ABSTRACT
Corneal endothelial cells (CEnCs) regulate corneal hydration and maintain tissue transparency through their barrier and pump function. However, these cells exhibit limited regenerative capacity following injury. Currently, corneal transplantation is the only established therapy for restoring endothelial function, and there are no pharmacologic interventions available for restoring endothelial function. This study investigated the efficacy of the neuropeptide α-melanocyte-stimulating hormone (α-MSH) in promoting endothelial regeneration during the critical window between ocular injury and the onset of endothelial decompensation using an established murine model of injury using transcorneal freezing. Local administration of α-MSH following injury prevented corneal edema and opacity, reduced leukocyte infiltration, and limited CEnC apoptosis while promoting their proliferation. These results suggest that α-MSH has a proregenerative and cytoprotective function on CEnCs and shows promise as a therapy for the prevention and management of corneal endothelial dysfunction.
Subject(s)
Cornea , Corneal Edema , alpha-MSH , Female , Pregnancy , Animals , Mice , Mice, Inbred BALB C , Humans , Cell Line , Cornea/cytology , Endothelial Cells , Corneal Edema/drug therapy , Corneal Edema/pathology , Tissue Preservation , alpha-MSH/therapeutic use , Cytoprotection , Neutrophil Infiltration , Monocytes/metabolism , Macrophages/metabolism , Wound Healing/drug effectsABSTRACT
The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.
Subject(s)
CD3 Complex/metabolism , Cell Membrane/metabolism , Cytoplasm/metabolism , Multiprotein Complexes/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , Animals , CD3 Complex/genetics , Humans , Hybridomas , Mechanotransduction, Cellular , Mice , Multiprotein Complexes/genetics , Protein Conformation , Protein Engineering , Protein Multimerization/genetics , Protein Multimerization/immunology , Protein Structure, Tertiary/genetics , Receptor Cross-Talk , Receptors, Antigen, T-Cell/genetics , Signal Transduction/geneticsABSTRACT
BACKGROUND: To evaluate and compare efficacy of pinhole surgical technique (PST) alone and with advanced platelet rich fibrin (A-PRF) in the management of bilateral multiple adjacent gingival recession defects (MAGRD). METHODS: One hundred and sixty five MAGRD were randomly assigned to control group (treated with PST) and test group (PST with A-PRF). Clinical parameters of gingival recession depth (GRD), gingival recession width (GRW), width of keratinised gingiva (WKG), complete root coverage (CRC) and gingival thickness (GT) on ST-CBCT was measured at 2, 4 and 6 mm apically from the gingival margin. Also, root coverage aesthetic score and patient satisfaction ratings were recorded at baseline, 6 and 12 months postoperatively. RESULTS: Substantial reduction in GRD (Test: 1.29 ± 0.69 mm and Control 0.98 ± 0.30 mm) (p < 0.001) and GRW (Test: 2.03 ± 0.90 mm and control 1.73 ± 0.99 mm) (p < 0.05) with associated gain in WKG and GT was observed (p < 0.001). Mean GT values were increased in both the groups at 2, 4 and 6 mm from the crest. Comparison of Test and Control groups yielded significant reductions in GRD (-0.17 ± 0.56 mm) and WKG (0.73 ± 1.07 mm) favoring the Test group (p < 0.05). Similar increase in GT was observed with better results in Test than control group. (p < 0.001). CONCLUSION: Both groups exhibited sound clinical outcomes with test group offering better resolution of MAGRD in comparison to control group. Also, it enhances clinical and therapeutic end results in terms of attaining reduction in GRD and GRW along with greater gain in KTW and GT. CLINICAL SIGNIFICANCE: PST as a minimally invasive approach has numerous benefits, some of which include the absence of scarring and improved aesthetics linked to faster wound healing. The addition of A-PRF enhances the intended therapy outcomes, which is beneficial for both patients and professionals in the field of periodontics.
