RESUMO
Neuroscientists studying the neural correlates of mouse behavior often lack access to the brain-wide activity patterns elicited during a specific task of interest. Fortunately, large-scale imaging is becoming increasingly accessible thanks to modalities such as Ca2+ imaging and functional ultrasound (fUS). However, these and other techniques often involve challenging cranial window procedures and are difficult to combine with other neuroscience tools. We address this need with an open-source 3D-printable cranial implant-the COMBO (ChrOnic Multimodal imaging and Behavioral Observation) window. The COMBO window enables chronic imaging of large portions of the brain in head-fixed mice while preserving orofacial movements. We validate the COMBO window stability using both brain-wide fUS and multisite two-photon imaging. Moreover, we demonstrate how the COMBO window facilitates the combination of optogenetics, fUS, and electrophysiology in the same animals to study the effects of circuit perturbations at both the brain-wide and single-neuron level. Overall, the COMBO window provides a versatile solution for performing multimodal brain recordings in head-fixed mice.
Assuntos
Encéfalo , Optogenética , Animais , Camundongos , Encéfalo/fisiologia , Encéfalo/diagnóstico por imagem , Optogenética/métodos , Neurônios/fisiologia , Camundongos Endogâmicos C57BL , Crânio/fisiologia , Masculino , Comportamento Animal/fisiologia , Imagem Multimodal/métodos , Ultrassonografia/métodos , Impressão TridimensionalRESUMO
α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).
Assuntos
Triticum , Inibidores da Tripsina , alfa-Amilases , Triticum/química , Cromatografia em Camada Fina/métodos , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/análise , Inibidores da Tripsina/análise , Inibidores da Tripsina/farmacologia , Proteínas de Plantas/análise , Farinha/análiseRESUMO
Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.
Assuntos
Hepatite C , Vírus de RNA , Humanos , Antivirais/farmacologia , Replicação Viral/fisiologia , RNA Viral/genética , Modelos TeóricosRESUMO
Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases.
Assuntos
Inteligência Artificial , Ligases , Ligases/química , Peptídeos/químicaRESUMO
Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage-like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest. While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags. To date - although highly desirable - no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides. In this study, a chemoenzymatic inâ vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners. This straightforward approach paves the way for customized protease inhibitors with built-in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition.
Assuntos
Peptídeos , Inibidores de Serina Proteinase , Peptídeos/química , Processamento de Proteína Pós-Traducional , Peptídeo Hidrolases , Lactamas , LactonasRESUMO
Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and show that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, especially polyprotein cleavage, and viral RNA synthesis may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the viral replication in vitro early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth. Author summary: Plus-strand RNA viruses comprise a large group of related and medically relevant viruses. The current global pandemic of COVID-19 caused by the SARS-coronavirus-2 as well as the constant spread of diseases such as dengue and chikungunya fever show the necessity of a comprehensive and precise analysis of plus-strand RNA virus infections. Plus-strand RNA viruses share similarities in their life cycle. To understand their within-host replication strategies, we developed a mathematical model that studies pan-viral similarities and virus-specific differences of three plus-strand RNA viruses, namely hepatitis C, dengue, and coxsackievirus. By fitting our model to in vitro data, we found that only small virus-specific variations in the model were required to describe the dynamics of all three viruses. Furthermore, our model predicted that ribosomes involved in viral RNA translation seem to be a key player in plus-strand RNA replication efficiency, which may determine acute or chronic infection outcome. Furthermore, our in-silico drug treatment analysis suggests that targeting viral proteases involved in polyprotein cleavage, in combination with viral RNA replication, may represent promising drug targets with broad-spectrum antiviral activity.
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A single sub-anesthetic dose of ketamine produces a rapid and sustained antidepressant response, yet the molecular mechanisms responsible for this remain unclear. Here, we identified cell-type-specific transcriptional signatures associated with a sustained ketamine response in mice. Most interestingly, we identified the Kcnq2 gene as an important downstream regulator of ketamine action in glutamatergic neurons of the ventral hippocampus. We validated these findings through a series of complementary molecular, electrophysiological, cellular, pharmacological, behavioral, and functional experiments. We demonstrated that adjunctive treatment with retigabine, a KCNQ activator, augments ketamine's antidepressant-like effects in mice. Intriguingly, these effects are ketamine specific, as they do not modulate a response to classical antidepressants, such as escitalopram. These findings significantly advance our understanding of the mechanisms underlying the sustained antidepressant effects of ketamine, with important clinical implications.
