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The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.
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COVID-19 , SARS-CoV-2 , Animais , Evolução Biológica , Vacinas contra COVID-19 , Humanos , National Institute of Allergy and Infectious Diseases (U.S.) , Pandemias/prevenção & controle , Variantes Farmacogenômicos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Estados Unidos/epidemiologia , VirulênciaRESUMO
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.
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Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Pandemias , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , Camelídeos Americanos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias/prevenção & controle , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/genética , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase family. It is a pseudophosphatase, which lacks the essential amino acids histidine and cysteine in the catalytic active signature motif (HCX5R). We previously reported that MK-STYX interacts with G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding-1] and reduces stress granules, stalled mRNA. To determine how MK-STYX reduces stress granules, truncated domains, CH2 (cell division cycle 25 phosphatase homology 2) and DUSP, of MK-STYX were used. Wild-type MK-STYX and the DUSP domain significantly decreased stressed granules that were induced by sodium arsenite, in which G3BP1 (a stress granule nucleator) was used as the marker. In addition, HEK/293 and HeLa cells co-expressing G3BP1-GFP and mCherry-MK-STYX, mCherry-MK-STYX-CH2, mCherry-MK-STYX-DUSP or mCherry showed that stress granules were significantly decreased in the presence of wild-type MK-STYX and the DUSP domain of MK-STYX. Further characterization of these dynamics in HeLa cells showed that the CH2 domain increased the number of stress granules within a cell, relative to wild-type and DUSP domain of MK-STYX. To further analyze the interaction of G3BP1 and the domains of MK-STYX, coimmunoprecipitation experiments were performed. Cells co-expressing G3BP1-GFP and mCherry, mCherry-MK-STYX, mCherry-MK-STYX-CH2, or mCherry-MK-STYX-DUSP demonstrated that the DUSP domain of MK-STYX interacts with both G3BP1-GFP and endogenous G3BP1, whereas the CH2 domain of MK-STYX did not coimmunoprecipitate with G3BP1. In addition, G3BP1 tyrosine phosphorylation, which is required for stress granule formation, was decreased in the presence of wild-type MK-STYX or the DUSP domain but increased in the presence of CH2. These data highlight a model for how MK-STYX decreases G3BP1-induced stress granules. The DUSP domain of MK-STYX interacts with G3BP1 and negatively alters its tyrosine phosphorylation- decreasing stress granule formation.
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DNA Helicases , Grânulos de Estresse , Humanos , Células HeLa , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , TirosinaRESUMO
Macrophages and other immune cells are important contributors to obesity-associated inflammation; however, the cellular identities of these specific populations remain unknown. In this study, we identified individual populations of myeloid cells found in mouse epididymal/visceral adipose tissue by single-cell RNA sequencing, immunofluorescence, and flow cytometry. Multiple canonical correlation analysis identified 11 unique myeloid and myeloid-associate cell populations. In obese mice, we detected an increased percentage of monocyte-derived pro-inflammatory cells expressing Cd9 and Trem2, as well as significantly decreased percentages of multiple cell populations, including tissue-resident cells expressing Lyve1, Mafb, and Mrc1. We have identified and validated a novel myeloid/macrophage population defined by Ly6a expression, exhibiting both myeloid and mesenchymal characteristics, which increased with obesity and showed high pro-fibrotic characteristics in vitro. Our mouse adipose tissue myeloid cell atlas provides an important resource to investigate obesity-associated inflammation and fibrosis.
