Distinct Functional Activities Research Articles

Exploring tumor endothelial cells heterogeneity in hepatocellular carcinoma: insights from single-cell sequencing and pseudotime analysis.

This study aimed to explore the heterogeneity of tumor endothelial cells (TECs) in hepatocellular carcinoma (HCC) and their role in tumor progression, with the goal of identifying new therapeutic targets and strategies to improve patient prognosis. Single-cell RNA sequencing data from nine primary liver cancer samples were analyzed, obtained from the Gene Expression Omnibus (GEO) database. Data preprocessing, normalization, dimensionality reduction, and batch effect correction were performed based on the Seurat package. HCC cell types were identified using uniform manifold approximation and projection (UMAP) and cluster analysis, and the different cell types were annotated using the CellMarker database. Pseudotime trajectory analysis was conducted with Monocle to explore the differentiation trajectory of TECs. MAPK signaling pathway activity and copy number variations (CNV) in TECs were analyzed in conjunction with data from The Cancer Genome Atlas (TCGA), the trans-well and wound healing assay was used for cell invasion and migration activity assessment. Two subgroups of TECs (TECs 1 and TECs 2) were identified, exhibiting distinct functional activities and signaling pathways. Specifically, TECs 1 may be involved in tumor cell proliferation and inflammatory responses, whereas TECs 2 is not only involved in cell proliferation pathways, but also enriched in pathways such as metabolic synthesis. Pseudotime analysis revealed dynamic changes in TECs subgroups during HCC progression, correlating specific gene expressions (such as PDGFRB, PGF, JUN, and NR4A1). Subsequently, the JUN gene was predicted by performing binding sites and was shown to act as a transcription factor that may regulate the expression of the PGF gene. CNV analysis highlighted key genes and pathways in TECs that might influence HCC progression, and the PGF as key regulatory factor mediated cell proliferation and migration. The study revealed the heterogeneity of TECs in HCC and their potential roles in tumor progression, offering new perspectives and potential therapeutic targets for HCC molecular mechanisms. The findings emphasize the importance of further exploring TECs heterogeneity for understanding HCC pathogenesis and developing personalized treatment strategies.

Effects of MhMYB1 and MhMYB2 transcription factors on the monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq.

Transcription factors MhMYB1 and MhMYB2 correlate with monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq, which could affect the contents of ( -)-menthol and ( -)-menthone. Mentha haplocalyx Briq., a plant with traditional medicinal and edible uses, is renowned for its rich essential oil content. The distinct functional activities and aromatic flavors of mint essential oils arise from various chemotypes. While the biosynthetic pathways of the main monoterpenes in mint are well understood, the regulatory mechanisms governing different chemotypes remain inadequately explored. In this investigation, we identified and cloned two transcription factor genes from the M. haplocalyx MYB family, namely MhMYB1 (PP236792) and MhMYB2 (PP236793), previously identified by our research group. Bioinformatics analysis revealed that MhMYB1 possesses two conserved MYB domains, while MhMYB2 contains a conserved SANT domain. Yeast one-hybrid (Y1H) analysis results demonstrated that both MhMYB1 and MhMYB2 interacted with the promoter regions of MhMD and MhPR, critical enzymes in the monoterpenoid biosynthesis pathway of M. haplocalyx. Subsequent virus-induced gene silencing (VIGS) of MhMYB1 and MhMYB2 led to a significant reduction (P < 0.01) in the relative expression levels of MhMD and MhPR genes in the VIGS groups of M. haplocalyx. In addition, there was a noteworthy decrease (P < 0.05) in the contents of ( -)-menthol and ( -)-menthone in the essential oil of M. haplocalyx. These findings suggest that MhMYB1 and MhMYB2 transcription factors play a positive regulatory role in ( -)-menthol biosynthesis, consequently influencing the essential oil composition in the l-menthol chemotype of M. haplocalyx. This study serves as a pivotal foundation for unraveling the regulatory mechanisms governing monoterpenoid biosynthesis in different chemotypes of M. haplocalyx.

Dissecting the roles of MBD2 isoforms and domains in regulating NuRD complex function during cellular differentiation

The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.

