RESUMO
Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.
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RNA Polimerases Dirigidas por DNA , Plastídeos , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/química , Plastídeos/enzimologia , Transcrição GênicaRESUMO
Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciclina D/metabolismo , Instabilidade Genômica , Fase S , Animais , Linhagem Celular , Proliferação de Células , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Knockout , Mutações Sintéticas LetaisRESUMO
The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.
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Adesão Celular , Epidermólise Bolhosa , Laminina , Lentivirus , Humanos , Laminina/metabolismo , Laminina/genética , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/terapia , Epidermólise Bolhosa/patologia , Criança , Lentivirus/genética , Masculino , Feminino , Pré-Escolar , Terapia Genética/métodos , Vetores Genéticos/genética , Células Epiteliais/metabolismo , Células Cultivadas , Expressão Gênica , Adolescente , LactenteRESUMO
Mammalian (or mechanistic) target of rapamycin complex 2 (mTORC2) is a kinase complex that targets predominantly Akt family proteins, SGK1 and protein kinase C (PKC), and has well-characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular K+ concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl-), which stimulates a previously unknown scaffolding activity of the protein 'with no lysine-1' (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2, resulting in enhanced SGK1 phosphorylation and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1-dependent regulatory mechanism that harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.
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Proteínas Imediatamente Precoces , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Cloretos/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Proteínas Serina-Treonina Quinases , Serina-Treonina Quinases TOR/metabolismo , Transporte de Íons , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mamíferos/metabolismoRESUMO
PURPOSE OF REVIEW: Maintenance of plasma K + concentration within a narrow range is critical to all cellular functions. The kidneys are the central organ for K + excretion, and robust renal excretory responses to dietary K + loads are essential for survival. Recent advances in the field have challenged the view that aldosterone is at the center of K + regulation. This review will examine recent findings and propose a new mechanism for regulating K + secretion. RECENT FINDINGS: Local aldosterone-independent response systems in the distal nephron are increasingly recognized as key components of the rapid response to an acute K + load, as well as playing an essential role in sustained responses to increased dietary K + . The master kinase mTOR, best known for its role in mediating the effects of growth factors and insulin on growth and cellular metabolism, is central to these aldosterone-independent responses. Recent studies have shown that mTOR, particularly in the context of the "type 2" complex (mTORC2), is regulated by K + in a cell-autonomous fashion. SUMMARY: New concepts related to cell-autonomous K + signaling and how it interfaces with aldosterone-dependent regulation are emerging. The underlying signaling pathways and effectors of regulated K + secretion, as well as implications for the aldosterone paradox and disease pathogenesis are discussed.
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Aldosterona , Néfrons , Potássio , Transdução de Sinais , Serina-Treonina Quinases TOR , Aldosterona/metabolismo , Humanos , Néfrons/metabolismo , Animais , Serina-Treonina Quinases TOR/metabolismo , Potássio/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismoRESUMO
SIGNIFICANCE STATEMENT: Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. BACKGROUND: Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. METHODS: We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. RESULTS: A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). CONCLUSIONS: The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K +in vivo .
Assuntos
Proteínas Imediatamente Precoces , Potássio , Camundongos , Animais , Fosforilação , Potássio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Potássio na Dieta , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Rim/metabolismo , Proteínas de Transporte/metabolismo , Camundongos Knockout , Transporte de ÍonsRESUMO
Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.
Assuntos
Proteínas Imediatamente Precoces , Proteínas Monoméricas de Ligação ao GTP , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-akt , Proteína Companheira de mTOR Insensível à Rapamicina , Humanos , Proteínas Imediatamente Precoces/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Sirolimo/farmacologia , Fatores de Transcrição/metabolismoRESUMO
Arctic soils are constantly subjected to microbial invasion from either airborne, marine, or animal sources, which may impact local microbial communities and ecosystem functioning. However, in winter, Arctic soils are isolated from outside sources other than snow, which is the sole source of microorganisms. Successful colonisation of soil by snow microorganisms depends on the ability to survive and compete of both, the invading and resident community. Using shallow shotgun metagenome sequencing and amplicon sequencing, this study monitored snow and soil microbial communities throughout snow melt to investigate the colonisation process of Arctic soils. Microbial colonisation likely occurred as all the characteristics of successful colonisation were observed. The colonising microorganisms originating from the snow were already adapted to the local environmental conditions and were subsequently subjected to many similar conditions in the Arctic soil. Furthermore, competition-related genes (e.g. motility and virulence) increased in snow samples as the snow melted. Overall, one hundred potentially successful colonisers were identified in the soil and, thus, demonstrated the deposition and growth of snow microorganisms in soils during melt.