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Gordura Intra-Abdominal/metabolismo , Células Mieloides/metabolismo , Obesidade/metabolismo , Análise de Sequência de RNA , Tecido Adiposo/metabolismo , Animais , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Receptores ImunológicosRESUMO
BACKGROUND: Patients with chronic inflammatory disease (CID) treated with immunosuppressive medications have increased risk for severe COVID-19. Although mRNA-based SARS-CoV-2 vaccination provides protection in immunocompetent persons, immunogenicity in immunosuppressed patients with CID is unclear. OBJECTIVE: To determine the immunogenicity of mRNA-based SARS-CoV-2 vaccines in patients with CID. DESIGN: Prospective observational cohort study. SETTING: Two U.S. CID referral centers. PARTICIPANTS: Volunteer sample of adults with confirmed CID eligible for early COVID-19 vaccination, including hospital employees of any age and patients older than 65 years. Immunocompetent participants were recruited separately from hospital employees. All participants received 2 doses of mRNA vaccine against SARS-CoV-2 between 10 December 2020 and 20 March 2021. Participants were assessed within 2 weeks before vaccination and 20 days after final vaccination. MEASUREMENTS: Anti-SARS-CoV-2 spike (S) IgG+ binding in all participants, and neutralizing antibody titers and circulating S-specific plasmablasts in a subset to assess humoral response after vaccination. RESULTS: Most of the 133 participants with CID (88.7%) and all 53 immunocompetent participants developed antibodies in response to mRNA-based SARS-CoV-2 vaccination, although some with CID developed numerically lower titers of anti-S IgG. Anti-S IgG antibody titers after vaccination were lower in participants with CID receiving glucocorticoids (n = 17) than in those not receiving them; the geometric mean of anti-S IgG antibodies was 357 (95% CI, 96 to 1324) for participants receiving prednisone versus 2190 (CI, 1598 to 3002) for those not receiving it. Anti-S IgG antibody titers were also lower in those receiving B-cell depletion therapy (BCDT) (n = 10). Measures of immunogenicity differed numerically between those who were and those who were not receiving antimetabolites (n = 48), tumor necrosis factor inhibitors (n = 39), and Janus kinase inhibitors (n = 11); however, 95% CIs were wide and overlapped. Neutralization titers seemed generally consistent with anti-S IgG results. Results were not adjusted for differences in baseline clinical factors, including other immunosuppressant therapies. LIMITATIONS: Small sample that lacked demographic diversity, and residual confounding. CONCLUSION: Compared with nonusers, patients with CID treated with glucocorticoids and BCDT seem to have lower SARS-CoV-2 vaccine-induced antibody responses. These preliminary findings require confirmation in a larger study. PRIMARY FUNDING SOURCE: The Leona M. and Harry B. Helmsley Charitable Trust, Marcus Program in Precision Medicine Innovation, National Center for Advancing Translational Sciences, and National Institute of Arthritis and Musculoskeletal and Skin Diseases.
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BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Saliva/virologia , COVID-19/epidemiologia , Colorimetria/métodos , Endopeptidase K/química , Humanos , Limite de Detecção , Pandemias , Testes Imediatos , SARS-CoV-2/químicaRESUMO
PURPOSE: The loss of nutrient supply is a suspected contributor of intervertebral disc degeneration. However, the extent to which low nutrition affects disc annulus fibrosus (AF) cells is unknown as nutrient deprivation has mainly been investigated in disc nucleus pulposus cells. Hence, an experimental study was designed to clarify the effects of limited nutrients on disc AF cell fate, including autophagy, the process by which cells recycle their own damaged components. METHODS: Rabbit disc AF cells were cultured in different media with varying serum concentrations under 5% oxygen. Cellular responses to changes in serum and nutrient concentrations were determined by measuring proliferation and metabolic activity. Autophagic flux in AF cells was longitudinally monitored using imaging cytometry and Western blotting for LC3, HMGB1, and p62/SQSTM1. Apoptosis (TUNEL staining and cleaved caspase-3 immunodetection) and cellular senescence (senescence-associated ß-galactosidase assay and p16/INK4A immunodetection) were measured. RESULTS: Markers of apoptosis and senescence increased, while cell proliferation and metabolic activity decreased under the withdrawal of serum and of nutrients other than oxygen, confirming cellular stress. Time-dependent increases in autophagy markers, including LC3 puncta number per cell, LC3-II expression, and cytoplasmic HMGB1, were observed under conditions of reduced nutrition, while an autophagy substrate, p62/SQSTM1, decreased over time. Collectively, these findings suggest increased autophagic flux in disc AF cells under serum and nutrient deprivation. CONCLUSION: Disc AF cells exhibit distinct responses to serum and nutrient deprivation. Cellular responses include cell death and quiescence in addition to reduced proliferation and metabolic activity, as well as activation of autophagy under conditions of nutritional stress. These slides can be retrieved under Electronic Supplementary Material.