Subject(s)
Gingival Recession , Platelet-Rich Fibrin , Humans , Gingiva/diagnostic imaging , Gingival Recession/surgery , Surgical Flaps/surgery , Tooth Root , Treatment OutcomeABSTRACT
Gene transcription is an essential and highly regulated process. In eukaryotic cells, the structural organization of nucleosomes with DNA wrapped around histone proteins impedes transcription. Chromatin remodelers, transcription factors, co-activators, and histone-modifying enzymes work together to make DNA accessible to RNA polymerase. Histone lysine methylation can positively or negatively regulate gene transcription. Methylation of histone 3 lysine 4 by SET-domain-containing proteins is evolutionarily conserved from yeast to humans. In higher eukaryotes, mutations in SET-domain proteins are associated with defects in the development and segmentation of embryos, skeletal and muscle development, and diseases, including several leukemias. Since histone methyltransferases are evolutionarily conserved, the mechanisms of gene regulation mediated by these enzymes are also conserved. Budding yeast Saccharomyces cerevisiae is an excellent model system to study the impact of histone 3 lysine 4 (H3K4) methylation on eukaryotic gene regulation. Unlike larger eukaryotes, yeast cells have only one enzyme that catalyzes H3K4 methylation, Set1. In this review, we summarize current knowledge about the impact of Set1-catalyzed H3K4 methylation on gene transcription in S. cerevisiae. We describe the COMPASS complex, factors that influence H3K4 methylation, and the roles of Set1 in gene silencing at telomeres and heterochromatin, as well as repression and activation at euchromatic loci. We also discuss proteins that "read" H3K4 methyl marks to regulate transcription and summarize alternate functions for Set1 beyond H3K4 methylation.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Histones/genetics , Histones/metabolism , Histone Methyltransferases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Lysine/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolismABSTRACT
Fuchs Endothelial Corneal Dystrophy (FECD), a late-onset oxidative stress disorder, is the most common cause of corneal endothelial degeneration and is genetically associated with CTG repeat expansion in Transcription Factor 4 (TCF4). We previously reported accumulation of nuclear (nDNA) and mitochondrial (mtDNA) damage in FECD. Specifically, mtDNA damage was a prominent finding in development of disease in the ultraviolet-A (UVA) induced FECD mouse model. We hypothesize that an aberrant DNA repair may contribute to the increased DNA damage seen in FECD. We analyzed differential expression profiles of 84 DNA repair genes by real-time PCR arrays using Human DNA Repair RT-Profiler plates using cDNA extracted from Descemet's membrane-corneal endothelium (DM-CE) obtained from FECD patients with expanded (>40) or non-expanded (<40) intronic CTG repeats in TCF4 gene and from age-matched normal donors. Change in mRNA expression of <0.5- or >2.0-fold in FECD relative to normal was set as cutoff for down- or upregulation. Downregulated mitochondrial genes were further validated using the UVA-based mouse model of FECD. FECD specimens exhibited downregulation of 9 genes and upregulation of 8 genes belonging to the four major DNA repair pathways, namely, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double strand break (DSB) repair, compared to normal donors. MMR gene MSH2 and BER gene POLB were preferentially upregulated in expanded FECD. BER genes LIG3 and NEIL2, DSB repair genes PARP3 and TOP3A, NER gene XPC, and unclassified pathway gene TREX1, were downregulated in both expanded and non-expanded FECD. MtDNA repair genes, Lig3, Neil2, and Top3a, were also downregulated in the UVA-based mouse model of FECD. Our findings identify impaired DNA repair pathways that may play an important role in DNA damage due to oxidative stress as well as genetic predisposition noted in FECD.
