ABSTRACT
The study objective is to evaluate the influence of attrition from a paediatric weight management program (PWM) on health indicators over a 2-year period. In this observational study, children and youth with obesity were recruited at entry into a family-based behaviour modification PWM and had four research study visits, independent of clinic visits, over 2 years. Participants were divided into attrition groups based on length of clinic enrolment. Body composition, cardiometabolic health and health-related quality of life (HRQoL) were assessed. Among 269 children enrolled, 19% had no clinic treatment visit, 16% had treatment visits only up to 6 months, 23% up to 1 year and 42% had at least one clinic visit after 1 year (No Attrition). Greater declines in BMI z-score and body fat were seen at 2 years in children with No Attrition, while improvements in HRQoL were similar for all attrition groups. Children who attended at least one treatment visit reported improved HRQoL up to 2 years, regardless of duration in clinic. In contrast, declines in body fat and BMI z-score were greater at 2 years for those with at least one visit after 1 year. Continued efforts to reduce attrition are likely to improve anthropometric health outcomes during PWM.
Subject(s)
Obesity , Quality of Life , Adolescent , Humans , Child , Body Mass Index , Obesity/therapy , Anthropometry , Body CompositionABSTRACT
We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.
Subject(s)
Neoplasms , Humans , Mice , Animals , Ion Channels/genetics , Membrane Proteins/geneticsABSTRACT
The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.
Subject(s)
Hydrogen Peroxide , Neisseria gonorrhoeae , Bacterial Proteins/genetics , Fimbriae, Bacterial , Hydrogen Peroxide/pharmacology , Iron , Neisseria gonorrhoeae/genetics , StreptonigrinABSTRACT
BACKGROUND: Pacifiers have been shown to affect maxillary growth related to the anatomic structure of the palate and forces placed upon it during sucking. This study compares and evaluates the mechanical behavior of pacifiers of different design and size (i.e., fit), identified by brand and size, positioned in age-specific palatal models with respect to both contact area and force when subjected to peristaltic tongue function and intraoral pressure related to non-nutritive sucking. METHODS: Nonlinear finite element analyses were used to simulate dynamic mechanical interaction between the pacifiers and palates. Time-varying, external pressure loads were applied which represent intraoral pressure arising from non-nutritive sucking and peristaltic behavior of the tongue. The silicone rubber pacifier bulb was represented using a hyperelastic material model. RESULTS: Results from the finite element analyses include deformation, stress, strain, contact area, and contact force. Mechanical interaction was evaluated in terms of the spatial distribution of the contact area and force between the pacifier and the palate. The resulting palatal interaction profiles were quantitatively compared to assess how pacifier fit specifically affects the support provided to two areas of the palate, the palatal vault and the Tektal wall. CONCLUSIONS: Pacifiers interact with the palate differently based on their fit (i.e., design and size) regardless of whether they are labeled conventional or orthodontic. Finite element analysis is an effective tool for evaluating how a pacifier's design affects functional mechanics and for providing guidance on biometric sizing.
Subject(s)
Malocclusion , Pacifiers , Finite Element Analysis , Humans , Infant , Pacifiers/adverse effects , Palate , Sucking Behavior , TongueABSTRACT
BACKGROUND: There is minimal objective data regarding adverse events related to endoscopic retrograde cholangio-pancreatography (ERCP) in patients with cirrhosis compared to those without cirrhosis and even fewer data comparing complications among cirrhosis patients based on severity of cirrhosis. AIM: To determine if patients with cirrhosis are at increased risk of adverse events related to ERCP: mainly pancreatitis, bleeding, perforation, cholangitis, and mortality; And to see if higher Child-Pugh (CP) score and Model for End-Stage Liver Disease (MELD) score are associated with higher post-ERCP complications. METHODS: We performed a retrospective analysis of 692 patients who underwent ERCP and analyzed the impact of cirrhosis etiology, gender, type of sedation used during procedure, interventions performed, and co-morbidities on the rate of complications in cirrhosis patients as compared to non-cirrhosis patients. RESULTS: Overall complications were higher in those with cirrhosis as compared to those without cirrhosis (P = 0.015 at significance level of 0.05). CP class, especially CP class C, was shown to be associated with a significantly higher rate of ERCP complications as compared to CP class A and CP class B (P = 0.010 at significance level of 0.05). CONCLUSION: The results of our study reaffirm that liver cirrhosis has an impact on the occurrence of complications during ERCP. Our study shows that CP class seems to be more reliable as compared to MELD score in predicting complications of ERCP in cirrhosis patients.