Assuntos
Ketamina , Animais , Antidepressivos/farmacologia , Hipocampo , Canal de Potássio KCNQ2/genética , Ketamina/farmacologia , Ketamina/uso terapêutico , Camundongos , Proteínas do Tecido Nervoso , NeurôniosRESUMO
Uncovering vulnerable steps in the life cycle of viruses supports the rational design of antiviral treatments. However, information on viral replication dynamics obtained from traditional bulk assays with host cell populations is inherently limited as the data represent averages over a multitude of unsynchronized replication cycles. Here, we use time-lapse imaging of virus replication in thousands of single cells, combined with computational inference, to identify rate-limiting steps for dengue virus (DENV), a widespread human pathogen. Comparing wild-type DENV with a vaccine candidate mutant, we show that the viral spread in the mutant is greatly attenuated by delayed onset of productive replication, whereas wild-type and mutant virus have identical replication rates. Single-cell analysis done after applying the broad-spectrum antiviral drug, ribavirin, at clinically relevant concentrations revealed the same mechanism of attenuating viral spread. We conclude that the initial steps of infection, rather than the rate of established replication, are quantitatively limiting DENV spread.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Ribavirina/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Células A549 , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Metilação , Microscopia de Vídeo , Mutação , RNA Viral/genética , RNA Viral/metabolismo , Análise de Célula Única , Fatores de Tempo , Imagem com Lapso de Tempo , Carga Viral , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue virus (DV) is a positive-strand RNA virus of the Flavivirus genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle in silico and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets.
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The research in biomedicine, cell signaling, diagnostics, and biocatalysis rely on selective protein binders that specifically capture a protein in a complex medium for either preparative or analytical use. These molecules are generally of biological origin and exposed to instability, denaturation, high cost, and inherently low binding capability. Imprinted polymers, serving as the artificial protein binders, demonstrate good potential to overcome these drawbacks. In this study, a novel epitope imprinting strategy is reported by employing double-cysteine-modified peptides as the templates and adsorbing the templates on a gold surface by means of forming self-assembled monolayer bridges, followed by electropolymerization to create a polymer network. The imprinted surface was initially designed to demonstrate specific affinity toward a short peptide (i.e., the epitope) or a target protein (i.e., neuron specific enolase) in buffer. This surface was subsequently used to measure the cancer biomarker in human serum that allows detecting 12 times lower concentration than threshold level of the biomarker. The molecular receptors exhibited a Kd < 65 pM for their respective target protein and low cross-reactivity with four nonspecific molecules. As compared to current strategies for the epitope imprinting, for example, through traditional, vertically adsorbed, or histidine-modified peptides, such a molecularly tunable system based on a surface-imprinting process may provide more efficient sensing systems with desirable affinity, sensitivity, and specificity in diagnostics applications.