Subject(s)
DNA Glycosylases , Fuchs' Endothelial Dystrophy , Animals , Mice , Humans , Fuchs' Endothelial Dystrophy/genetics , Fuchs' Endothelial Dystrophy/metabolism , Endothelium, Corneal/metabolism , Genetic Predisposition to Disease , DNA Repair/genetics , DNA, Mitochondrial/genetics , DNA Glycosylases/genetics , DNA Glycosylases/metabolismABSTRACT
Over the past decades, reactions involving C-H functionalization have become a hot theme in organic transformations because they have a lot of potential for the streamlined synthesis of complex molecules. C(sp3)-H bonds are present in most organic species. Since organic molecules have massive significance in various aspects of life, the exploitation and functionalization of C(sp3)-H bonds hold enormous importance. In recent years, the first-row transition metal-catalyzed direct and selective functionalization of C-H bonds has emerged as a simple and environmentally friendly synthetic method due to its low cost, unique reactivity profiles and easy availability. Therefore, research advancements are being made to conceive catalytic systems that foster direct C(sp3)-H functionalization under benign reaction conditions. Cobalt-based catalysts offer mild and convenient reaction conditions at a reasonable expense compared to conventional 2nd and 3rd-row transition metal catalysts. Consequently, the probing of Co-based catalysts for C(sp3)-H functionalization is one of the hot topics from the outlook of an organic chemist. This review primarily focuses on the literature from 2018 to 2022 and sheds light on the substrate scope, selectivity, benefits and limitations of cobalt catalysts for organic transformations.
ABSTRACT
T cells express clonotypic T cell receptors (TCRs) that recognize peptide antigens in the context of class I or II MHC molecules (pMHCI/II). These receptor modules associate with three signaling modules (CD3γε, δε, and ζζ) and work in concert with a coreceptor module (either CD8 or CD4) to drive T cell activation in response to pMHCI/II. Here, we describe a first-generation biomimetic five-module chimeric antigen receptor (5MCAR). We show that 1) chimeric receptor modules built with the ectodomains of pMHCII assemble with CD3 signaling modules into complexes that redirect cytotoxic T lymphocyte (CTL) specificity and function in response to the clonotypic TCRs of pMHCII-specific CD4+ T cells, and 2) surrogate coreceptor modules enhance the function of these complexes. Furthermore, we demonstrate that adoptively transferred 5MCAR-CTLs can mitigate type I diabetes by targeting autoimmune CD4+ T cells in NOD mice. This work provides a framework for the construction of biomimetic 5MCARs that can be used as tools to study the impact of particular antigen-specific T cells in immune responses, and may hold potential for ameliorating diseases mediated by pathogenic T cells.
Subject(s)
Antigens/metabolism , Biomimetics/methods , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/metabolism , Animals , Antigens/immunology , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Female , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Pancreas/immunology , Pancreas/pathology , Receptors, Antigen, T-Cell , T-Lymphocytes/immunology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.
Subject(s)
Cytochrome P-450 CYP1B1/metabolism , DNA Adducts/radiation effects , DNA Damage/radiation effects , Estrogens/metabolism , Fuchs' Endothelial Dystrophy/etiology , Ultraviolet Rays/adverse effects , Acetylcysteine/administration & dosage , Animals , Aqueous Humor/drug effects , Aqueous Humor/metabolism , Aqueous Humor/radiation effects , DNA Adducts/metabolism , DNA Damage/drug effects , DNA, Mitochondrial/metabolism , DNA, Mitochondrial/radiation effects , Disease Models, Animal , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Endothelium, Corneal/radiation effects , Female , Free Radical Scavengers/administration & dosage , Fuchs' Endothelial Dystrophy/diagnosis , Fuchs' Endothelial Dystrophy/drug therapy , Fuchs' Endothelial Dystrophy/pathology , Humans , Male , Mice , Oxidative Stress/radiation effects , Reactive Oxygen Species/metabolism , Severity of Illness IndexABSTRACT
