Transcription Factor PU Research Articles

PD-L1 enhances migration and invasion of trophoblasts by upregulating ARHGDIB via transcription factor PU.1

As the main constituent cells of the human placenta, trophoblasts proliferate, differentiate, and invade the uterine endometrium via a series of processes, which are regulated exquisitely through intercellular signaling mediated by hormones, cytokines, and growth factors. Programmed cell death ligand 1 (PD-L1) is a biomarker of the response to immune checkpoint inhibitors and can regulate maternal-fetal immune tolerance during pregnancy progression. Recently, it was found that PD-L1 may regulate obstetric complications by affecting the function of trophoblasts. Therefore, we examined the expression and localization of PD-L1 in the human placenta and observed the effects of PD-L1 on trophoblasts migration and invasion in both the trophoblasts line HTR-8/SVneo and an extravillous explant culture model. Finally, we explored the molecular mechanisms underlying PD-L1-regulated trophoblasts migration and invasion through RNA sequencing and bioinformatics analysis. Our data showed that PD-L1 was mainly expressed in syncytiotrophoblasts and that its protein levels increased with gestational age. Interestingly, the protein expression of PD-L1 was significantly decreased in placentas from pregnancies with preeclampsia compared with normal placentas. Importantly, the migration and invasion abilities of trophoblasts were significantly changed after knockdown or overexpression of PD-L1 in HTR-8/SVneo cells and an extravillous explant culture model, which was partially mediated through the transcription factor PU.1 (encoded by Spi1)-regulated Rho GDP-dissociation inhibitor beta (ARHGDIB) expression. These results suggested that PD-L1 was highly involved in the regulation of trophoblasts migration and invasion, providing a potential target for the diagnosis and treatment of placenta-derived pregnancy disorders.

PU.1-c-Jun interaction is crucial for PU.1 function in myeloid development

The Ets transcription factor PU.1 is essential for inducing the differentiation of monocytes, macrophages, and B cells in fetal liver and adult bone marrow. PU.1 controls hematopoietic differentiation through physical interactions with other transcription factors, such as C/EBPα and the AP-1 family member c-Jun. We found that PU.1 recruits c-Jun to promoters without the AP-1 binding sites. To address the functional importance of this interaction, we generated PU.1 point mutants that do not bind c-Jun while maintaining normal DNA binding affinity. These mutants lost the ability to transactivate a target reporter that requires a physical PU.1-c-Jun interaction, and did not induce monocyte/macrophage differentiation of PU.1-deficient cells. Knock-in mice carrying these point mutations displayed an almost complete block in hematopoiesis and perinatal lethality. While the PU.1 mutants were expressed in hematopoietic stem and early progenitor cells, myeloid differentiation was severely blocked, leading to an almost complete loss of mature hematopoietic cells. Differentiation into mature macrophages could be restored by expressing PU.1 mutant fused to c-Jun, demonstrating that a physical PU.1-c-Jun interaction is crucial for the transactivation of PU.1 target genes required for myeloid commitment and normal PU.1 function in vivo during macrophage differentiation.

Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator

BackgroundImmune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host...

The Il6 -39 kb enhancer containing clustered GATA2- and PU.1-binding sites is essential for Il6 expression in murine mast cells

SummaryMast cells serve as a first-line defense of innate immunity. Interleukin-6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1-clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.

Direct TLR2 Signaling Through mTOR and TBK1 Induces C/EBPβ and IRF7-Dependent Macrophage Differentiation in Hematopoietic Stem and Progenitor Cells.

During an infection, hematopoiesis is altered to increase the output of mature myeloid cells to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, more mechanistic studies are needed to reveal whether pattern recognition receptor (PRR) signaling initiates myeloid development directly, or indirectly through the production of cytokines by HSPCs that can act in an autocrine/paracrine manner, or by a combination of both direct and indirect mechanisms. In this study, we have used an in vitro model of murine HSPCs to study myeloid differentiation in response to the TLR2 ligand Pam3CSK4 and showed that, besides indirect mechanisms, TLR2 stimulation of HSPCs promotes myelopoiesis directly by initiating a MyD88-dependent signaling. This direct differentiation program involves a combined activation of the transcription factors PU.1, C/EBPβ, and IRF7 driven by TBK1 and PI3K/mTOR. Notably, downstream of MyD88, the activated TBK1 kinase can activate mTOR directly and IRF7 induction is mediated by both TBK1 and mTOR. TLR2 signaling also induces NF-κB dependent IL-6 production that may further induce indirect myeloid differentiation. Our results have identified the direct signaling pathways and the transcription factors involved in macrophage development from HSPCs in response to TLR2 engagement, a critical process to trigger a rapid immune response during infection.