Nanocellulose as a Functional Ingredient in the Management of Metabolic Syndrome: A Review

Alternative treatments in the management of metabolic syndrome are required because multiple drugs for individual components was found to have negative side effects on other components. Functional ingredients, particularly fiber, has shown great benefits in improving metabolic syndrome. Nanocellulose is a novel type of fiber, derived from cellulose through various processes that result in a nanoscale fiber with the dimension below 100 nm. Its smaller size brought improvements to the physicochemical properties of cellulose and consequently its biological activities. Nanocellulose appear to exhibit distinct functional activities that affect various processes in the gastrointestinal tract, including interference in lipid and carbohydrate digestion and reinforcement of gut microflora. These properties may ameliorate abdominal obesity, dyslipidemia, hyperglycemia, and high blood pressure through similar mechanisms of both soluble and insoluble fibers. In this review, we first introduce nanocellulose and its particular characteristics that makes it separate from cellulose. With the limited studies available, we try to go in depth into its activity in the gastrointestinal tract followed by the possible implications of those functional properties on health, especially on the components of metabolic syndrome. Lastly, we discuss the potential applications and advantages of incorporating nanocellulose in functional food for the management of metabolic syndrome.

Stat3 Tyrosine 705 and Serine 727 Phosphorylation Associate With Clinicopathological Characteristics and Distinct Tumor Cell Phenotypes in Triple-Negative Breast Cancer.

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.

Abstract 664: Immune inhibitory receptors ILT2 and ILT4 exhibit both distinct and overlapping biology in vitro and in vivo

Abstract Background: Immune suppression in the tumor microenvironment is known to contribute to tumor immune evasion. ILT2 (aka LILRB1) and ILT4 (aka LILRB2) are distinct ITIM-containing immunosuppressive receptors that recognize the shared ligand MHC-I. Both ILT2 and ILT4 are highly expressed on tumor infiltrating myeloid cells, while ILT2 is also expressed on tumor-associated T and NK cells. Given their common expression on myeloid cells, we anticipated that ILT2 and ILT4 blockade would be redundant. However, assays showed distinct functional activities of ILT2 or ILT4, with limited overlap. Here we explore the relative contribution of ILT2 and ILT4 on in vitro immune activation and in vivo anti-tumor activity. Methods: Anti-ILT2 and anti-ILT4 antibodies were assessed on T cells, NK cells and various myeloid cell subsets, including monocytes, macrophages, myeloid-derived suppressor cells (MDSC) and dendritic cells. ILT2 and ILT4 do not have clear murine orthologs; thus, to characterize in vivo activity, we utilized humanized murine tumor models, including PBMC-reconstituted and CD34-reconstituted mice. Mice were engrafted with tumors and treated with anti-ILT2, anti-ILT4, dual antagonist NGM707 and/or anti-PD-1 antagonist antibodies. Results: The in vitro and in vivo biology of ILT2 and ILT4 was explored using mono-specific antagonist antibodies, as well as the ILT2/ILT4 dual antagonist NGM707. Surprisingly, ILT2- and ILT4-specific antibodies showed distinct activities on myeloid cell populations, despite expression of both receptors. Using assays designed to induce optimal MHC-I engagement, both ILT2 and ILT4 contributed to myeloid suppression. ILT2 blockade further enhanced macrophage phagocytosis, CD8+ T cell cytolytic activity and activation of NKG2C+ memory-like NK cells. Humanized mouse models were used to characterize the in vivo anti-tumor activity of ILT2 and ILT4 blockade. Consistent with our in vitro findings, we observed a distinct effect of ILT2 and ILT4 blockade on tumor growth inhibition. Combination with an anti-PD-1 antibody further enhanced tumor suppression. Conclusions: Despite high expression of ILT2 and ILT4 on myeloid cell populations, evidence from standard in vitro assays suggested that ILT2 and ILT4 have distinct functional activities. Here, we provide evidence that both ILT2 and ILT4 play key roles in myeloid immune suppression, and that blockade of these two receptors can be additive or synergistic. ILT2 blockade additionally enhanced T cell and NK cell function. Furthermore, in vivo studies delineated a role for both ILT2 and ILT4 blockade in promoting anti-tumor immune responses and demonstrated that blockade of ILT2 and ILT4 is complementary to PD-1 inhibition. These data support the clinical evaluation of the dual ILT2/ILT4 antagonist antibody NGM707 alone and in combination with PD-1 blockade. Citation Format: Jane Tian, Samir Qurashi, Christina Song, Lauren Rocha, Kalyani Mondal, Suzanne Crawley, Sara Medfisch, Peirong Krisney Chen, Julie Roda, Jon Sitrin, Carmence Ho, Jonathan Aguayo, Lee Rivera, Jiawei Huang, Vicky Lin, David Shen, Yan Wang, Hui Tian, Alan Kutach, James Sissons, Daniel D. Kaplan, Geoffrey W. Stone. Immune inhibitory receptors ILT2 and ILT4 exhibit both distinct and overlapping biology in vitro and in vivo [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 664.