Mono-methylation of the fourth lysine on the N-terminal tail of histone H3 was found to support the induction of RNA polymerase II transcription in S. cerevisiae during nutrient stress. In S. cerevisiae, the mono-, di- and tri-methylation of lysine 4 on histone H3 (H3K4) is catalyzed by the protein methyltransferase, Set1. The three distinct methyl marks on H3K4 act in discrete ways to regulate transcription. Nucleosomes enriched with tri-methylated H3K4 are usually associated with active transcription whereas di-methylated H3K4 is associated with gene repression. Mono-methylated H3K4 has been shown to repress gene expression in S. cerevisiae and is detected at enhancers and promoters in eukaryotes. S. cerevisiae set1Δ mutants unable to methylate H3K4 exhibit growth defects during histidine starvation. The growth defects are rescued by either a wild-type allele of SET1 or partial-function alleles of set1, including a mutant that predominantly generates H3K4me1 and not H3K4me3. Rescue of the growth defect is associated with induction of the HIS3 gene. Growth defects observed when set1Δ cultures were starved for isoleucine and valine were also rescued by wild-type SET1 or partial-function set1 alleles. The results show that H3K4me1, in the absence of H3K4me3, supports transcription of the HIS3 gene and expression of one or more of the genes required for biosynthesis of isoleucine and valine during nutrient stress. Set1-like methyltransferases are evolutionarily conserved, and research has linked their functions to developmental gene regulation and several cancers in higher eukaryotes. Identification of mechanisms of H3K4me1-mediated activation of transcription in budding yeast will provide insight into gene regulation in all eukaryotes.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Lysine/metabolism , Nutrients , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
A key difference that distinguishes viral infections from protein immunizations is the recognition of viral nucleic acids by cytosolic pattern recognition receptors (PRRs). Insights into the functions of cytosolic PRRs such as the RNA-sensing Rig-I-like receptors (RLRs) in the instruction of adaptive immunity are therefore critical to understand protective immunity to infections. West Nile virus (WNV) infection of mice deficent of RLR-signaling adaptor MAVS results in a defective adaptive immune response. While this finding suggests a role for RLRs in the instruction of adaptive immunity to WNV, it is difficult to interpret due to the high WNV viremia, associated exessive antigen loads, and pathology in the absence of a MAVS-dependent innate immune response. To overcome these limitations, we have infected MAVS-deficient (MAVSKO) mice with a single-round-of-infection mutant of West Nile virus. We show that MAVSKO mice failed to produce an effective neutralizing antibody response to WNV despite normal antibody titers against the viral WNV-E protein. This defect occurred independently of antigen loads or overt pathology. The specificity of the antibody response in infected MAVSKO mice remained unchanged and was still dominated by antibodies that bound the neutralizing lateral ridge (LR) epitope in the DIII domain of WNV-E. Instead, MAVSKO mice produced IgM antibodies, the dominant isotype controlling primary WNV infection, with lower affinity for the DIII domain. Our findings suggest that RLR-dependent signals are important for the quality of the humoral immune response to WNV.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Receptors, Pattern Recognition/immunology , Adaptive Immunity/immunology , Adaptor Proteins, Signal Transducing/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , DEAD Box Protein 58/immunology , DEAD Box Protein 58/metabolism , Female , Immunity, Humoral , Immunity, Innate/immunology , Immunoglobulin M , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Pattern Recognition/metabolism , Signal Transduction/immunology , West Nile Fever/immunology , West Nile Fever/virology , West Nile virus/pathogenicityABSTRACT
The developmental programme of epithelial-mesenchymal transition (EMT), involving loss of epithelial and acquisition of mesenchymal properties, plays an important role in the invasion-metastasis cascade of cancer cells. In the present study, we show that activation of AMP-activated protein kinase (AMPK) using A769662 led to a concomitant induction of EMT in multiple cancer cell types, as observed by enhanced expression of mesenchymal markers, decrease in epithelial markers, and increase in migration and invasion. In contrast, inhibition or depletion of AMPK led to a reversal of EMT. Importantly, AMPK activity was found to be necessary for the induction of EMT by physiological cues such as hypoxia and TGFß treatment. Furthermore, AMPK activation increased the expression and nuclear localization of Twist1, an EMT transcription factor. Depletion of Twist1 impaired AMPK-induced EMT phenotypes, suggesting that AMPK might mediate its effects on EMT, at least in part, through Twist1 upregulation. Inhibition or depletion of AMPK also attenuated metastasis. Thus, our data underscore a central role for AMPK in the induction of EMT and in metastasis, suggesting that strategies targeting AMPK might provide novel approaches to curb cancer spread.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Epithelial-Mesenchymal Transition , Neoplasms/metabolism , Neoplasms/physiopathology , Nuclear Proteins/metabolism , Twist-Related Protein 1/metabolism , AMP-Activated Protein Kinases/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/enzymology , Neoplasms/genetics , Nuclear Proteins/genetics , Protein Transport , Transforming Growth Factor beta/metabolism , Twist-Related Protein 1/genetics , Up-RegulationABSTRACT