Isolevuglandins disrupt PU.1-mediated C1q expression and promote autoimmunity and hypertension in systemic lupus erythematosus.

We describe a mechanism responsible for systemic lupus erythematosus (SLE). In humans with SLE and in 2 SLE murine models, there was marked enrichment of isolevuglandin-adducted proteins (isoLG adducts) in monocytes and dendritic cells. We found that antibodies formed against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduced transcription of all C1q subunits. Treatment of SLE-prone mice with the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA) ameliorated parameters of autoimmunity, including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowered blood pressure, attenuated renal injury, and reduced inflammatory gene expression uniquely in C1q-expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.

The Myeloid-Specific Transcription Factor PU.1 Upregulates Mannose Receptor Expression but Represses Basal Activity of the HIV-LTR Promoter.

Human mannose receptor 1 (MRC1) is a cell surface receptor expressed in macrophages and other myeloid cells that inhibits human immunodeficiency virus type 1 (HIV-1) particle release by tethering virions to producer cell membranes. HIV-1 counteracts MRC1 expression by inhibiting mrc1 transcription. Here, we investigated the mechanism of MRC1 downregulation in HIV-1-infected macrophages. We identified the myeloid cell-specific transcription factor PU.1 as critical for regulating MRC1 expression. In the course of our study, we recognized a complex interplay between HIV-1 Tat and PU.1 transcription factors: Tat upregulated HIV-1 gene expression but inhibited mrc1 transcription, whereas PU.1 inhibited HIV-1 transcription but activated MRC1 expression. Disturbing this equilibrium by silencing PU.1 resulted in increased HIV-1 gene expression and reduced MRC1 promoter activity. Our study identified PU.1 as a central player in transcriptional control, regulating a complex interplay between viral and host gene expression in HIV-infected macrophages. IMPORTANCE HIV-1 replication in primary human cells depends on the activity of virus-encoded proteins but also involves cellular factors that can either promote (viral dependency factors) or inhibit (host restriction factors) virus replication. In previous work, we identified human MRC1 as a macrophage-specific host restriction factor that inhibits the detachment of viral particles from infected cells. Here, we report that HIV-1 counteracts this effect of MRC1 by imposing a transcriptional block on cellular MRC1 gene expression. The transcriptional inhibition of the MRC1 gene is accomplished by Tat, an HIV-1 factor whose best-described function actually is the enhancement of HIV-1 gene expression. Thus, HIV-1 has evolved to use the same protein for (i) activation of its own gene expression while (ii) inhibiting expression of MRC1 and other host factors.

Combined Approach to Leukemic Differentiation Using Transcription Factor PU.1-Enhancing Agents.

The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.

Extracellular ST6GAL1 regulates monocyte-macrophage development and survival.

Interaction of immune cells with the systemic environment is necessary for the coordinated development and execution of immune responses. Monocyte-macrophage lineage cells reside at the junction of innate and adaptive immunity. Previously we reported that the sialyltransferase ST6GAL1 in the extracellular milieu modulates B cell development and IgG production, granulocyte production, and attenuates acute airway inflammation to bacterial challenge in mouse models. Here, we report that extracellular ST6GAL1 also elicits profound responses in monocyte-macrophage lineage cells. We show that recombinant ST6GAL1 adheres to subsets of thioglycolate-elicited inflammatory cells in the mouse peritoneum and to cultured human monocyte THP-1 cells. Exposure of the inflammatory cells to recombinant ST6GAL1 elicited wholesale changes in the gene expression profile of primary mouse myeloid cells; most notable was the striking up-regulation of monocyte-macrophage and monocyte-derived dendritic cell development pathway signature genes and transcription factors PU.1, NFκB and their target genes, driving increased monocyte-macrophage population and survival ex vivo. In the cultured human monocyte cells, the essential cell surface receptor of the monocyte-macrophage lineage, the M-CSF receptor (M-CSF-R, Csfr1) was a target of extracellular ST6GAL1 catalytic activity. Extracellular ST6GAL1 activated the M-CSF-R and initiated intracellular signaling events, namely, the nuclear translocation of NFκB subunit p65, and phosphorylation of ERK 1/2 and AKT. The findings implicate extracellular ST6GAL1 in monocyte development by a mechanism initiated at the cell surface and support an emerging paradigm of an extracellular glycan-modifying enzyme as a central regulator coordinating immune hematopoietic cell development and function.

BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia

BackgroundThe BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth.MethodsMicroglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches.ResultsHere, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3.ConclusionsOur convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.Graphical

Contribution of PU.1 and STAT6 to fibrosis-mediated growth suppression of the Diphyllobothriidae family cestode, Schistocephalus solidus.

Abstract Threespine stickleback (Gasterosteus aculeatus) fish are naturally infected with the Diphyllobothriidae family tapeworm, Schistocephalus solidus. The anti-parasite response to this body cavity infection varies greatly among wild stickleback populations. Some populations generate extensive peritoneal fibrosis which suppresses cestode grown, thereby preventing the parasite from reaching sexual maturity and blocking its ability to infect its terminal host. In extreme cases this response can encapsulate the cestode. On the other hand, other fish populations tolerate S. solidus, carry heavy parasite burdens, grow parasites 34-fold larger than resistant fish, and do not produce fibrosis. Combined genetic mapping and population genomics identified regions of the genome associated with this resistance-tolerance axis. Quantitative trait loci (QTL) mapping of the fibrosis phenotype led us to a 78bp deletion in SPI1b which encodes the transcription factor PU.1 which is associated with the initiation of tissue fibrosis. This deletion was fixed in tolerant fish, but not resistant fish. SPI1b expression increases with infection and is higher in fibrotic fish compared to non-fibrotic controls. QTL mapping of cestode mass detected STAT6 on Ch 12, which is associated with alternatively activated macrophages and fibrosis. In tolerant fish, but not resistant, a deletion in stat6 exon 5 is nearly fixed. Several additional QTL were also found containing genes associated with fibrosis initiation or regulation, evidence for strong selection on this anti-parasite mechanism and identifying pathways involved in the regulation of a pathologic fibrotic response against a Diphyllobothriidae cestode.

Cytokine responses to LPS in reprogrammed monocytes are associated with the transcription factor PU.1.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that arise and expand during extensive inflammatory conditions by increased hematopoietic output or reprogramming of immune cells. In sepsis, an increase of circulating MDSCs is associated with adverse outcomes, but unique traits that can be used to identify increased activity of MDSCs are lacking. By using endotoxin tolerance as a model of sepsis-induced monocytic MDSCs (M-MDSC-like cells), this study aims to identify the mediator and transcriptional regulator profile associated with M-MDSC activity. After analyzing 180 inflammation-associated proteins, a profile of differentially expressed cytokines was found in M-MDSC-like cells versus normal monocytes stimulated with LPS. These cytokines were associated with 5 candidate transcription factors, where particularly PU.1 showed differential expression on both transcriptional and protein levels in M-MDSC-like cells. Furthermore, inhibition of PU.1 led to increased production of CXCL5 and CCL8 in M-MDSC-like cells indicating its role in regulating the ability of M-MDSC-like cells to recruit other immune cells. Taken together, the study identifies a unique profile in the pattern of immune mediators defining M-MDSC activity upon LPS stimulation, which offers a functional link to their contribution to immunosuppression.

Rat Spinal Cord Injury Associated with Spasticity Leads to Widespread Changes in the Regulation of Retained Introns.

To determine molecular changes that correlate with long-term physiological changes after spinal cord injury associated with spasticity, we used a complete transection model with an injury at sacral spinal level S2, wherein tail spasms develop in rats weeks to months post-injury. Using Illumina and nanopore sequencing, we found that from 12,266 expressed genes roughly 11% (1,342) change expression levels in the rats with spasticity. The transcription factor PU.1 (Spi-1 proto-oncogene) and several of its known regulated genes were upregulated during injury, possibly reflecting changes in cellular composition. In contrast to widespread changes in gene expression, only a few changes in alternative exon usage could be detected because of injury. There were more than 1,000 changes in retained intron usage, however. Unexpectedly, most of these retained introns have not been described yet but could be validated using direct RNA nanopore sequencing. In addition to changes from injury, our model allowed regional analysis of gene expression. Comparing the segments rostral and caudal to the injury site in naïve animals showed 525 differentially regulated genes and differential regional use of retained introns. We did not detect changes in the serotonin receptor 2C editing that were implicated previously in this spinal cord injury model. Our data suggest that regulation of intron retention of polyadenylated pre-mRNA is an important regulatory mechanism in the spinal cord under both physiological and pathophysiological conditions.

