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Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.
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Glutationa , Ratos , Transcobalaminas , Vitamina B 12 , Animais , Cristalografia por Raios X , Glutationa/metabolismo , Glutationa/análogos & derivados , Glutationa/química , Ligação Proteica , Transcobalaminas/metabolismo , Transcobalaminas/química , Vitamina B 12/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMO
Oxford Nanopore Technologies sequencing, also referred to as Nanopore sequencing, stands at the forefront of a revolution in clinical genetics, offering the potential for rapid, long read, and real-time DNA and RNA sequencing. This technology is currently making sequencing more accessible and affordable. In this comprehensive review, we explore its potential regarding precision cancer diagnostics and treatment. We encompass a critical analysis of clinical cases where Nanopore sequencing was successfully applied to identify point mutations, splice variants, gene fusions, epigenetic modifications, non-coding RNAs, and other pivotal biomarkers that defined subsequent treatment strategies. Additionally, we address the challenges of clinical applications of Nanopore sequencing and discuss the current efforts to overcome them.
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Cancer is one of the three leading causes of death worldwide. Even after successful therapy and achieving remission, the risk of relapse often remains. In this context, dormant residual cancer cells in secondary organs such as the bone marrow constitute the cellular reservoir from which late tumor recurrences arise. This dilemma leads the term of minimal residual disease, which reflects the presence of tumor cells disseminated from the primary lesion to distant organs in patients who lack any clinical or radiological signs of metastasis or residual tumor cells left behind after therapy that eventually lead to local recurrence. Disseminated tumor cells have the ability to survive in a dormant state following treatment and linger unrecognized for more than a decade before emerging as recurrent disease. They are able to breakup their dormant state and to readopt their proliferation under certain circumstances, which can finally lead to distant relapse and cancer-associated death. In recent years, extensive molecular and genetic characterization of disseminated tumor cells and blood-based biomarker has contributed significantly to our understanding of the frequency and prevalence of tumor dormancy. In this article, we describe the clinical relevance of disseminated tumor cells and highlight how latest advances in different liquid biopsy approaches can be used to detect, characterize, and monitor minimal residual disease in breast cancer, prostate cancer, and melanoma patients.
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Neoplasias da Mama , Detecção Precoce de Câncer , Masculino , Humanos , Neoplasia Residual/diagnóstico , Neoplasias da Mama/patologia , Biópsia Líquida , RecidivaRESUMO
INTRODUCTION: We evaluated the prognostic role of pre-salvage prostate-specific membrane antigen-radioguided surgery (PSMA-RGS) serum levels of alkaline phosphatase (AP), carcinoembryonic antigen (CEA), lactate dehydrogenase (LDH), and neuron-specific enolase (NSE). MATERIALS AND METHODS: Patients who consecutively underwent PSMA-RGS for prostate cancer (PCa) oligorecurrence between January 2019 and January 2022 were selected. Biomarkers were assessed one day before surgery. Cox regression and logistic regression models tested the relationship between biochemical recurrence-free survival (BFS), 6- and 12-month biochemical recurrence (BCR), and several independent variables, including biomarkers. RESULTS: 153 consecutive patients were analyzed. In the univariable Cox regression analysis, none of the biomarkers achieved predictor status (AP: hazard ratio [HR] = 1.03, 95% CI 0.99, 1.01; p = 0.19; CEA: HR = 1.73, 95% CI 0.94, 1.21; p = 0.34; LDH: HR = 1.01, 95% CI 1.00, 1.01; p = 0.05; NSE: HR = 1.02, 95% CI 0.98, 1.06; p = 0.39). The only independent predictor of BFS was the number of positive lesions on PSMA PET (HR = 1.17, 95% CI 1.02, 1.30; p = 0.03). The number of positive lesions was confirmed as independent predictor for BCR within 6 and 12 months (BCR < 6 months: odds ratio [OR] = 1.1, 95% CI 1.0, 1.3; p = 0.04; BCR < 12 months: OR = 1.1, 95% CI 1.0, 1.3; p = 0.04). CONCLUSION: The assessment of AP, CEA, LDH, and NSE before salvage PSMA-RGS showed no prognostic impact. Further studies are needed to identify possible predictors that will optimize patient selection for salvage PSMA-RGS.