Corneal endothelium (CE) is a monolayer of mitochondria-rich cells, critical for maintaining corneal transparency compatible with clear vision. Fuchs endothelial corneal dystrophy (FECD) is a heterogeneous, genetically complex disorder, where oxidative stress plays a key role in the rosette formation during the degenerative loss of CE. Increased mitochondrial fragmentation along with excessive mitophagy activation has been detected in FECD; however, the mechanism of aberrant mitochondrial dynamics in CE cell loss is poorly understood. Here, the role of oxidative stress in mitophagy activation in FECD is investigated. Immunoblotting of FECD ex vivo specimens revealed an accumulation of PINK1 and phospho-Parkin (Ser65) along with loss of total Parkin and total Drp1. Similarly, modeling of rosette formation with menadione (MN), led to phospho-Parkin accumulation in fragmented mitochondria resulting in mitophagy-induced mitochondrial clearance, albeit possibly in a PINK1-independent manner. Loss of PINK1, phospho-Drp1, and total Drp1 was prominent after MN-induced oxidative stress, but not after mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone. Moreover, MN-induced mitophagy led to degradation of Parkin along with sequestration of Drp1 and PINK1 that was rescued by mitophagy inhibition. This study shows that in FECD, intracellular oxidative stress induces Parkin-mediated mitochondrial fragmentation where endogenous Drp1 and PINK1 are sequestered and degraded by mitophagy during degenerative loss of post-mitotic cells of ocular tissue.
Subject(s)
Endothelium, Corneal/pathology , Fuchs' Endothelial Dystrophy/pathology , Mitochondria/pathology , Mitochondrial Proteins/metabolism , Mitophagy , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Antifibrinolytic Agents/pharmacology , Endothelium, Corneal/drug effects , Endothelium, Corneal/metabolism , Fuchs' Endothelial Dystrophy/genetics , Fuchs' Endothelial Dystrophy/metabolism , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Oxidative Stress , Protein Kinases/genetics , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Vitamin K 3/pharmacologyABSTRACT
How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR-pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4(+) or CD8(+) T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR-MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or "shaved" peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR-MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.
Subject(s)
Histocompatibility Antigens Class II/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Cell Antigen Receptor Specificity , Amino Acid Sequence , Animals , Antigen Presentation , Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , CHO Cells , Cell Line , Cell Membrane/immunology , Coculture Techniques , Cricetulus , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Hybridomas , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Immunological , Molecular Sequence Data , Peptides/chemistry , Peptides/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Recombinant Fusion Proteins/immunology , Sequence DeletionABSTRACT
TCRs relay information about peptides embedded within MHC molecules (pMHC) to the ITAMs of the associated CD3γε, CD3δε, and CD3ζζ signaling modules. CD4 then recruits the Src kinase p56(Lck) (Lck) to the TCR-CD3 complex to phosphorylate the ITAMs, initiate intracellular signaling, and drive CD4(+) T cell fate decisions. Whereas the six ITAMs of CD3ζζ are key determinants of T cell development, activation, and the execution of effector functions, multiple models predict that CD4 recruits Lck proximal to the four ITAMs of the CD3 heterodimers. We tested these models by placing FRET probes at the cytosolic juxtamembrane regions of CD4 and the CD3 subunits to evaluate their relationship upon pMHC engagement in mouse cell lines. The data are consistent with a compact assembly in which CD4 is proximal to CD3δε, CD3ζζ resides behind the TCR, and CD3γε is offset from CD3δε. These results advance our understanding of the architecture of the TCR-CD3-pMHC-CD4 macrocomplex and point to regions of high CD4-Lck + ITAM concentrations therein. The findings thus have implications for TCR signaling, as phosphorylation of the CD3 ITAMs by CD4-associated Lck is important for CD4(+) T cell fate decisions.