Assuntos
Ecossistema , Solo , Neve , Regiões ÁrticasRESUMO
Nonshivering thermogenesis is essential for mammals to maintain body temperature. According to the canonical view, temperature is sensed by cutaneous thermoreceptors and nerve impulses transmitted to the hypothalamus, which generates sympathetic signals to ß-adrenergic receptors in brown adipocytes. The energy for heat generation is primarily provided by the oxidation of fatty acids derived from triglyceride hydrolysis and cellular uptake. Fatty acids also activate the uncoupling protein, UCP1, which creates a proton leak that uncouples mitochondrial oxidative phosphorylation from ATP production, resulting in energy dissipation as heat. Recent evidence supports the idea that in response to mild cold, ß-adrenergic signals stimulate not only lipolysis and fatty acid oxidation, but also act through the mTORC2-Akt signaling module to stimulate de novo lipogenesis. This opposing anabolic effect is thought to maintain lipid fuel stores during increased catabolism. We show here, using brown fat-specific Gs-alpha knockout mice and cultured adipocytes that, unlike mild cold, severe cold directly cools brown fat and bypasses ß-adrenergic signaling to inhibit mTORC2. This cell-autonomous effect both inhibits lipogenesis and augments UCP1 expression to enhance thermogenesis. These findings suggest a novel mechanism for overriding ß-adrenergic-stimulated anabolic activities while augmenting catabolic activities to resolve the homeostatic crisis presented by severe cold.
Assuntos
Tecido Adiposo Marrom/metabolismo , Cromograninas/fisiologia , Temperatura Baixa , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Termogênese , Tecido Adiposo Marrom/citologia , Animais , Lipogênese , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Regulated Na+ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na+ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na+ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K+, Ca++, Mg++, Cl-, and HCO3-, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na+ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na+ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components-kinases, ubiquitin ligases, phosphatases, transcription factors, and others-have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na+ and K+ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K+ concentrations are involved in the reciprocal regulation of Na+-Cl- cotransporter (NCC) and ENaC activity to adjust renal K+ secretion to dietary intake.
Assuntos
Canais Epiteliais de Sódio , Túbulos Renais Distais , Aldosterona/metabolismo , Eletrólitos/metabolismo , Canais Epiteliais de Sódio/metabolismo , Homeostase , Transporte de Íons , Túbulos Renais Distais/metabolismo , Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Doenças Pulmonares Intersticiais , Humanos , Camundongos , Animais , Bleomicina/farmacologia , Plasmócitos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/metabolismo , Doenças Pulmonares Intersticiais/induzido quimicamenteRESUMO
mTORC2 lies at the intersection of signaling pathways that control metabolism and ion transport through phosphorylation of the AGC-family kinases, the Akt and SGK1 proteins. How mTORC2 targets these functionally distinct downstream effectors in a context-specific manner is not known. Here, we show that the salt- and blood pressure-regulatory hormone, angiotensin II (AngII) stimulates selective mTORC2-dependent phosphorylation of SGK1 (S422) but not Akt (S473 and equivalent sites). Conventional PKC (cPKC), a critical mediator of the angiotensin type I receptor (AT1R, also known as AGTR1) signaling, regulates the subcellular localization of SIN1 (also known as MAPKAP1) and SGK1. Inhibition of cPKC catalytic activity disturbs SIN1 and SGK1 subcellular localization, re-localizing them from the nucleus and a perinuclear compartment to the plasma membrane in advance of hormonal stimulation. Surprisingly, pre-targeting of SIN1 and SGK1 to the plasma membrane prevents SGK1 S422 but not Akt S473 phosphorylation. Additionally, we identify three sites on SIN1 (S128, S315 and S356) that are phosphorylated in response to cPKC activation. Collectively, these data demonstrate that SGK1 activation occurs at a distinct subcellular compartment from that of Akt and suggests a mechanism for the selective activation of these functionally distinct mTORC2 targets through subcellular partitioning of mTORC2 activity.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de SinaisRESUMO