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Fosfatase Alcalina , Biomarcadores Tumorais , Antígeno Carcinoembrionário , L-Lactato Desidrogenase , Recidiva Local de Neoplasia , Fosfopiruvato Hidratase , Neoplasias da Próstata , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatase Alcalina/sangue , Antígenos de Superfície/sangue , Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/sangue , Glutamato Carboxipeptidase II/sangue , L-Lactato Desidrogenase/sangue , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/diagnóstico por imagem , Fosfopiruvato Hidratase/sangue , Prognóstico , Prostatectomia/métodos , Neoplasias da Próstata/sangue , Neoplasias da Próstata/cirurgia , Neoplasias da Próstata/terapia , Estudos RetrospectivosRESUMO
Clinical tumor dormancy is specified as an extended latency period between removal of the primary tumor and subsequent relapse in a cancer patient who has been clinically disease-free. In particular, patients with estrogen receptor-positive breast cancer can undergo extended periods of more than five years before they relapse with overt metastatic disease. Recent studies have shown that minimal residual disease in breast cancer patients can be monitored by different liquid biopsy approaches like analysis of circulating tumor cells or cell-free tumor DNA. Even though the biological principles underlying tumor dormancy in breast cancer patients remain largely unknown, clinical observations and experimental studies have identified emerging mechanisms that control the state of tumor dormancy. In this review, we illustrate the latest discoveries on different molecular aspects that contribute to the control of tumor dormancy and distant metastatic relapse, then discuss current treatments affecting minimal residual disease and dormant cancer cells, and finally highlight how novel liquid biopsy based diagnostic methodologies can be integrated into the detection and molecular characterization of minimal residual disease.
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Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Microambiente Tumoral , Neoplasias da Mama/etiologia , Tomada de Decisão Clínica , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , HumanosRESUMO
Nanozymes are nanomaterials with biocatalytic properties under physiological conditions and are one class of artificial enzymes to overcome the high cost and low stability of natural enzymes. However, surface ligands on nanomaterials will decrease the catalytic activity of the nanozymes by blocking the active sites. To address this limitation, ligand-free PtAg nanoclusters (NCs) are synthesized and applied as nanozymes for various enzyme-mimicking reactions. By taking advantage of the mutual interaction of zeolitic imidazolate frameworks (ZIF-8) and Pt precursors, a good dispersion of PtAg bimetal NCs with a diameter of 1.78 ± 0.1 nm is achieved with ZIF-8 as a template. The incorporation of PtAgNCs in the voids of ZIF-8 is confirmed with structural analysis using the atomic pair-distribution function and powder X-ray diffraction. Importantly, the PtAgNCs present good catalytic activity for various enzyme-mimicking reactions, including peroxidase-/catalase- and oxidase-like reactions. Further, this work compares the catalytic activity between PtAg NCs and PtAg nanoparticles with different compositions and finds that these two nanozymes present a converse dependency of Ag-loading on their activity. This study contributes to the field of nanozymes and presents a potential option to prepare ligand-free bimetal biocatalysts with sizes in the nanocluster regime.
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Nanopartículas Metálicas , Mimetismo Molecular , Peroxidase/química , Peroxidase/metabolismo , Nanopartículas Metálicas/química , Platina/química , Prata/química , Ligas/químicaRESUMO
The aim of this study was to assess prosodic features in Finnish speakers with (n = 16) and without (n = 20) Parkinson's disease (PD), as there are no published studies to date of prosodic features in Finnish speakers with PD. Chosen metrics were articulation rate (syllables/second), pitch (mean F0) and pitch variability (standard deviation F0), energy proportion below 1 kHz (epb1kHz), normalised pairwise variability index (nPVI), and a novel syllabic prosody index (SPI). Four statistically significant results were found: (1) energy was distributed more to lower frequencies in speakers with PD compared to control speakers, (2) male PD speakers had higher pitch and (3) higher syllabic prosody index compared to control males, and (4) female PD speakers had narrower pitch variability than controls. In this study, PD was manifested as less emphatic and breathier voice. Interestingly, male PD speakers' dysprosody was manifested as an effortful speaking style, whereas female PD speakers exhibited dysprosody with a monotonous speaking style. A novel syllable-based prosody index could be a potentially useful tool in analysing prosody in disordered speech.