Subject(s)
CD4 Antigens/immunology , Cell Membrane/immunology , Cytosol/immunology , Major Histocompatibility Complex/immunology , Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Cell Line, Tumor , Fluorescence Resonance Energy Transfer , Humans , MiceABSTRACT
BACKGROUND: Trauma is the leading nonobstetric cause of death in women of reproductive age, and pregnant women in particular may be at increased risk of violent trauma. Management of trauma in pregnancy is complicated by altered maternal physiology, provider expertise, potential disparate imaging, and distorted anatomy. Little is known about the impact of trauma on maternal mortality. OBJECTIVE: We sought to: (1) characterize nonviolent and violent trauma among pregnant women; (2) determine whether pregnancy is associated with increased mortality following traumatic injury; and (3) identify risk factors for trauma-related death in pregnant women. STUDY DESIGN: We studied 1148 trauma events among pregnant girls and women and 43,608 trauma events among nonpregnant girls and women of reproductive age (14-49 years) who presented to any accredited trauma center in Pennsylvania for treatment of trauma-related injuries from 2005 through 2015, as captured in the Pennsylvania Trauma Outcome Study. Traumas were categorized as violent (eg, homicide or assault) or nonviolent (eg, motor vehicle accident or accidental fall). We used modified Poisson regression to estimate relative rate of trauma-related death, adjusting for demographic characteristics and severity of trauma. RESULTS: Compared to nonpregnant women, pregnant women and girls had a lower injury severity score (8.9 vs 10.9, P < .001) and were significantly more likely to experience violent trauma (15.9% vs 9.8%, P < .001). Pregnant trauma victims had a 1.6-fold higher rate of mortality compared to their nonpregnant counterparts (P < .001), and were both more likely to be dead on arrival and to die during their hospital course (adjusted relative risk, 2.33, P < .001, and adjusted relative risk, 1.79, P = .004, respectively). Pregnancy was associated with increased mortality in both victims of nonviolent and violent trauma (adjusted relative risk, 1.69, P = .002, and adjusted relative risk, 1.60, P = .007, respectively). Pregnant trauma victims were less likely to undergo surgery (adjusted relative risk, 0.70, P = .001) and more likely to be transferred to another facility (adjusted relative risk, 1.72, P < .001). Even after adjusting for demographics and injury severity score, violent trauma was associated with 3.14-fold higher mortality in pregnant women and girls compared to nonviolent trauma (adjusted relative risk, 3.14, P = .003). CONCLUSION: Pregnant women and girls are nearly twice as likely to die after trauma and twice as likely to experience violent trauma. Universal screening for violence and trauma during pregnancy may provide an opportunity to identify women at risk for death during pregnancy.
Subject(s)
Homicide/statistics & numerical data , Pregnancy Complications/mortality , Wounds and Injuries/mortality , Accidental Falls/mortality , Accidents, Traffic/mortality , Adolescent , Adult , Case-Control Studies , Female , Humans , Maternal Mortality , Middle Aged , Pennsylvania/epidemiology , Pregnancy , Regression Analysis , Risk , Violence/statistics & numerical data , Young AdultABSTRACT
BACKGROUND: For many female cancer survivors, the preservation of reproductive potential is central to quality of life (QOL), and concerns regarding infertility may affect treatment decisions. Despite the existence of several consensus guidelines supporting routine fertility preservation consultation, to the authors' knowledge little is known regarding psychological outcomes in female cancer patients who undergo fertility preservation counseling/consultation (FPC), with or without fertility preservation (FP). METHODS: This literature review examined the effect of FPC alone, or with FP, on psychological outcomes including satisfaction, decisional regret, and QOL. PubMed and PsychINFO were systematically searched for English-language publications from the earliest available publication date of each database through March 2015. Among 111 unique articles concerning oncofertility, 13 met inclusion criteria: peer-reviewed articles reporting primary data regarding satisfaction and psychological outcomes among women who underwent FPC alone or with FP. RESULTS: A majority of women receiving FPC reported that the possibility of FP was instrumental to improved coping. Receiving FPC reduced long-term regret and dissatisfaction concerning fertility, and was associated with improved physical QOL and trends toward improved psychological QOL. Women also desired prompt, standardized, and written information addressing perceived unmet needs specific to oncofertility. Offering FPC was perceived as critical regardless of age or parity. CONCLUSIONS: To the best of the authors' knowledge, little research to date has addressed the impact of FPC alone, or with FP, on QOL in women with cancer. Clinicians should recognize the existing evidence base supporting the psychological benefit of prompt FPC. Future research must be conducted to elucidate the long-term psychosocial effects of FP.