Assuntos
Biomarcadores Tumorais/sangue , Epitopos/química , Neoplasias Pulmonares/sangue , Impressão Molecular , Oligopeptídeos/sangue , Carcinoma de Pequenas Células do Pulmão/sangue , Técnicas Biossensoriais , Técnicas Eletroquímicas , Eletrodos , Humanos , Modelos Moleculares , Conformação Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Telomerase is best known for its function in maintaining telomeres but has also multiple additional, non-canonical functions. One of these functions is the decrease of oxidative stress and DNA damage due to localisation of the telomerase protein TERT into mitochondria under oxidative stress. However, the exact molecular mechanisms behind these protective effects are still not well understood. We had shown previously that overexpression of human telomerase reverse transcriptase (hTERT) in human fibroblasts results in a decrease of mitochondrial DNA (mtDNA) damage after oxidative stress. MtDNA damage caused by oxidative stress is removed via the base excision repair (BER) pathway. Therefore we aimed to analyse whether telomerase is able to improve this pathway. We applied different types of DNA damaging agents such as irradiation, arsenite treatment (NaAsO2) and treatment with hydrogen peroxide (H2O2). Using a PCR-based assay to evaluate mtDNA damage, we demonstrate that overexpression of hTERT in MRC-5 fibroblasts protects mtDNA from H2O2 and NaAsO2 induced damage, compared with their isogenic telomerase-negative counterparts. However, overexpression of hTERT did not seem to increase repair of mtDNA after oxidative stress, but promoted increased levels of manganese superoxide dismutase (MnSOD) and forkhead-box-protein O3 (FoxO3a) proteins during incubation in serum free medium as well as under oxidative stress, while no differences were found in protein levels of catalase. Together, our results suggest that rather than interfering with mitochondrial DNA repair mechanisms, such as BER, telomerase seems to increase antioxidant defence mechanisms to prevent mtDNA damage and to increase cellular resistance to oxidative stress. However, the result has to be reproduced in additional cellular systems in order to generalise our findings.
Assuntos
Meios de Cultura Livres de Soro/química , Mitocôndrias/genética , Superóxido Dismutase/genética , Telomerase/genética , Arsenitos/efeitos adversos , Células Cultivadas , Reparo do DNA , DNA Mitocondrial/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Peróxido de Hidrogênio/efeitos adversos , Mitocôndrias/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Telomerase/metabolismo , Raios Ultravioleta/efeitos adversos , Regulação para CimaRESUMO
The E. coli siderophore enterobactin, the strongest FeIII chelator known to date, forms hexacoordinate complexes with SiIV , GeIV , and TiIV . Synthetic protocols have been developed to prepare non-symmetric enterobactin analogues with varying denticities. Various benzoic acid residues were coupled to the macrocyclic lactone to afford a diverse library of ligands. These enterobactin analogues were bound to SiIV , GeIV , and TiIV , and the complexes were investigated through experimental and computational techniques. The binding behavior of the synthesized chelators enabled assessment of the contribution of each of the phenolic hydroxy groups in enterobactin to metal-ion complexation. It was found that at least four O-donors are needed for enterobactin derivatives to act as metal binders. Density functional theory calculations indicate that the strong binding behavior of enterobactin can be ascribed to a diminished translational entropy penalty, a common feature of the chelate effect, coupled with the structural arrangement of the three catechol moieties, which allows the triseryl base to be installed without distorting the preferred local metal-binding geometry of the catecholate ligands.
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This research aims to engineer molecularly imprinted polymer (MIP)-based synthetic receptors for the molecular recognition of neuron specific enolase (NSE) biomarker. The synthetic peptide derived from the NSE was synthesized along with its cysteine and histidine modified versions. The modified peptides were utilized as templates for molecular imprinting, which was achieved by combination of epitope- and electrochemical surface imprinting strategy. The subsequently generated imprinted cavities were used for the detection of the NSE derived peptide and NSE. The imprints created with cysteine (CME) and histidine modified epitopes (HME) could detect the peptide in a concentration range of 2-128⯵M and 15.6â¯nM to 128⯵M, respectively. The recognition of NSE was achieved by the same imprints in a linear range of 1-64â¯ngâ¯mL-1 (CME) and 0.25-64â¯ngâ¯mL-1 (HME), respectively. The target molecules bound to the control polymer very weakly, confirming the high selectivity of the MIP cavities. Selectivity studies resulted in imprinting factors of 8.8 and 11 for the CME and HME imprints, respectively. The affinity analyses provided dissociation constants of 2.3â¯×â¯10-10 M and 3â¯×â¯10-11 M for NSE recognition using the corresponding epitope imprints. Cross-reactivity studies with non-specific molecules proved high specificity of the artificial receptors for the targets.