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Doença de Parkinson , Humanos , Masculino , Adulto , Feminino , Finlândia , Distúrbios da Fala , Idioma , Medida da Produção da FalaRESUMO
BACKGROUND: Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer. METHODS: We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes. RESULTS: Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1. CONCLUSIONS: The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells.
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Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Neoplásicas Circulantes , Neoplasias da Próstata , Linhagem Celular Tumoral , Proteínas Correpressoras , Humanos , Masculino , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Tretinoína/farmacologiaRESUMO
Immunotherapeutic treatment approaches are now an integral part of the treatment of many solid tumors. However, attempts to integrate immunotherapy into the treatment of prostate cancer have been disappointing so far. This is due to a highly immunosuppressive, "cold" tumor microenvironment, which is characterized, for example, by the absence of cytotoxic T cells, an increased number of myeloid-derived suppressor cells or regulatory T cells, a decreased number of tumor antigens, or a defect in antigen presentation. The consequence is a reduced efficacy of many established immunotherapeutic treatments such as checkpoint inhibitors. However, a growing understanding of the underlying mechanisms of tumor-immune system interactions raises hopes that immunotherapeutic strategies can be optimized in the future. The aim of this review is to provide an overview of the current status and future directions of immunotherapy development in prostate cancer. Background information on immune response and tumor microenvironment will help to better understand current therapeutic strategies under preclinical and clinical development.
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Imunoterapia , Neoplasias da Próstata , Antígenos de Neoplasias , Humanos , Fatores Imunológicos , Masculino , Neoplasias da Próstata/patologia , Linfócitos T Citotóxicos/patologia , Microambiente TumoralRESUMO
BACKGROUND: Osteoclast activation is a hallmark of breast cancer-induced bone disease while little is known about the role of osteoblasts in this process. Recently, we identified the homeodomain protein TG-interacting factor-1 (Tgif1) as a crucial regulator of osteoblast function. In this study, we demonstrate that lack of Tgif1 also restricts the progression of breast cancer bone metastases. METHODS: Transwell migration assays were used to investigate the osteoblast-breast cancer cell interaction in vitro. Molecular analyses included RNA sequencing, immunoblotting, and qRT-PCR. To determine the role of Tgif1 in metastatic bone disease, 4T1 breast cancer cells were injected intracardially into mice with a germ line deletion of Tgif1 (Tgif1-/-) or control littermates (Tgif1+/+). Progression of bone metastases and alterations in the bone microenvironment were assessed using bioluminescence imaging, immunofluorescence staining, confocal microscopy, and histomorphometry. RESULTS: Medium conditioned by osteoblasts stimulated breast cancer cell migration, indicating a potential role of osteoblasts during bone metastasis progression. Tgif1 expression was strongly increased in osteoblasts upon stimulation by breast cancer cells, demonstrating the implication of Tgif1 in the osteoblast-breast cancer cell interaction. Indeed, conditioned medium from osteoblasts of Tgif1-/- mice failed to induce breast cancer cell migration compared to control, suggesting that Tgif1 in osteoblasts augments cancer cell motility. Semaphorin 3E (Sema3E), which is abundantly secreted by Tgif1-/- osteoblasts, dose-dependently reduced breast cancer cell migration while silencing of Sema3E expression in Tgif1-/- osteoblasts partially restored the impaired migration. In vivo, we observed a decreased number of breast cancer bone metastases in Tgif1-/- mice compared to control littermates. Consistently, the presence of single breast cancer cells or micro-metastases in the tibiae was reduced in Tgif1-/- mice. Breast cancer cells localized in close proximity to Endomucin-positive vascular cells as well as to osteoblasts. Although Tgif1 deficiency did not affect the bone marrow vasculature, the number and activity of osteoblasts were reduced compared to control. This suggests that the protective effect on bone metastases might be mediated by osteoblasts rather than by the bone marrow vasculature. CONCLUSION: We propose that the lack of Tgif1 in osteoblasts increases Sema3E expression and attenuates breast cancer cell migration as well as metastases formation.