Bacterial community composition is largely influenced by environmental factors, and this applies to the Arctic region. However, little is known about the role of spatial factors in structuring such communities. In this study, we evaluated the influence of spatial scale on bacterial community structure across an Arctic landscape. Our results showed that spatial factors accounted for approximately 10% of the variation at the landscape scale, equivalent to observations across the whole Arctic region, suggesting that while the role and magnitude of other processes involved in community structure may vary, the role of dispersal may be stable globally in the region. We assessed dispersal limitation by identifying the spatial autocorrelation distance, standing at approximately 60 m, which would be required in order to obtain fully independent samples and may inform future sampling strategies in the region. Finally, indicator taxa with strong statistical correlations with environment variables were identified. However, we showed that these strong taxa-environment associations may not always be reflected in the geographical distribution of these taxa.IMPORTANCE The significance of this study is threefold. It investigated the influence of spatial scale on the soil bacterial community composition across a typical Arctic landscape and demonstrated that conclusions reached when examining the influence of specific environmental variables on bacterial community composition are dependent upon the spatial scales over which they are investigated. This study identified a dispersal limitation (spatial autocorrelation) distance of approximately 60 m, required to obtain samples with fully independent bacterial communities, and therefore, should serve to inform future sampling strategies in the region and potentially elsewhere. The work also showed that strong taxa-environment statistical associations may not be reflected in the observed landscape distribution of the indicator taxa.
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PURPOSE OF REVIEW: The novel corona virus (SARS-CoV2) has been demonstrated to cause acute kidney injury due to direct cellular toxicity as well as due to a variety of autoimmune glomerular diseases. The concept of a surge of infected patients resulting in an overwhelming number of critical patients has been a central concern in healthcare planning during the COVID-19 era. RECENT FINDINGS: One crucial question remains as to how to manage patients with end stage renal disease and acute kidney injury in case of a massive surge of critically ill infected patients. Some publications address practical and ingenious solutions for just such a surge of need for renal replacement therapy. We present a plan for using a blood pump, readily available dialysis filter, and a prefilter and postfilter replacement fluid set up. This is in conjunction with multiple intravenous pumps to develop a simple hemofiltration apparatus. SUMMARY: The current set up may be a readily available option for use in critical situations where the need for renal replacement therapy outstrips the capacity of traditional hemodialysis services in a hospital or region.
Assuntos
Injúria Renal Aguda/terapia , COVID-19/epidemiologia , Terapia de Substituição Renal Contínua , Desastres , Hemodiafiltração , SARS-CoV-2 , Injúria Renal Aguda/etiologia , COVID-19/complicações , HumanosRESUMO
Accumulated evidence indicates that the gut microbiota affects brain function and may be altered in neurological diseases. In this study, we analyzed the gut microbiota in Cln1R151X and Cln2R207X mice, models of the childhood neurodegenerative disorders, infantile CLN1 and late infantile CLN2 Batten diseases. Significant alterations were found in the overall gut microbiota composition and also at the individual taxonomic ranks as compared to wild-type mice. The disease-specific alterations in the gut microbiota of Cln1R151X and Cln2R207X mice may contribute to the disease phenotypes observed in these mouse models. We also compared the gut microbiota composition of three wild-type mouse strains frequently used in transgenic studies: 129S6/SvEv, C57BL/6J and mixed 129S6/SvEv × C57BL/6J. Our results show that the gut microbiota of 129S6/SvEv and C57BL/6J mice differs remarkably, which likely contributes to the known, pronounced differences in behavior and disease susceptibility between these two wild-type mouse strains.