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Anel Fibroso , Autofagia/fisiologia , Animais , Anel Fibroso/citologia , Anel Fibroso/metabolismo , Apoptose/fisiologia , Células Cultivadas , Senescência Celular , Meios de Cultura , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Oxigênio/metabolismo , CoelhosRESUMO
Many forms of neurodegenerative disease are characterized by Wallerian degeneration, an active program of axonal destruction. Recently, the important player which enacts Wallerian degeneration was discovered, the multidomain protein SARM1. Since the SARM1 protein has classically been thought of as an innate immune molecule, its role in Wallerian degeneration has raised questions on the evolutionary forces acting on it. Here, we synthesize a picture of SARM1's evolution through various organisms by examining the molecular and genetic changes of SARM1 and the genes around it. Using proteins that possess domains homologous to SARM1, we established distances and Ka/Ks values through 5671 pairwise species-species comparisons. We demonstrate that SARM1 diverged across species in a pattern similar to other SAM domain-containing proteins. This is surprising, because it was expected that SARM1 would behave more like its TIR domain relatives. Going along with this divorce from TIR, we also noted that SARM1's TIR is under stronger purifying selection than the rest of the TIR domain-containing proteins (remaining highly conserved). In addition, SARM1's synteny analysis reveals that the surrounding gene cluster is highly conserved, functioning as a potential nexus of gene functionality across species. Taken together, SARM1 demonstrates a unique evolutionary pattern, separate from the TIR domain protein family.
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Proteínas do Domínio Armadillo/genética , Proteínas do Citoesqueleto/genética , Águias/genética , Esocidae/genética , Cavalos/genética , Degeneração Walleriana/genética , Sequência de Aminoácidos/genética , Animais , Axônios/patologia , Composição de Bases/genética , Evolução Biológica , Bases de Dados GenéticasRESUMO
The integrity of long axons is essential for neural communication. Unfortunately, relatively minor stress to a neuron can cause extensive loss of this integrity. Axon degeneration is the cell-intrinsic program that actively deconstructs an axon after injury or damage. Although ultrastructural examination has revealed signs of axon degeneration in vivo, the occurrence and progression of axon degeneration in avian species have not yet been documented in vitro. Here, we use a novel cell culture system with primary embryonic zebra finch retinal ganglion cells to interrogate the properties of avian axon degeneration. First, we establish that both axotomy and a chemically induced injury (taxol and vincristine) are sufficient to initiate degeneration. These events are dependent on a late influx of calcium. In addition, as in mammals, the NAD pathway is involved, since a decrease in NMN with FK866 can reduce degeneration. Importantly, these retinal ganglion cell axons were sensitive to a pressure-induced injury, which may mimic the effect of high intraocular pressure associated with glaucoma. We have demonstrated that avian neurons undergo Wallerian degeneration in response to both physical and chemical injury. Subsequent avian studies will investigate whether blocking the degeneration pathway can protect individuals from neurodegenerative disease.