ABSTRACT
Leukemic stem cells (LSCs) comprise a very rare cell population that results in the development of acute myeloid leukemia. The selective targeting of drivers in LSCs with small molecule inhibitors holds promise for treatment of acute myeloid leukemia. Recently, we reported the identification of inhibitors of the histone lysine demethylase JMJD1C that preferentially kill MLL rearranged acute leukemia cells. Here, we report the identification of jumonji domain modulator #7 (JDM-7). Surface plasmon resonance analysis showed that JDM-7 binds to JMJD1C and its family homolog JMJD1B. JDM-7 did not significantly suppress cell proliferation in liquid cell culture at higher doses, although it led to a significant decrease in semi-solid colony formation experiments at lower concentrations. Moreover, low doses of JDM-7 did not suppress the proliferation of erythroid progenitor cells. We identified that JDM-7 downregulates the LSC self-renewal gene HOXA9 in leukemia cells. We further found that the structure of JDM-7 is similar to that of tadalafil, a drug approved by the US Food and Drug Administration. Molecular docking and surface plasmon resonance analysis showed that tadalafil binds to JMJD1C. Moreover, similar to JDM-7, tadalafil suppressed colony formation of leukemia cells in semi-solid cell culture at a concentration that did not affect primary umbilical cord blood cells. In summary, we have identified JDM-7 and tadalafil as potential JMJD1C modulators that selectively inhibit the growth of LSCs.
Subject(s)
Antineoplastic Agents/pharmacology , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Neoplastic Stem Cells/drug effects , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Drug Screening Assays, Antitumor , Gene Expression Regulation, Leukemic/drug effects , Homeodomain Proteins/genetics , Human Umbilical Vein Endothelial Cells , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Primary Cell Culture , Tadalafil/pharmacology , Tadalafil/therapeutic useABSTRACT
Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain.IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.
Subject(s)
Bacterial Proteins/genetics , Fimbriae, Bacterial/physiology , Neisseria gonorrhoeae/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , DNA, Bacterial/genetics , Fimbriae, Bacterial/classification , Gene Expression Regulation, Bacterial , Neisseria gonorrhoeae/metabolism , Phenotype , Protein Domains , VirulenceABSTRACT
Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.
Subject(s)
Antineoplastic Agents/pharmacology , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Leukemia, Biphenotypic, Acute/drug therapy , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Adult , Aged , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Bone Marrow/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Demethylation/drug effects , DNA Methylation/drug effects , Drug Screening Assays, Antitumor , Drug Synergism , Female , Histones/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Leukemia, Biphenotypic, Acute/pathology , Male , Middle Aged , Molecular Docking Simulation , Oxidoreductases, N-Demethylating/chemistry , Oxidoreductases, N-Demethylating/metabolism , Protein Domains , Structure-Activity Relationship , Surface Plasmon Resonance , Tretinoin/pharmacology , Tretinoin/therapeutic useABSTRACT
Compensation among paralogous transcription factors (TFs) confers genetic robustness of cellular processes, but how TFs dynamically respond to paralog depletion on a genome-wide scale in vivo remains incompletely understood. Using single and double conditional knockout of myocyte enhancer factor 2 (MEF2) family TFs in granule neurons of the mouse cerebellum, we find that MEF2A and MEF2D play functionally redundant roles in cerebellar-dependent motor learning. Although both TFs are highly expressed in granule neurons, transcriptomic analyses show MEF2D is the predominant genomic regulator of gene expression in vivo. Strikingly, genome-wide occupancy analyses reveal upon depletion of MEF2D, MEF2A occupancy robustly increases at a subset of sites normally bound to MEF2D. Importantly, sites experiencing compensatory MEF2A occupancy are concentrated within open chromatin and undergo functional compensation for genomic activation and gene expression. Finally, motor activity induces a switch from non-compensatory to compensatory MEF2-dependent gene regulation. These studies uncover genome-wide functional interdependency between paralogous TFs in the brain.