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.

Bridging the macroscopic and microscopic: a volumetric approach to DNA recognition

Living cells adapt to osmotically variable conditions in their environment. To understand the molecular basis of these adaptations, it is necessary to study the hydration properties of the biomolecular interactions that mediate the osmotic stress response. Gene expression studies have shown that the transcription factor PU.1 regulates target genes in response to cellular hyperosmotic stress, and osmotic pressure studies in vitro have shown that the high-affinity and low-affinity DNA binding modes of PU.1 are differentially hydrated.

Inhibiting KDM6A Demethylase Represses Long Non-Coding RNA Hotairm1 Transcription in MDSC During Sepsis.

Myeloid-derived suppressor cells (MDSCs) prolong sepsis by promoting immunosuppression. We reported that sepsis MDSC development requires long non-coding RNA Hotairm1 interactions with S100A9. Using a mouse model that simulates the immunobiology of sepsis, we find that histone demethylase KDM6A promotes Hotairm1 transcription by demethylating transcription repression H3K27me3 histone mark. We show that chemical targeting of KDM6A by GSK-J4 represses Hotairm1 transcription, which coincides with decreases in transcription activation H3K4me3 histone mark and transcription factor PU.1 binding to the Hotairm1 promoter. We further show that immunosuppressive IL-10 cytokine promotes KDM6A binding at the Hotairm1 promoter. IL-10 knockdown repletes H3K27me3 and reduces Hotairm1 transcription. GSK-J4 treatment also relocalizes nuclear S100A9 protein to the cytosol. To support translation to human sepsis, we demonstrate that inhibiting H3K27me3 demethylation by KDM6A ex vivo in MDSCs from patients with protracted sepsis decreases Hotairm1 transcription. These findings suggest that epigenetic targeting of MDSCs in human sepsis might resolve post-sepsis immunosuppression and improve sepsis survival.

Expression of the transcription factor PU.1 induces the generation of microglia-like cells in human cortical organoids

Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-β (Aβ). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aβ. Furthermore, in mhCOs, we observed reduced expression of Aβ-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer’s disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aβ using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.

PU.1 Regulates Cathepsin S Expression in Large Yellow Croaker (Larimichthys crocea) Macrophages

Different morphologies have been detected in teleost macrophages. In this study, two macrophage cell lines were sub-cloned from a large yellow croaker head kidney cell line, LYCK. One type of sub-cloned cells was fusiform but the other was round, named LYC-FM and LYC-RM cells respectively, based on their morphologies. Both types showed the characteristics of macrophages, including expression of macrophage-specific marker genes, possession of phagocytic and bactericidal activities, and production of reactive oxygen species (ROS) and nitric oxide (NO). The transcription factor PU.1, crucial for the development of macrophages in mammals, was found to exist in two transcripts, PU.1a and PU.1b, in large yellow croaker, and constitutively expressed in LYC-FM and LYC-RM cells. The expression levels of PU.1a and PU.1b could be upregulated by recombinant large yellow croaker IFN-γ protein (rLcIFN-γ). Further studies showed that both PU.1a and PU.1b increased the expression of cathepsin S (CTSS) by binding to different E26−transformation−specific (Ets) motifs of the CTSS promoter. Additionally, we demonstrated that all three domains of PU.1a and PU.1b were essential for initiating CTSS expression by truncated mutation experiments. Our results therefore provide the first evidence that teleost PU.1 has a role in regulating the expression of CTSS.