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Neoplasias Ósseas/prevenção & controle , Osso e Ossos/patologia , Neoplasias da Mama/prevenção & controle , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/fisiologia , Semaforinas/genética , Microambiente Tumoral , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Osso e Ossos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
The traditional model of metastatic progression postulates that the ability to form distant metastases is driven by random mutations in cells of the primary tumor.
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Metástase Neoplásica/genética , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Progressão da Doença , Humanos , MutaçãoRESUMO
Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.
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Dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS) allows binding analysis of biomolecules. Combining cross- and autocorrelation amplitudes yields binding degrees and concentrations of bound and unbound species. However, non-ideal detection volume overlap reduces the cross-correlation, causing overestimation of the Kd . The overlap quality factor that relates measured and true cross-correlation amplitudes has been difficult to determine, because neither a perfect 1 : 1 labeled sample nor perfectly overlapping volumes are readily accomplished. Here, we describe how a stochastically labeled sample can be used for quantitative calibration. Lipid vesicles doped with green and red fluorescent dyes yield highly reproducible relative cross-correlations and allow determination of the setup-dependent overlap quality factor. This reliable, affordable and quick-to-prepare calibration standard expedites any quantitative co-localization or binding analysis by dcFCCS.
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The present study attempts to extend the research on the effects of phonetic training on the production and perception of second-language (L2) vowels. We also examined whether success in learning L2 vowels through high-variability intensive phonetic training is related to the learners' general musical abilities. Forty Azerbaijani learners of Standard Southern British English participated in a pre-test/training/post-test setting. Discrimination and production tests were used in pre- and post-tests. The participants' musical ability was evaluated through three musical aptitude tests. Results revealed a significant improvement in the discrimination and production of L2 vowels after training. However, the lack of a one-to-one relationship between production and perception gains suggests distinct representations underlying L2 speech perception and production. There was no significant correlation between overall musical ability scores and L2 vowel learning, however, results revealed a correlation between discrimination improvements and tonal memory. This suggests tonal memory facilitates the perceptual learning of the novel phonological structure of L2.
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Idioma , Multilinguismo , Música , Fonética , Estimulação Acústica , Adulto , Feminino , Humanos , Aprendizagem , MasculinoRESUMO
Fluorescence correlation spectroscopy has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners, however, comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding, and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color fluorescence cross-correlation spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants, KD. The total protein concentration, but not the fraction of labeled protein, needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds "major-minor-mix" liposomes. A Langmuir isotherm model yields KD=(2.1±1.1)µM as the single-site dissociation constant. The dcFCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.
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Lipossomos/química , Ligação Proteica , Espectrometria de Fluorescência/métodos , GTP Fosfo-Hidrolases/química , Microscopia Confocal , Microscopia de Fluorescência , Modelos Químicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The characterization of circulating tumor cells (CTC) has the potential not only to provide important insights into molecular alterations of advanced tumor disease but also to facilitate risk prediction. Epithelial mesenchymal transition (EMT) has been discovered as important process for the development of metastases and the dissemination of tumor cells into the blood stream. In different tumor types, CTC with a mesenchymal phenotype have been reported that have presumably underwent EMT. Moreover, CTC with stem-cell like characteristics have been postulated as important drivers of tumor progression. Different platforms have been introduced to allow CTC enrichment independent of expression of epithelial antigens, as these may be downregulated in EMT- or stem-cell-like CTC. Both for CTCs with EMT- or stem-cell features different markers have been proposed. However, there is still a lack of evidence on the association of these markers with functional features and characteristics for stem cells and cells undergoing EMT.
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Biomarcadores Tumorais/sangue , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células Neoplásicas Circulantes/metabolismo , Células-Tronco Neoplásicas/metabolismo , Biomarcadores Tumorais/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
A novel class of rigid-rod bolapolyphilic molecules with three philicities (rigid aromatic core, mobile aliphatic side chains, polar end groups) has recently been demonstrated to incorporate into and span lipid membranes, and to exhibit a rich variety of self-organization modes, including macroscopically ordered snowflake structures with 6-fold symmetry. In order to support a structural model and to better understand the self-organization on a molecular scale, we here report on proton and carbon-13 high-resolution magic-angle spinning solid-state NMR investigations of two different bolapolyphiles (BPs) in model membranes of two different phospholipids (DPPC, DOPC). We elucidate the changes in molecular dynamics associated with three new phase transitions detected by calorimetry in composite membranes of different composition, namely, a change in π-π-packing, the melting of lipid tails associated with the superstructure, and the dissolution and onset of free rotation of the BPs. We derive dynamic order parameters associated with different H-H and C-H bond directions of the BPs, demonstrating that the aromatic cores are well packed below the final phase transition, showing only 180° flips of the phenyl ring, and that they perform free rotations with additional oscillations of the long axis when dissolved in the fluid membrane. Our data suggests that BPs not only form ordered superstructures, but also rather homogeneously dispersed π-packed filaments within the lipid gel phase, thus reducing the corrugation of large vesicles.