PU.1 Expression Defines Distinct Functional Activities in the Phenotypic HSC Compartment of a Murine Inflammatory Stress Model.

The transcription factor PU.1 is a critical regulator of lineage fate in blood-forming hematopoietic stem cells (HSC). In response to pro-inflammatory signals, such as the cytokine IL-1β, PU.1 expression is increased in HSC and is associated with myeloid lineage expansion. To address potential functional heterogeneities arising in the phenotypic HSC compartment due to changes in PU.1 expression, here, we fractionated phenotypic HSC in mice using the SLAM surface marker code in conjunction with PU.1 expression levels, using the PU.1-EYFP reporter mouse strain. While PU.1lo SLAM cells contain extensive long-term repopulating activity and a molecular signature corresponding to HSC activity at steady state, following IL-1β treatment, HSCLT induce PU.1 expression and are replaced in the PU.1lo SLAM fraction by CD41+ HSC-like megakaryocytic progenitors (SL-MkP) with limited long-term engraftment capacity. On the other hand, the PU.1hi SLAM fraction exhibits extensive myeloid lineage priming and clonogenic activity and expands rapidly in response to IL-1β. Furthermore, we show that EPCR expression, but not CD150 expression, can distinguish HSCLT and SL-MkP under inflammatory conditions. Altogether, our data provide insights into the dynamic regulation of PU.1 and identify how PU.1 levels are linked to HSC fate in steady state and inflammatory stress conditions.

Synthesis and evaluation of peptide–fentanyl analogue conjugates as dual µ/δ-opioid receptor agonists for the treatment of pain

Novel peptide-fentanyl analogue conjugates were synthesized by the covalent coupling of carfentanyl derivatives to the C-terminus or N-terminus of the conformationally constrained dermorphin tetrapeptide BVD03 via a chemical linker. The carfentanyl-related analogues displayed distinct binding and functional activities at µ/δ opioid receptors (MOR/DOR) and antinociceptive effects when conjugated to the peptide. The most potent compound, SW-LJ-11, displayed mixed MOR/DOR agonist properties in the low nanomolar range and significant analgesic efficacy in vivo in four classic mouse models of pain. Interestingly, SW-LJ-11 did not exhibit any physical dependence or respiratory depression, in contrast to an equipotent analgesic dose of morphine or BVD03, indicating that the use of opioid peptide–fentanyl analogue conjugates as dual MOR/DOR agonists may be a promising strategy for obtaining safer opioids.

Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via β2-gp1 (β2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation.

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand-receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in-situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.