A novel PU.1 inhibitor for the treatment of Alzheimer’s disease

AbstractBackgroundIn 2015, we identified the microglia‐specific transcription factor PU.1, encoded for by SPI1, as a master regulator of neuroinflammation in a mouse model of Alzheimer’s Disease and in human brain tissue from deceased Alzheimer’s Disease patients1. In 2017, a GWAS SNP that delays onset of Alzheimer’s Disease, was shown to reduce PU.1 expression levels2. Here, we describe a high‐throughput screen for small molecules that inhibit PU.1 activity. Our goal is to develop novel agents that can rein in the abnormal neuroimmune response associated with Alzheimer’s dementia.MethodWe used a stable immortalized mouse microglia (BV2) reporter line that expresses luciferase in a PU.1‐dependent manner. We screened a total of 58’100 molecules from 3 separate chemical libraries and obtained 264 hits. We further validated these hits using multiple assays in human stem cell derived microglia. Finally, we administered our top hit (A11) to transgenic mouse models of Alzheimer’s Disease.ResultA11 exhibits anti‐inflammatory effects at a concentration of 20 nM in activated microglia, as assessed by RTqPCR and ELISA for IL1β, morphological analyses and lipid accumulation quantification. In mice, A11 preferentially distributed to brain tissues after systemic (intraperitoneal) application and reached a peak concentration at 4 hours. When dosing mice that develop severe neurodegeneration (CK‐p25 transgenic mice), we found a reduction of microglial proliferation, astrogliosis and retention of p25‐positive neurons in the hippocampus. No abnormalities were seen in the treated control littermates.ConclusionOur novel PU.1 inhibitor (A11) shows nanomolar potency and efficacy in multiple assays and cell lines. Mouse experiments demonstrate suitability for systemic application. Administration of A11 in CK‐p25 transgenic mice show an amelioration of neurodegeneration‐associated hallmarks, such as microglial and astrocytic activation and loss of p25‐positive neurons.

A Study to Assess the Effectiveness of Self Instructional Module on Knowledge Regarding Prevention of Osteoporosis Among the Middle Aged Women in Selected Hospital, Bangalore

Bone is living, growing tissue. It is made mostly of collagen, a protein that provides a soft framework, and calcium phosphate, a mineral that adds strength and hardens the framework. This combination of collagen and calcium makes bone both flexible and strong, which in turn helps bone to withstand stress.1 More than 99 percent of the body’s calcium is contained in the bones and teeth. The remaining 1 percent is found in the blood. Throughout one’s lifetime, old bone is removed (resorption) and new bone is added to the skeleton (formation). During childhood and teenage years, new bone is added faster than old bone is removed. As a result, bones become larger, heavier, and denser. Bone formation outpaces resorption until peak bone mass (maximum bone density and strength) is reached around age 30. After that time, bone resorption slowly begins to exceed bone formation. For women, bone loss is fastest in the first few years after menopause, and it continues into the postmenopausal years. Osteoporosis, or porous bone, is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased risk of fractures of the hip, spine, and wrist. Osteoporosis is more likely to develop if you did not reach optimal peak bone mass during your bone-building years. Women are at a greater risk than men, especially women who are thin or have a small frame, as are those of advanced age. Women who are postmenopausal, including those who have had early or surgically induced menopause, or abnormal or absence of menstrual periods, are at greater risk. Cigarette smoking, eating disorders such as anorexia nervosa or bulimia, low amounts of calcium in the diet, heavy alcohol consumption, inactive lifestyle, and use of certain medications, such as corticosteroids and anticonvulsants, are also risk factors for osteoporopsis.2 The underlying mechanism in all cases of osteoporosis is an imbalance between bone resorption and bone formation. In normal bone, matrix remodeling of bone is constant; up to 10% of all bone mass may be undergoing remodeling at any point in time. The process takes place in bone multicellular units (BMUs) as first described by Frost & Thomas in 1963. Osteoclasts are assisted by transcription factor PU.1 to degrade the bone matrix, while osteoblasts rebuild the bone matrix. Low bone mass density can then occur when osteoclasts are degrading the bone matrix faster than the osteoblasts are rebuilding the bone. The three main mechanisms by which osteoporosis develops are an inadequate peak bone mass (the skeleton develops insufficient mass and strength during growth), excessive bone resorption, and inadequate formation of new bone during remodeling. An interplay of these three mechanisms underlies the development of fragile bone tissue. Hormonal factors strongly determine the rate of bone resorption; lack of estrogen (e.g. as a result of menopause) increases bone resorption, as well as decreasing the deposition of new bone that normally takes place in weight-bearing bones.this leads to weakening and softening of bones the bones become soft and it will prone to get fracture or collapse.