Assuntos
Biomarcadores/química , Técnicas Biossensoriais , Epitopos/isolamento & purificação , Fosfopiruvato Hidratase/isolamento & purificação , Materiais Biomiméticos/química , Cisteína/química , Epitopos/química , Histidina/química , Impressão Molecular , Peptídeos/química , Fosfopiruvato Hidratase/química , Polímeros/químicaRESUMO
Alzheimer's disease (AD) is characterized by amyloid-ß plaques, tau pathology and vascular impairment including pericyte damage. Pericytes are perivascular cells of the blood-brain barrier and can differentiate into different cell types in vitro including microglia. The aim of the present study is to explore if primary mouse brain pericytes isolated and cultured from transgenic AD (APP_SweDI) mice can differentiate into CD11b+ (integrin alpha M) microglia in vitro. We show that primary pericytes (passage 5) isolated from wildtype C57BL6 mice differentiated into CD11b+ microglia (Type B, >90%), when exposed to a differentiation factor mix of FGF-2, cAMP and fibronectin. This differentiation was time-dependent and seen as a large 80â¯kDa CD11b fragment (days 1-8) and a smaller 50â¯kDA CD11b fragment (>4â¯days). These pericytes did not differentiate into neurons, astroglia or oligodendroglia. However, pericytes isolated from transgenic AD mice differentiated into CD11b+ microglia (Type A, <10%) without addition of exogenous differentiation factors, displayed moderate Iba1+ immunostaining and phagocytic activity, but were still positive for PDGFRß. In conclusion, we show for the first time that primary mouse pericytes from AD mice have the potential to spontanously differentiate in vitro into a CD11b+ microglial-like (Type A) cell type, but we do not provide evidence that these pericytic microglia display a full active microglial cell.
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Doença de Alzheimer/metabolismo , Antígeno CD11b/metabolismo , Microglia/metabolismo , Pericitos/metabolismo , Animais , Astrócitos/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Pericitos/patologiaRESUMO
The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release.
Assuntos
Tonsila do Cerebelo/fisiologia , Ansiedade/psicologia , Hormônio Liberador da Corticotropina/deficiência , Dopamina/metabolismo , Neurônios GABAérgicos/fisiologia , Tonsila do Cerebelo/citologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Hormônio Liberador da Corticotropina/farmacologia , Espinhas Dendríticas/ultraestrutura , Injeções , Masculino , Camundongos , Camundongos Knockout , Atividade Motora , Optogenética , Percepção da Dor , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/fisiologiaRESUMO
Natural products and their semisynthetic derivatives are an important source of drugs for the pharmaceutical industry. Bacteria are prolific producers of natural products and encode a vast diversity of natural product biosynthetic gene clusters. However, much of this diversity is inaccessible to natural product discovery. Here, we use a combination of phylogenomic analysis of the microviridin biosynthetic pathway and chemo-enzymatic synthesis of bioinformatically predicted microviridins to yield new protease inhibitors. Phylogenomic analysis demonstrated that microviridin biosynthetic gene clusters occur across the bacterial domain and encode three distinct subtypes of precursor peptides. Our analysis shed light on the evolution of microviridin biosynthesis and enabled prioritization of their chemo-enzymatic production. Targeted one-pot synthesis of four microviridins encoded by the cyanobacterium Cyanothece sp. PCC 7822 identified a set of novel and potent serine protease inhibitors, the most active of which had an IC50 value of 21.5 nM. This study advances the genome mining techniques available for natural product discovery and obviates the need to culture bacteria.