Targeting AML: The Development of Two Functionally and Mechanistically Distinct Classes of Cereblon-Mediated Protein Homeostatic Modulators

Targeting disease-relevant proteins by exploiting the cells' very own protein homeostasis machinery is the next generation drug discovery platform that has come center stage for treatment of hematological malignancies. We present our novel and unique Protein Homeostatic Modulators (PHMsTM) - that promote ubiquitination and subsequent proteasomal degradation of known substrates as well as neosubstrates via Cereblon - as therapeutic candidates for acute myeloid leukemia (AML). Specifically, we report the discovery of two functionally and mechanistically distinct classes of Cereblon-mediated PHMsTM from phenotypic screens of our proprietary PHM®library. Unlike classical IMiDs, our oncology candidates, BTX508 and BTX1119 demonstrate significant cytotoxicity with IC50s in low-nanomolar range in a panel of AML cell lines. BTX508 is a purely cytotoxic compound that does not display immune modulatory activity. In contrast, BTX1119 is not only cytotoxic, but is also immunomodulatory in that it inhibits inflammatory cytokines such as IL-1β, IL-6 and TNF-α as well as induces IL-2, an indicator of T cell activation. BTX1119's immune modulatory activity is a 100-fold more potent when compared to Pomalidomide. The distinct functional activities exhibited by BTX508 and BTX1119 is a result of strategically engineered substrate degradation profiles. Importantly, BTX508 and BTX1119 exhibit a large safety window wherein the concentrations at which they target AML cells do not affect normal healthy cells such as liver epithelial cells or lung fibroblasts. To further establish BTX508 as a clinical candidate for AML, we performed in vivo efficacy studies in the MV-4-11 human AML xenograft model using athymic nude mice. A single daily dose of BTX508 results in a significant reduction in tumor volume. BTX508 exhibits significant oral bioavailability, thus making it a promising clinical candidate for treatment of AML as well as other potential hematological malignancies. With regard to BTX1119, we believe that incorporation of both the potent cytotoxic and immunomodulatory properties into a single molecule holds great therapeutic promise; however, to evaluate the synergistic potential of this combination will require use of a humanized mouse model - this work is ongoing. Disclosures No relevant conflicts of interest to declare.

The Nucleosome Remodelling and Deacetylation complex suppresses transcriptional noise during lineage commitment

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often found as multiple paralogous proteins in vertebrate complexes. In some cases, these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here, we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within embryonic stem (ES) cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. ES cells lacking all three MTA proteins exhibit complete NuRD loss of function and are viable, allowing us to identify a previously unreported function for NuRD in reducing transcriptional noise, which is essential for maintaining a proper differentiation trajectory during early stages of lineage commitment.

P63 isoforms in triple-negative breast cancer: ΔNp63 associates with the basal phenotype whereas TAp63 associates with androgen receptor, lack of BRCA mutation, PTEN and improved survival.

The TP63 gene encodes two major protein variants that differ in their N-terminal sequences and have opposing effects. In breast, ΔNp63 is expressed by immature stem/progenitor cells and mature myoepithelial/basal cells and is a characteristic feature of basal-like triple-negative breast cancers (TNBCs). The expression and potential role of TAp63 in the mammary gland and breast cancers is less clear, partly due to the lack of studies that employ p63 isoform-specific antibodies. We used immunohistochemistry with ΔNp63-specific or TAp63-specific monoclonal antibodies to investigate p63 isoforms in 236 TNBCs. TAp63, but not ΔNp63, was seen in tumour-associated lymphocytes and other stromal cells. Tumour cells showed nuclear staining for ΔNp63 in 17% of TNBCs compared to 7.3% that were positive for TAp63. Whilst most TAp63+ tumours also contained ΔNp63+ cells, the levels of the two isoforms were independent of each other. ΔNp63 associated with metaplastic and medullary cancers, and with a basal phenotype, whereas TAp63 associated with androgen receptor, BRCA1/2 wild-type status and PTEN positivity. Despite the proposed effects of p63 on proliferation, Ki67 did not correlate with either p63 isoform, nor did they associate with p53 mutation status. ΔNp63 showed no association with patient outcomes, whereas TAp63+ patients showed fewer recurrences and improved overall survival. These findings indicate that both major p63 protein isoforms are expressed in TNBCs with different tumour characteristics, indicating distinct functional activities of p63 variants in breast cancer. Analysis of individual p63 isoforms provides additional information into TNBC biology, with TAp63 expression indicating improved prognosis.

Biphasic regulation of the transcription factor ABORTED MICROSPORES (AMS) is essential for tapetum and pollen development in Arabidopsis.