Assuntos
Biodiversidade , Microbioma Gastrointestinal , Lipofuscinoses Ceroides Neuronais/microbiologia , Animais , Modelos Animais de Doenças , Microbioma Gastrointestinal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Tripeptidil-Peptidase 1RESUMO
ISSUE ADDRESSED: People living with mental illness die on average 15 years earlier than the general population, primarily due to preventable and premature cardiovascular disease. Lifestyle interventions can be effective in reducing cardiovascular risk, yet mental health services do not routinely provide targeted lifestyle interventions. Exposing mental health staff to lifestyle interventions prior to targeting patients may be critical to changing culture and improving patient outcomes. This study aimed to improve the physical health of mental health staff through a targeted lifestyle intervention. METHODS: A pragmatic single-arm intervention study was conducted in a public mental health service, including inpatient and community settings, in Sydney, Australia. Participants in this study were n = 212 clinical and non-clinical staff. A five-session individualised lifestyle intervention (delivered over 5 weeks) incorporating physical activity and nutritional counselling was delivered by multidisciplinary teams. Participants were assessed at baseline, following the intervention, and at follow-up (mean = 16.7 weeks). The primary outcome was the barriers, attitudes, knowledge and confidence regarding screening, promoting and intervening to improve physical health outcomes of patients (M-BACK questionnaire). Secondary outcomes included anthropometric measures, cardiorespiratory fitness, sedentary time and nutritional intake. Repeated measures ANCOVAs were performed. RESULTS: A total of 212 staff (79% female) participated in this study. M-BACK total score significantly increased from baseline to follow-up (P < .001). Waist circumference, sedentary time and total energy intake all significantly decreased (all P's < .001) and cardiorespiratory fitness significantly increased (P < .001). CONCLUSION: A brief lifestyle intervention for staff of a public mental health service may increase the capability of the participants to improve their own physical health. SO WHAT?: Improving staff health may be an important strategy in improving the uptake and/or the effectiveness of lifestyle interventions targeting mental health service users.
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Transtornos Mentais , Serviços de Saúde Mental , Atitude Frente a Saúde , Feminino , Mão de Obra em Saúde , Humanos , Masculino , Transtornos Mentais/terapia , Saúde MentalRESUMO
BACKGROUND: Previous animal experiments and small human studies suggest that urinary plasmin can activate the epithelial sodium channel (ENaC) and contribute to sodium retention in nephrotic syndrome (NS), but this however is not well studied in clinical settings, and its relevance to edema formation is not well characterized in humans. We have investigated the association between urinary plasmin and clinical phenotypes in a large group of patients with NS from multiple etiologies, aiming to assess the role of urinary plasmin in sodium handling and edema formation. METHODS: Two hundred and three NS patients with urine and blood samples were divided into mild and severe symptom groups based on their edema severity. Twenty six of them had serial samples collected during the course of immunosuppressive therapy. The plasminogen-plasmin level and other key parameters were assayed, and their association with clinical manifestations were analyzed. RESULTS: One hundred and one of the 203 patients had renal biopsies performed, the results of which had included all the common types of primary NS and various types of secondary NS. Quantitative comparison and multivariate logistic regression analysis identified urinary plasminogen-plasmin to creatinine ratio (uPLG-PL/C), serum albumin, D-Dimer, and cardiac dysfunction history, but not albuminuria or 24-h urine protein, as independent risk factors for edema (p < 0.01). In patients who were treated and had serial samples, a decrease in uPLG-PL/C was identified as an independent influencing factor of edema remission (p < 0.01). Finally, the urinary fractional excretion of sodium (FENa) in patients was inversely correlated with the fractional excretion of potassium (FEK; p< 0.001), and FEK/FENa ratio was positively correlated with uPLG-PL/C (p < 0.001), suggesting a close association between uPLG-PL and ENaC activation. CONCLUSIONS: Our study identifies uPLG-PL abundance as an independent influencing factor of edema in adult NS patients, and supports the conclusion that plasmin-dependent ENaC activation is an important pathophysiological mechanism of sodium retention and edema formation in humans with NS.