Assuntos
Vias Biossintéticas/genética , Depsipeptídeos/biossíntese , Genoma Bacteriano , Filogenia , Inibidores de Serina Proteinase/biossíntese , Proteínas de Bactérias/genética , Biologia Computacional , Cianobactérias/enzimologia , Cianobactérias/genética , Mineração de Dados , Genômica , Família MultigênicaRESUMO
Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is mainly characterized by beta-amyloid (Aß) plaque deposition, Tau pathology and dysfunction of the cholinergic system causing memory impairment. The aim of the present study was to examine (1) anxiety and cognition, (2) Aß plaque deposition and (3) degeneration of cholinergic neurons in the nucleus basalis of Meynert (nbM) and cortical cholinergic innervation in an Alzheimer mouse model (APP_SweDI; overexpressing amyloid precursor protein (APP) with the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations). Our results show that 12-month-old APP_SweDI mice were more anxious and had more memory impairment. A large number of Aß plaques were already visible at the age of 6 months and increased with age. A significant decrease in cholinergic neurons was seen in the transgenic mouse model in comparison to the wild-type mice, identified by immunohistochemistry against choline acetyltransferase (ChAT) and p75 neurotrophin receptor as well as by in situ hybridization. Moreover, a significant decrease in cortical cholinergic fiber density was found in the transgenic mice as compared to the wild-type. In the cerebral cortex of APP_SweDI mice, swollen cholinergic varicosities were seen in the vicinity of Aß plaques. In conclusion, the present study shows that in an AD mouse model (APP_SweDI mice) a high Aß plaque load in the cortex causes damage to cholinergic axons in the cortex, followed by subsequent retrograde-induced cell death of cholinergic neurons and some forms of compensatory processes. This degeneration was accompanied by enhanced anxiety and impaired cognition.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide/metabolismo , Ansiedade/fisiopatologia , Núcleo Basal de Meynert , Comportamento Animal/fisiologia , Córtex Cerebral , Neurônios Colinérgicos/patologia , Modelos Animais de Doenças , Placa Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Núcleo Basal de Meynert/metabolismo , Núcleo Basal de Meynert/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
A series of novel palladium(ii) acetylacetonato complexes bearing mesoionic carbenes (MICs) have been synthesized and characterized. The synthesis of the complexes of type (MIC)Pd(acac)I (MIC = 1-mesityl-3-methyl-4-phenyl-1,2,3-triazol-5-ylidene (1), 1,4-(2,4,6-methyl)-phenyl-3-methyl-1,2,3-triazol-5-ylidene (2), 1,4-(2,6-diisopropyl)-phenyl-3-methyl-1,2,3-triazol-5-ylidene (3); acac = acetylacetonato) via direct metalation starting from the corresponding triazolium iodides and palladium(ii) acetylacetonate is described herein. All complexes were characterized by ¹H- and 13C-NMR spectroscopy and high resolution mass spectrometry. Additionally, two of the complexes were characterized by single crystal X-ray crystallography confirming a square-planar coordination geometry of the palladium(ii) center. A delocalized bonding situation was observed within the triazolylidene rings as well as for the acac ligand respectively. Complex 2 was found to be an efficient pre-catalyst for the Suzuki-Miyaura cross coupling reaction between aryl-bromides or -chlorides with phenylboronic acid.
Assuntos
Complexos de Coordenação/síntese química , Catálise , Química Click , Cristalografia por Raios X , Hidroxibutiratos/química , Metano/análogos & derivados , Metano/química , Conformação Molecular , Paládio/química , Pentanonas/químicaRESUMO
Alzheimer's disease (AD) is a severe neurodegenerative disorder of the brain, characterized by extracellular beta-amyloid (Aß) plaques, intracellular tau pathology, neurodegeneration and inflammation. There is clear evidence that the blood-brain barrier is damaged in AD and that vessel function is impaired. Alpha-smooth muscle actin (αSMA) is a prominent protein expressed on brain vessels, especially in cells located closer to the arteriole end of the capillaries, which possibly influences the blood vessel contraction. The aim of the present study was to observe αSMA protein and mRNA expression in isolated brain vessel extracts and cortex in an Alzheimer mouse model with strong Aß plaque deposition. Our data revealed a prominent expression of αSMA protein in isolated brain vessel extracts of AD mice by Western blot analysis. Immunostaining showed that these vessels were associated with Aß plaques. Quantitative real-time PCR analysis confirmed this increase at the mRNA expression level and showed a significant increase of transforming growth factor beta-1 mRNA expression in AD mice. In situ hybridization demonstrated a strong expression pattern of αSMA mRNA in the whole cortex and hippocampus. In conclusion, our data provide evidence that αSMA protein and mRNA are enhanced in vessels in an AD mouse model, possibly counteracting vessel malfunction in AD.
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Actinas/biossíntese , Doença de Alzheimer/metabolismo , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , RNA Mensageiro/biossíntese , Animais , Barreira Hematoencefálica/metabolismo , Circulação Cerebrovascular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient inâ vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient inâ vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.