Summary Viable pollen is essential for plant reproduction and crop yield. Its production requires coordinated expression at specific stages during anther development, involving early meiosis‐associated events and late pollen wall formation. The ABORTED MICROSPORES (AMS) transcription factor is a master regulator of sporopollenin biosynthesis, secretion and pollen wall formation in Arabidopsis. Here we show that it has complex regulation and additional essential roles earlier in pollen formation.An inducible‐AMS reporter was created for functional rescue, protein expression pattern analysis, and to distinguish between direct and indirect targets. Mathematical modelling was used to create regulatory networks based on wild‐type RNA and protein expression.Dual activity of AMS was defined by biphasic protein expression in anther tapetal cells, with an initial peak around pollen meiosis and then later during pollen wall development. Direct AMS‐regulated targets exhibit temporal regulation, indicating that additional factors are associated with their regulation.We demonstrate that AMS biphasic expression is essential for pollen development, and defines distinct functional activities during early and late pollen development. Mathematical modelling suggests that AMS may competitively form a protein complex with other tapetum‐expressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation.

Genomic and Transcriptomic Resolution of Organic Matter Utilization Among Deep-Sea Bacteria in Guaymas Basin Hydrothermal Plumes.

Microbial chemosynthesis within deep-sea hydrothermal vent plumes is a regionally important source of organic carbon to the deep ocean. Although chemolithoautotrophs within hydrothermal plumes have attracted much attention, a gap remains in understanding the fate of organic carbon produced via chemosynthesis. In the present study, we conducted shotgun metagenomic and metatranscriptomic sequencing on samples from deep-sea hydrothermal vent plumes and surrounding background seawaters at Guaymas Basin (GB) in the Gulf of California. De novo assembly of metagenomic reads and binning by tetranucleotide signatures using emergent self-organizing maps (ESOM) revealed 66 partial and nearly complete bacterial genomes. These bacterial genomes belong to 10 different phyla: Actinobacteria, Bacteroidetes, Chloroflexi, Deferribacteres, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Verrucomicrobia. Although several major transcriptionally active bacterial groups (Methylococcaceae, Methylomicrobium, SUP05, and SAR324) displayed methanotrophic and chemolithoautotrophic metabolisms, most other bacterial groups contain genes encoding extracellular peptidases and carbohydrate metabolizing enzymes with significantly higher transcripts in the plume than in background, indicating they are involved in degrading organic carbon derived from hydrothermal chemosynthesis. Among the most abundant and active heterotrophic bacteria in deep-sea hydrothermal plumes are Planctomycetes, which accounted for seven genomes with distinct functional and transcriptional activities. The Gemmatimonadetes and Verrucomicrobia also had abundant transcripts involved in organic carbon utilization. These results extend our knowledge of heterotrophic metabolism of bacterial communities in deep-sea hydrothermal plumes.

Increased dysbindin-1B isoform expression in schizophrenia and its propensity in aggresome formation.

Genetic variations in the human dysbindin-1 gene (DTNBP1) have been associated with schizophrenia. As a result of alternative splicing, the human DTNBP1 gene generates at least three distinct protein isoforms, dysbindin-1A, -1B and -1C. Significant effort has focused on dysbindin-1A, an important player in multiple steps of neurodevelopment. However, the other isoforms, dysbindin-1B and dysbindin-1C have not been well characterized. Nor have been associated with human diseases. Here we report an increase in expression of DTNBP1b mRNA in patients with paranoid schizophrenia as compared with healthy controls. A single-nucleotide polymorphism located in intron 9, rs117610176, has been identified and associated with paranoid schizophrenia, and its C allele leads to an increase of DTNBP1b mRNA splicing. Our data show that different dysbindin splicing isoforms exhibit distinct subcellular distribution, suggesting their distinct functional activities. Dysbindin-1B forms aggresomes at the perinuclear region, whereas dysbindin-1A and -1C proteins exhibit diffused patterns in the cytoplasm. Dysbindin-1A interacts with dysbindin-1B, getting recruited to the aggresome structure when co-expressed with dysbindin-1B. Moreover, cortical neurons over-expressing dysbindin-1B show reduction in neurite outgrowth, suggesting that dysbindin-1B may interfere with dysbindin-1A function in a dominant-negative manner. Taken together, our study uncovers a previously unknown association of DTNBP1b expression with schizophrenia in addition to its distinct biochemical and functional properties.