Subject(s)
Counseling , Fertility Preservation , Neoplasms/psychology , Adult , Female , Humans , Mental Health , Quality of Life , Referral and ConsultationABSTRACT
PURPOSE: Copy-number variants have been associated with a variety of diseases, especially cancer, autism, schizophrenia, and developmental delay. The majority of clinically relevant events occur de novo, necessitating the interpretation of novel events. In this light, we present the Scripps Genome ADVISER CNV annotation pipeline and Web server, which aims to fill the gap between copy number variant detection and interpretation by performing in-depth annotations and functional predictions for copy number variants. METHODS: The Scripps Genome ADVISER CNV suite includes a Web server interface to a high-performance computing environment for calculations of annotations and a table-based user interface that allows for the execution of numerous annotation-based variant filtration strategies and statistics. RESULTS: The annotation results include details regarding location, impact on the coding portion of genes, allele frequency information (including allele frequencies from the Scripps Wellderly cohort), and overlap information with other reference data sets (including ClinVar, DGV, DECIPHER). A summary variant classification is produced (ADVISER score) based on the American College of Medical Genetics and Genomics scoring guidelines. We demonstrate >90% sensitivity/specificity for detection of pathogenic events. CONCLUSION: Scripps Genome ADVISER CNV is designed to allow users with no prior bioinformatics expertise to manipulate large volumes of copy-number variant data. Scripps Genome ADVISER CNV is available at http://genomics.scripps.edu/ADVISER/.
Subject(s)
Computational Biology/methods , DNA Copy Number Variations , Internet , Molecular Sequence Annotation/methods , Software , Databases, Genetic , Genome, Human , Genomic Structural Variation , HumansABSTRACT
Fuchs endothelial corneal dystrophy is a heterogenous disease with multifactorial etiology, and genetic, epigenetic, and exogenous factors contributing to its pathogenesis. DNA damage plays a significant role, with ultraviolet-A (UV-A) emerging as a key contributing factor. We investigate the potential application of neuropeptide α-melanocyte stimulating hormone (α-MSH) in mitigating oxidative stress induced endothelial damage. First, we examined the effects of α-MSH on a cultured human corneal endothelial cell line (HCEnC-21T) exposed to hydrogen peroxide (H2O2) induced oxidative DNA damage. We performed immunofluorescence and flow cytometry to assess DNA damage and cell death in the cultured cells. Additionally, we used an established mouse model that utilizes ultraviolet light to induce corneal endothelial cell damage resulting in decreased CEnC number, increased cell size variability, and decreased percentage of hexagonal cells. This endothelial decompensation leads to an increase in corneal thickness. Following UV-A exposure, the mice were systemically treated with α-MSH, either immediately after exposure (early treatment) or beginning two weeks post-exposure (delayed treatment). To evaluate treatment efficacy, we analyzed CEnC density and morphology using in vivo confocal microscopy, and central corneal thickness using anterior segment optical coherence tomography. Our findings demonstrated that α-MSH treatment effectively protects HCEnC-21T from free-radical induced oxidative DNA damage and subsequent cell death. In vivo, α-MSH treatment, mitigated the loss of CEnC density, deterioration of cell morphology and suppression of the resultant corneal swelling. These results underline the potential application of α-MSH as a therapeutic agent for mitigating corneal endothelial damage.
Subject(s)
DNA Damage , Disease Models, Animal , Endothelium, Corneal , Fuchs' Endothelial Dystrophy , Oxidative Stress , alpha-MSH , Animals , alpha-MSH/pharmacology , Mice , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Humans , Fuchs' Endothelial Dystrophy/pathology , Fuchs' Endothelial Dystrophy/drug therapy , DNA Damage/drug effects , Oxidative Stress/drug effects , Ultraviolet Rays/adverse effects , Cell Line , Hydrogen Peroxide/pharmacologyABSTRACT
Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.