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Axônios/fisiologia , Tentilhões/fisiologia , Degeneração Neural/fisiopatologia , Células Ganglionares da Retina/fisiologia , Estresse Fisiológico/fisiologia , Degeneração Walleriana/fisiopatologia , Análise de Variância , Animais , Axônios/patologia , Axotomia , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Microscopia Confocal , Microscopia de Fluorescência , NAD/metabolismo , Degeneração Neural/tratamento farmacológico , Degeneração Neural/etiologia , Degeneração Neural/patologia , Paclitaxel , Pressão , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Estresse Fisiológico/efeitos dos fármacos , Vincristina , Degeneração Walleriana/tratamento farmacológico , Degeneração Walleriana/etiologia , Degeneração Walleriana/patologiaRESUMO
Background: Low back pain is a global health problem that originated mainly from intervertebral disc degeneration (IDD). Autophagy, negatively regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, prevents metabolic and degenerative diseases by removing and recycling damaged cellular components. Despite growing evidence that autophagy occurs in the intervertebral disc, the regulation of disc cellular autophagy is still poorly understood. Methods: Annulus fibrosus (rAF) cell cultures derived from healthy female rabbit discs were used to test the effect of autophagy inhibition or activation on disc cell fate and matrix homeostasis. Specifically, different chemical inhibitors including rapamycin, 3-methyladenine, MK-2206, and PP242 were used to modulate activities of different proteins in the PI3K/Akt/mTOR signaling pathway to assess IL-1ß-induced cellular senescence, apoptosis, and matrix homeostasis in rAF cells grown under nutrient-poor culture condition. Results: Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), reduced the phosphorylation of mTOR and its effector p70/S6K in rAF cell cultures. Rapamycin also induced autophagic flux as measured by increased expression of key autophagy markers, including LC3 puncta number, LC3-II expression, and cytoplasmic HMGB1 intensity and decreased p62/SQSTM1 expression. As expected, IL-1ß stimulation promoted rAF cellular senescence, apoptosis, and matrix homeostatic imbalance with enhanced aggrecanolysis and MMP-3 and MMP-13 expression. Rapamycin treatment effectively mitigated IL-1ß-mediated inflammatory stress changes, but these alleviating effects of rapamycin were abrogated by chemical inhibition of Akt and mTOR complex 2 (mTORC2). Conclusions: These findings suggest that rapamycin blunts adverse effects of inflammation on disc cells by inhibiting mTORC1 to induce autophagy through the PI3K/Akt/mTOR pathway that is dependent on Akt and mTORC2 activities. Hence, our findings identify autophagy, rapamycin, and PI3K/Akt/mTOR signaling as potential therapeutic targets for IDD treatment.
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Objective: Lateral flow assays (LFA) are sensitive for detecting antibodies to SARS-CoV-2 proteins within weeks after infection. This study tested samples from immunocompetent adults, and those receiving treatments for chronic inflammatory diseases (CID), before and after mRNA SARS-CoV-2 vaccination. Methods: We compared results obtained with the COVIBLOCK Covid-19 LFA to those obtained by anti-spike (S) ELISA. Results: The LFA detected anti-S antibodies in 29 of 29 (100%) of the immunocompetent and 110 of 126 (87.3%) of the CID participants after vaccination. Semiquantitative LFA scores were statistically significantly lower in samples from immunosuppressed participants, and were significantly correlated with anti-S antibody levels measured by ELISA. Conclusions: This simple LFA test is a practical alternative to laboratory-based assays for detecting anti-S antibodies after infection or vaccination. This type of test may be most useful for testing people in outpatient or resource-limited settings.
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Neurons in the peripheral nervous system (PNS) display a higher capacity to regenerate after injury than those in the central nervous system, suggesting cell specific transcriptional modules underlying axon growth and inhibition. We report a systems biology based search for PNS specific transcription factors (TFs). Messenger RNAs enriched in dorsal root ganglion (DRG) neurons compared to cerebellar granule neurons (CGNs) were identified using subtractive hybridization and DNA microarray approaches. Network and transcription factor binding site enrichment analyses were used to further identify TFs that may be differentially active. Combining these techniques, we identified 32 TFs likely to be enriched and/or active in the PNS. Twenty-five of these TFs were then tested for an ability to promote CNS neurite outgrowth in an overexpression screen. Real-time PCR and immunohistochemical studies confirmed that one representative TF, STAT3, is intrinsic to PNS neurons, and that constitutively active STAT3 is sufficient to promote CGN neurite outgrowth.