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.

The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

Low Frequency of Circulating CD8+ T Stem Cell Memory Cells in Chronic Chagasic Patients with Severe Forms of the Disease

BackgroundCD8+ T cells have been shown to play a crucial role in Trypanosoma cruzi infection. Memory CD8+ T cells can be categorised based on their distinct differentiation stages and functional activities as follows: stem cell memory (TSCM), central memory (TCM), transitional memory (TTM), effector memory (TEM) and terminal effector (TTE) cells. Currently, the immune mechanisms that control T. cruzi in the chronic phase of the infection are unknown.Methodology/Principal FindingsTo characterise the CD8+ T cell subsets that could be participating in the control of T. cruzi infection, in this study, we compared total and T. cruzi-specific circulating CD8+ T cells with distinctive phenotypic and functional features in chronic chagasic patients (CCPs) with different degrees of cardiac dysfunction. We observed a decreased frequency of total TSCM along with an increased frequency of TTE in CCPs with severe disease. Antigen-specific TSCM cells were not detectable in CCPs with severe forms of the disease. A functional profile of CD8+ T cell subsets among CCPs revealed a high frequency of monofunctional CD8+ T cells in the most severe patients with IFN-γ+- or TNF-α+-producing cells.Conclusions/SignificanceThese findings suggest that CD8+ TSCM cells may be associated with the immune response to T. cruzi and outcome of Chagas disease, given that these cells may be involved in repopulating the T cell pool that controls infection.

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment.

BackgroundParacoccidioidomycosis (PCM) is systemic mycosis caused by the thermal dimorphic fungus of genus Paracoccidioides, leading to either acute/subacute (AF) or chronic (CF) clinical forms. Numerous CF patients after treatment exhibit sequels, such as pulmonary and adrenal fibrosis. Monocytes are cells that are involved in the inflammatory response during active infection as well as in the fibrogenesis. These cells comprise a heterogeneous population with distinct phenotypic and functional activities. The scope of this study was to identify changes regarding functional and phenotypical aspects in monocytes comparing CF PCM patients on antifungal treatment versus non-treated patients (PMC-p).MethodsTwenty-three CF PCM composed of 11 non-treated patients (NTG) and 12 patients in apparent cure (ACG) were studied. Sixteen healthy individuals were used as control group (CG). Monocyte subsets were determined by immunophenotyping based on CD14 and CD16 expression. Cellular function was measured in vitro with and without stimulation with lipopolysaccharide (LPS) and P. brasiliensis exoantigen (PbAg) for 24 hours. Independent samples were compared using unpaired t tests, dependent samples were analyzed by paired t-test. Groups of more than two independent samples were analyzed using an ANOVA, with Tukey's post-test. Significance was set up at p <0.05.ResultsOur results showed high counts of peripheral blood CD14+CD16+ and CD14+CD16++ monocytes in untreated PCM-p accompanied by intense production of pro-inflammatory cytokines (IL-1β and TNF-α) and profibrotic growth factors (TGF-β1 and bFGF) by monocytes challenged with P. brasiliensis antigens. After the introduction of antifungal therapy, the counts of CD14+CD16+ cells returned to baseline while CD14+CD16++ counts remained high. Interestingly, counts of CD14+CD16++ monocytes remained elevated even 52 ± 7 months after successful antifungal treatment. Furthermore, the ACG-patients showed preserved pro-inflammatory activity in the presence of specific antigen stimuli and high spontaneous production of TNF-α by monocytes.ConclusionsInfection with Paracoccidioides leads to initiation of a specific proinflammatory response by monocytes of PCM-p during active disease and in the apparent cure. A profibrotic profile by monocytes was observed only at admission. Furthermore, PCM-p with apparent cure showed high spontaneous production of TNF-α and high counts of CD14+CD16++ monocytes, probably induced by hypoxia duo to fibrotic sequelae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0552-x) contains supplementary material, which is available to authorized users.