Subject(s)
Cerebellum/metabolism , Chromatin/metabolism , Gene Expression Regulation , Neurons/metabolism , Animals , Cerebellum/cytology , Chromatin/genetics , Genome-Wide Association Study , MEF2 Transcription Factors/genetics , MEF2 Transcription Factors/metabolism , Mice , Neurons/cytologyABSTRACT
Neuronal activity-dependent transcription is tuned to ensure precise gene induction during periods of heightened synaptic activity, allowing for appropriate responses of activated neurons within neural circuits. The consequences of aberrant induction of activity-dependent genes on neuronal physiology are not yet clear. Here, we demonstrate that, in the absence of synaptic excitation, the basic-helix-loop-helix (bHLH)-PAS family transcription factor ARNT2 recruits the NCoR2 co-repressor complex to suppress neuronal activity-dependent regulatory elements and maintain low basal levels of inducible genes. This restricts inhibition of excitatory neurons, maintaining them in a state that is receptive to future sensory stimuli. By contrast, in response to heightened neuronal activity, ARNT2 recruits the neuronal-specific bHLH-PAS factor NPAS4 to activity-dependent regulatory elements to induce transcription and thereby increase somatic inhibitory input. Thus, the interplay of bHLH-PAS complexes at activity-dependent regulatory elements maintains temporal control of activity-dependent gene expression and scales somatic inhibition with circuit activity.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Regulation , Neurons/metabolism , Nuclear Receptor Co-Repressor 2/metabolism , Animals , Mice , Neural Inhibition , Regulatory Elements, Transcriptional , Transcriptional ActivationABSTRACT
Staphylococcus aureus, an opportunistic pathogen member of the nasal and skin microbiota, can also be found in human oral samples and has been linked to infectious diseases of the oral cavity. As the nasal and oral cavities are anatomically connected, it is currently unclear whether S. aureus can colonize the oral cavity and become part of the oral microbiota, or if its presence in the oral cavity is simply transient. To start addressing this question, we assessed S. aureus ability to directly bind selected members of the oral microbiota as well as its ability to integrate into a human-derived complex oral microbial community in vitro. Our data show that S. aureus forms aggregates with Fusobacterium nucleatum and Porphyromonas gingivalis and that it can incorporate into the human-derived in vitro oral community. Further analysis of the F. nucleatum-S. aureus interaction revealed that the outer-membrane adhesin RadD is partially involved in aggregate formation and that the RadD-mediated interaction leads to an increase in expression of the staphylococcal global regulator gene sarA. Our findings lend support to the notion that S. aureus can become part of the complex microbiota of the human mouth, which could serve as a reservoir for S. aureus. Furthermore, direct interaction with key members of the oral microbiota could affect S. aureus pathogenicity contributing to the development of several S. aureus associated oral infections.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Fusobacterium nucleatum/metabolism , Microbiota , Mouth/microbiology , Staphylococcus aureus/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Biofilms , Fusobacterium nucleatum/genetics , Humans , Protein Binding , Staphylococcus aureus/geneticsABSTRACT
Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to Nε-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.
Subject(s)
Acetyltransferases/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , RNA, Small Untranslated/genetics , Transcription, Genetic , Acetyltransferases/chemistry , Biocatalysis , Escherichia coli Proteins/metabolism , Models, Molecular , Multienzyme Complexes/metabolism , Phosphoprotein Phosphatases/metabolism , Promoter Regions, Genetic/genetics , Protein Domains , Protein Kinases/metabolismABSTRACT
Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.