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Edema/epidemiologia , Canais Epiteliais de Sódio/metabolismo , Fibrinolisina/urina , Síndrome Nefrótica/complicações , Plasminogênio/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Edema/etiologia , Edema/patologia , Edema/urina , Feminino , Fibrinolisina/metabolismo , Taxa de Filtração Glomerular/fisiologia , Humanos , Rim/patologia , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Síndrome Nefrótica/patologia , Síndrome Nefrótica/fisiopatologia , Síndrome Nefrótica/urina , Potássio/metabolismo , Eliminação Renal/fisiologia , Fatores de Risco , Sódio/metabolismo , Adulto JovemRESUMO
Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a downstream mediator of PI3K, which is essential for maintaining the functional integrity of podocytes. However, little is known about the role of SGK3 in podocyte function. Herein, we demonstrated that SGK3 contributes to the maintenance of podocyte integrity. Conditionally immortalized mouse podocyte cells (MPCs) were treated with puromycin aminonucleoside (PAN). PAN treatment inhibited the activity of SGK3 and the expression of podocin. Short hairpin RNA (shRNA)-mediated knockdown of SGK3 also reduced podocin expression in the absence of PAN. Adriamycin (ADR)-treated mice developed proteinuria and had decreased renal glomerular SGK3 expression in comparison to control mice. Consistent with a role for SGK3 in the ADR effect, SGK3 knockout (KO) mice had markedly reduced kidney podocin expression and significantly elevated proteinuria compared with wild-type mice. Electron microscopy revealed that SGK3 KO mice displayed partial effacement of podocyte foot processes. Further, a SGK3 target protein, glycogen synthase kinase-3 (GSK3), was discovered to be dramatically activated in PAN and SGK3 shRNA-treated MPCs and in SGK3 KO mice. Taken together, these data strongly suggest that SGK3 plays a significant role in regulating podocyte function, likely by controlling the expression and activity of GSK3.-Peng, L.-Q., Zhao, H., Liu, S., Yuan, Y.-P., Yuan, C.-Y., Mwamunyi, M.-J., Pearce, D., Yao, L.-J. Lack of serum- and glucocorticoid-inducible kinase 3 leads to podocyte dysfunction.
Assuntos
Podócitos/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Animais , Linhagem Celular Transformada , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Podócitos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Puromicina Aminonucleosídeo/efeitos adversos , Puromicina Aminonucleosídeo/farmacologiaRESUMO
A growing need for sampling environmental spaces in high detail is driving the rapid development of non-destructive three-dimensional (3D) sensing technologies. LiDAR sensors, capable of precise 3D measurement at various scales from indoor to landscape, still lack affordable and portable products for broad-scale and multi-temporal monitoring. This study aims to configure a compact and low-cost 3D fusion scanning system (FSS) with a multi-segment Leddar (light emitting diode detection and ranging, LeddarTech), a monocular camera, and rotational robotics to recover hemispherical, colored point clouds. This includes an entire framework of calibration and fusion algorithms utilizing Leddar depth measurements and image parallax information. The FSS was applied to scan a cottonwood (Populus spp.) stand repeatedly during autumnal leaf drop. Results show that the calibration error based on bundle adjustment is between 1 and 3 pixels. The FSS scans exhibit a similar canopy volume profile to the benchmarking terrestrial laser scans, with an r2 between 0.5 and 0.7 in varying stages of leaf cover. The 3D point distribution information from FSS also provides a valuable correction factor for the leaf area index (LAI) estimation. The consistency of corrected LAI measurement demonstrates the practical value of deploying FSS for canopy foliage monitoring.
Assuntos
Monitoramento Ambiental/métodos , Dispositivos Ópticos , Folhas de Planta , Tecnologia de Sensoriamento Remoto/instrumentação , Árvores , Algoritmos , Calibragem , Canadá , Monitoramento Ambiental/instrumentação , Desenho de Equipamento , Populus , Tecnologia de Sensoriamento Remoto/métodos , Robótica/instrumentação , Análise Espaço-Temporal , Gravação em VídeoRESUMO
In recent years, studies on psychrophilic lipases have been an emerging area of research in the field of enzymology. This study focuses on bacterial strains isolated from anthropogenically-influenced soil samples collected around Signy Island Research Station (South Orkney Islands, maritime Antarctic). Limited information on lipase activities from bacteria isolated from Signy station is currently available. The presence of lipase genes was determined using real time quantification PCR (qPCR) in samples obtained from three different locations on Signy Island. Twenty strains from the location with highest lipase gene detection were screened for lipolytic activities at a temperature of 4 °C, and from this one strain was selected for further examination based on the highest enzymatic activities obtained. Analysis of 16S rRNA sequence data of this strain showed the highest level of sequence similarity (98%) to a Pseudomonas sp. strain also isolated from Antarctica. In order to increase lipase production of this psychrophilic strain, optimisation of different parameters of physical and nutritional factors were investigated. Optimal production was obtained at 10 °C and pH 7.0, at 150 rev/min shaking rate over 36 h incubation.