cellular and molecular landscape of cardiac non-myocytes in murine diabetic cardiomyopathy

Diabetes is associated with a significantly elevated risk of heart failure. However, despite extensive efforts to characterize the phenotype of the diabetic heart, the molecular and cellular protagonists that underpin cardiac pathological remodeling in diabetes remain unclear, with a notable paucity of data regarding the impact of diabetes on non-myocytes within the heart. Here we aimed to define key differences in cardiac non-myocytes between spontaneously type-2 diabetic (db/db) and healthy control (db/h) mouse hearts. Single-cell transcriptomic analysis revealed a concerted diabetes-induced cellular response contributing to cardiac remodeling. These included cell-specific activation of gene programs relating to fibroblast hyperplasia and cell migration, and dysregulation of pathways involving vascular homeostasis and protein folding. This work offers a new perspective for understanding the cellular mediators of diabetes-induced cardiac pathology, and pathways that may be targeted to address the cardiac complications associated with diabetes.