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1,2-Dipalmitoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Transição de FaseRESUMO
During our continuous search for new resistance-breaking insecticides applicable to malaria vector control, a new class of α,ß-unsaturated imines was identified by applying the principle of conformational rigidification as a powerful tool for compound optimisation. Herein we describe the successful synthesis of these compounds and their biological test results. Our lead compound 16 from this insecticidal class outperforms market standards, notably for the control of mosquito strains that exhibit either metabolic or target-site resistance to these established insecticides. In our model system for insecticide-treated mosquito nets the compound reveals long-lasting efficacy for up to several months.
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Iminas/farmacologia , Insetos Vetores , Inseticidas/farmacologia , Malária/prevenção & controle , Controle de Mosquitos/métodos , Animais , Descoberta de Drogas , Resistência a Inseticidas , Inseticidas/síntese químicaRESUMO
Agrotransfection with viral vectors is an effective solution for the transient production of valuable proteins in plants grown in contained facilities. Transfection methods suitable for field applications are desirable for the production of high-volume products and for the transient molecular reprogramming of plants. The use of genetically modified (GM) Agrobacterium strains for plant transfections faces substantial biosafety issues. The environmental biosafety of GM Agrobacterium strains could be improved by regulating their T-DNA transfer via chemically inducible expression of virE2, one of the essential Agrobacterium virulence genes. In order to identify strong and stringently regulated promoters in Agrobacterium strains, we evaluated isopropyl-ß-d-thiogalactoside-inducible promoters Plac, Ptac, PT7/lacO, and PT5/lacOlacO and cumic acid-inducible promoters PlacUV5/CuO, Ptac/CuO, PT5/CuO, and PvirE/CuO. Nicotiana benthamiana plants were transfected with a virE2-deficient A. tumefaciens strain containing transient expression vectors harboring inducible virE2 expression cassettes and containing a marker green fluorescent protein (GFP) gene in their T-DNA region. Evaluation of T-DNA transfer was achieved by counting GFP expression foci on plant leaves. The virE2 expression from cumic acid-induced promoters resulted in 47 to 72% of wild-type T-DNA transfer. Here, we present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a novel approach to address environmental biosafety concerns in agrobiotechnology.
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Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Canais Iônicos/genética , Nicotiana/genética , Agrobacterium tumefaciens/patogenicidade , Benzoatos/farmacologia , Fluorescência , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isopropiltiogalactosídeo/farmacologia , Medições Luminescentes/métodos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Transfecção/métodos , Virulência/genéticaRESUMO
A novel class of bolapolyphile (BP) molecules are shown to integrate into phospholipid bilayers and self-assemble into unique sixfold symmetric domains of snowflake-like dendritic shapes. The BPs comprise three philicities: a lipophilic, rigid, π-π stacking core; two flexible lipophilic side chains; and two hydrophilic, hydrogen-bonding head groups. Confocal microscopy, differential scanning calorimetry, XRD, and solid-state NMR spectroscopy confirm BP-rich domains with transmembrane-oriented BPs and three to four lipid molecules per BP. Both species remain well organized even above the main 1,2-dipalmitoyl-sn-glycero-3-phosphocholine transition. The BP molecules only dissolve in the fluid membrane above 70 °C. Structural variations of the BP demonstrate that head-group hydrogen bonding is a prerequisite for domain formation. Independent of the head group, the BPs reduce membrane corrugation. In conclusion, the BPs form nanofilaments by π stacking of aromatic cores, which reduce membrane corrugation and possibly fuse into a hexagonal network in the dendritic domains.