Subject(s)
Geniculate Bodies/metabolism , Retinal Ganglion Cells/metabolism , TWEAK Receptor/biosynthesis , Visual Perception/physiology , Animals , Female , Gene Expression , Geniculate Bodies/growth & development , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Optic Tract/growth & development , Optic Tract/metabolism , Retina/metabolism , TWEAK Receptor/geneticsABSTRACT
Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/ß-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
Subject(s)
Kinesins/metabolism , Signal Transduction , Wnt-5a Protein/metabolism , Animals , Cell Line , Embryonic Development/physiology , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Kinesins/genetics , Mice , Mice, Inbred C57BL , Morphogenesis/drug effects , Proteomics , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Wnt Signaling Pathway , Wnt-5a Protein/pharmacology , beta Catenin/metabolismABSTRACT
An estimated 1.5 billion microbial infections occur globally each year and result in â¼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of â¼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE: This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.
Subject(s)
Bacterial Infections/diagnosis , Cross Infection/diagnosis , DNA, Bacterial/analysis , Lab-On-A-Chip Devices , Microfluidics/methods , Point-of-Care Systems , Acinetobacter baumannii/classification , Acinetobacter baumannii/genetics , Bacterial Infections/microbiology , Cross Infection/microbiology , DNA Primers/genetics , DNA, Bacterial/genetics , Drug Resistance, Multiple, Bacterial , Enterobacter/classification , Enterobacter/genetics , Enterococcus faecium/classification , Enterococcus faecium/genetics , Humans , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/genetics , Microfluidics/instrumentation , Pseudomonas aeruginosa/classification , Pseudomonas aeruginosa/genetics , Staphylococcus aureus/classification , Staphylococcus aureus/geneticsABSTRACT
Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.
Subject(s)
Evolution, Molecular , Muscle Proteins/metabolism , Neocortex/metabolism , Neurons/metabolism , Transcription Factors/metabolism , Transcriptome , Animals , Base Sequence , Bone and Bones/metabolism , Dendrites/metabolism , Enhancer Elements, Genetic/genetics , Female , Humans , MEF2 Transcription Factors/metabolism , Macaca mulatta , Male , Mice , Molecular Sequence Data , Muscle Proteins/genetics , Muscles/metabolism , Neocortex/cytology , Neurons/cytology , Organ Specificity , Species Specificity , Transcription Factors/geneticsABSTRACT
In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.
Subject(s)
Acetyltransferases/metabolism , Carbon Cycle , Carbon/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Processing, Post-Translational , Acetates/metabolism , Acetylation , Acetyltransferases/genetics , Carbon Cycle/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Glucose/metabolism , Lysine/metabolism , Metabolic Networks and Pathways , ProteomicsABSTRACT
N(ε) -lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ε-amino group of a lysine residue can be acetylated either catalytically by acetyl-coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA-dependent N(ε) -lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass spectrometric, structural and immunological evidence that CobB, a deacetylase of the sirtuin family of NAD(+) -dependent deacetylases, can reverse acetylation regardless of acetyl donor or acetylation mechanism. We analyzed 69 lysines on 51 proteins that we had previously detected as robustly, reproducibly, and significantly more acetylated in a cobB mutant than in its wild-type parent. Functional and pathway enrichment analyses supported the hypothesis that CobB regulates protein function in diverse and often essential cellular processes, most notably translation. Combined mass spectrometry, bioinformatics, and protein structural data provided evidence that the accessibility and three-dimensional microenvironment of the target acetyllysine help determine CobB specificity. Finally, we provide evidence that CobB is the predominate deacetylase in E. coli.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Lysine/metabolism , Sirtuins/metabolism , Acetylation , Substrate SpecificityABSTRACT
The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Organophosphates/metabolism , Proteomics/methods , Acetylation/drug effects , Amino Acid Sequence , Binding Sites , Blotting, Western , Crystallography, X-Ray , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli Proteins/chemistry , Glucose/pharmacology , Kinetics , Lysine/metabolism , Mass Spectrometry , Molecular Sequence Data , Mutant Proteins/metabolism , Staining and LabelingABSTRACT
While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.