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Neurônios/fisiologia , Sistema Nervoso Periférico/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Cerebelo/citologia , Gânglios Espinais/citologia , Perfilação da Expressão Gênica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries/métodos , Neurônios/citologia , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
The dual specificity phosphatase (DUSP) family has catalytically inactive members, called pseudophosphatases. They have mutations in their catalytic motifs that render them enzymatically inactive. This study analyzes the significance of two pseudophosphatases, MK-STYX [MAPK (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein]) and STYX (serine/threonine/tyrosine-interacting protein), throughout their evolution and provides measurements and comparison of their evolutionary conservation. Phylogenetic trees were constructed to show any deviation from various species evolutionary paths. Data was collected on a large set of proteins that have either one of the two domains of MK-STYX, the DUSP domain or the cdc-25 homology (CH2) /rhodanese-like domain. The distance between species pairs for MK-STYX or STYX and Ka/Ks ratio were calculated. In addition, both pseudophosphatases were ranked among a large set of related proteins, including the active homologs of MK-STYX, MKP (MAPK phosphatase)-1 and MKP-3. MK-STYX had one of the highest species-species protein distances and was under weaker purifying selection pressure than most proteins with its domains. In contrast, the protein distances of STYX were lower than 82% of the DUSP-containing proteins and was under one of the strongest purifying selection pressures. However, there was similar selection pressure on the N-terminal sequences of MK-STYX, STYX, MKP-1, and MKP-3. We next perform statistical coupling analysis, a process that reveals interconnected regions within the proteins. We find that while MKP-1,-3, and STYX all have 2 functional units (sectors), MK-STYX only has one, and that MK-STYX is similar to MKP-3 in the evolutionary coupling of the active site and KIM domain. Within those two domains, the mean coupling is also most similar for MK-STYX and MKP-3. This study reveals striking distinctions between the evolutionary patterns of MK-STYX and STYX, suggesting a very specific role for each pseudophosphatase, further highlighting the relevance of these atypical members of DUSP as signaling regulators. Therefore, our study provides computational evidence and evolutionary reasons to further explore the properties of pseudophosphatases, in particular MK-STYX and STYX.
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Fosfatases de Especificidade Dupla , Proteínas Quinases Ativadas por Mitógeno , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Filogenia , Treonina/genética , Tirosina/metabolismoRESUMO
Cellular behaviors emerge from layers of molecular interactions: proteins interact to form complexes, pathways, and phenotypes. We show that hierarchical networks of protein interactions can be defined from the statistical pattern of proteome variation measured across thousands of diverse bacteria and that these networks reflect the emergence of complex bacterial phenotypes. Our results are validated through gene-set enrichment analysis and comparison to existing experimentally derived databases. We demonstrate the biological utility of our approach by creating a model of motility in Pseudomonas aeruginosa and using it to identify a protein that affects pilus-mediated motility. Our method, SCALES (Spectral Correlation Analysis of Layered Evolutionary Signals), may be useful for interrogating genotype-phenotype relationships in bacteria.
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Mapas de Interação de Proteínas , Proteoma , Bactérias/genética , Fímbrias Bacterianas , FenótipoRESUMO
Objectives: Neuroinflammation signaling has been identified as an important hallmark of Alzheimer's disease (AD) in addition to amyloid ß plaques (Aß) and neurofibrillary tangles (NFTs). However, the molecular mechanisms and biological processes of neuroinflammation remain unclear and have not well delineated using transcriptomics data available. Our objectives are to uncover the core neuroinflammation signaling pathways in AD using integrative network analysis on the transcriptomics data. Materials and methods: From a novel perspective, i.e., investigating weakly activated molecular signals (rather than the strongly activated molecular signals), we developed integrative and systems biology network analysis to uncover potential core neuroinflammation signaling targets and pathways in AD using the two large-scale transcriptomics datasets, i.e., Mayo Clinic (77 controls and 81 AD samples) and ROSMAP (97 controls and 260 AD samples). Results: Our analysis identified interesting core neuroinflammation signaling pathways, which are not systematically reported in the previous studies of AD. Specifically, we identified 7 categories of signaling pathways implicated on AD and related to virus infection: immune response, x-core signaling, apoptosis, lipid dysfunctional, biosynthesis and metabolism, and mineral absorption signaling pathways. More interestingly, most of the genes in the virus infection, immune response, and x-core signaling pathways are associated with inflammation molecular functions. The x-core signaling pathways were defined as a group of 9 signaling proteins: MAPK, Rap1, NF-kappa B, HIF-1, PI3K-Akt, Wnt, TGF-beta, Hippo, and TNF, which indicated the core neuroinflammation signaling pathways responding to the low-level and weakly activated inflammation and hypoxia and leading to the chronic neurodegeneration. It is interesting to investigate the detailed signaling cascades of these weakly activated neuroinflammation signaling pathways causing neurodegeneration in a chronic process, and consequently uncover novel therapeutic targets for effective AD treatment and prevention. Conclusions: The potential core neuroinflammation and associated signaling targets and pathways were identified using integrative network analysis on two large-scale transcriptomics datasets of AD.
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Most human genetic variation is classified as variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator Mitofusin 2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.
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Edição de Genes , Genômica , Humanos , Mutação , Genômica/métodos , Fenótipo , Mitocôndrias/genéticaRESUMO
Perturbed gut microbiome development has been linked to childhood malnutrition. Here, we characterize bacterial Toll/interleukin-1 receptor (TIR) protein domains that metabolize nicotinamide adenine dinucleotide (NAD), a co-enzyme with far-reaching effects on human physiology. A consortium of 26 human gut bacterial strains, representing the diversity of TIRs observed in the microbiome and the NAD hydrolase (NADase) activities of a subset of 152 bacterial TIRs assayed in vitro, was introduced into germ-free mice. Integrating mass spectrometry and microbial RNA sequencing (RNA-seq) with consortium membership manipulation disclosed that a variant of cyclic-ADPR (v-cADPR-x) is a specific product of TIR NADase activity and a prominent, colonization-discriminatory, taxon-specific metabolite. Guided by bioinformatic analyses of biochemically validated TIRs, we find that acute malnutrition is associated with decreased fecal levels of genes encoding TIRs known or predicted to generate v-cADPR-x, as well as decreased levels of the metabolite itself. These results underscore the need to consider microbiome TIR NADases when evaluating NAD metabolism in the human holobiont.
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Microbioma Gastrointestinal , Desnutrição , Animais , Bactérias/metabolismo , Criança , ADP-Ribose Cíclica , Vida Livre de Germes , Humanos , Camundongos , NAD/metabolismo , NAD+ Nucleosidase/metabolismo , Receptores de Interleucina-1RESUMO
Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase alpha (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.
Assuntos
Forma Celular , Hipocampo/enzimologia , Regeneração Nervosa , Neurônios/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genótipo , Hipocampo/embriologia , Humanos , Camundongos , Regeneração Nervosa/genética , Fenótipo , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Transfecção , Regulação para CimaRESUMO
Neuro-inflammation signaling has been identified as an important hallmark of Alzheimer's disease (AD) in addition to amyloid ß plaques (Aß) and neurofibrillary tangles (NFTs). However, our knowledge of neuro-inflammation is very limited; and the core signaling pathways associated with neuro-inflammation are missing. From a novel perspective, i.e., investigating weakly activated molecular signals (rather than the strongly activated molecular signals), in this study, we uncovered the core neuro-inflammation signaling pathways in AD. Our novel hypothesis is that weakly activated neuro-inflammation signaling pathways can cause neuro-degeneration in a chronic process; whereas, strongly activated neuro-inflammation often cause acute disease progression like in COVID-19. Using the two large-scale genomics datasets, i.e., Mayo Clinic (77 control and 81 AD samples) and RosMap (97 control and 260 AD samples), our analysis identified 7 categories of signaling pathways implicated on AD and related to virus infection: immune response, x-core signaling, apoptosis, lipid dysfunctional, biosynthesis and metabolism, and mineral absorption signaling pathways. More interestingly, most of genes in the virus infection, immune response and x-core signaling pathways, are associated with inflammation molecular functions. Specifically, the x-core signaling pathways were defined as a group of 9 signaling proteins: MAPK, Rap1, NF-kappa B, HIF-1, PI3K-Akt, Wnt, TGF-beta, Hippo and TNF, which indicated the core neuro-inflammation signaling pathways responding to the low-level and weakly activated inflammation and hypoxia, and leading to the chronic neuro-degeneration. The core neuro-inflammation signaling pathways can be used as novel therapeutic targets for effective AD treatment and prevention.