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Introduction: Protease activity can serve as a highly specific biomarker for application in health, biotech, and beyond. The aim of this study was to develop a protease cleavable synthetic protein platform to detect protease activity in a rapid cell-free setting. Methods: The protease sensor is modular, with orthogonal peptide tags at the N and C terminal ends, which can be uncoupled via a protease responsive module located in between. The sensor design allows for several different readouts of cleavage signal. A protein 'backbone' [Green fluorescent protein (GFP)] was designed in silico to have both a C-terminal Flag-tag and N-Terminal 6x histidine tag (HIS) for antibody detection. A protease cleavage site, which can be adapted for any known protease cleavage sequence, enables the uncoupling of the peptide tags. Three different proteases-Tobacco, Etch Virus (TEV), the main protease from coronavirus SARS-COV-2 (Mpro) and Matrix Metallopeptidase 9 (MMP9)-a cancer-selective human protease-were examined. A sandwich Enzyme-Linked Immunosorbent Assay (ELISA) was developed based on antibodies against the HIS and Flag tags. As an alternative readout, a C-terminal quencher peptide separable by protease cleavage from the GFP was also included. Purified proteins were deployed in cell-free cleavage assays with their respective protease. Western blots, fluorescence assays and immunoassay were performed on samples. Results: Following the design, build and validation of protein constructs, specific protease cleavage was initially demonstrated by Western blot. The novel ELISA proved to afford highly sensitive detection of protease activity in all cases. By way of alternative readout, activation of fluorescence signal upon protease cleavage was also demonstrated but did not match the sensitivity provided by the ELISA method. Discussion: This platform, comprising a protease-responsive synthetic protein device and accompanying readout, is suitable for future deployment in a rapid, low-cost, lateral flow setting. The modular protein device can readily accommodate any desired protease-response module (target protease cleavage site). This study validates the concept with three disparate proteases and applications-human infectious disease, cancer and agricultural crop infection.
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Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.
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Barreira Hematoencefálica , Transportador 1 de Aminoácidos Catiônicos , Animais , Humanos , Camundongos , Barreira Hematoencefálica/metabolismo , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Transportador 1 de Aminoácidos Catiônicos/genética , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Semaglutide is the first oral glucagon-like peptide-1 (GLP-1) analog commercially available for the treatment of type 2 diabetes. In this work, semaglutide was incorporated into poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (NPs) to improve its delivery across the intestinal barrier. The nanocarriers were surface-decorated with either a peptide or an affibody that target the human neonatal Fc receptor (hFcRn), located on the luminal cell surface of the enterocytes. Both ligands were successfully conjugated with the PLGA-PEG via maleimide-thiol chemistry and thereafter, the functionalized polymers were used to produce semaglutide-loaded NPs. Monodisperse NPs with an average size of 170 nm, neutral surface charge and 3% of semaglutide loading were obtained. Both FcRn-targeted NPs exhibited improved interaction and association with Caco-2 cells (cells that endogenously express the hFcRn), compared to non-targeted NPs. Additionally, the uptake of FcRn-targeted NPs was also observed to occur in human intestinal organoids (HIOs) expressing hFcRn through microinjection into the lumen of HIOs, resulting in potential increase of semaglutide permeability for both ligand-functionalized nanocarriers. Herein, our study demonstrates valuable data and insights that the FcRn-targeted NPs has the capacity to promote intestinal absorption of therapeutic peptides.
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Diabetes Mellitus Tipo 2 , Peptídeos Semelhantes ao Glucagon , Lactatos , Nanopartículas , Polietilenoglicóis , Recém-Nascido , Humanos , Células CACO-2 , Peptídeos , Receptores FcRESUMO
BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201. METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1). RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.
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Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Bovinos , Camundongos , Suínos , Barreira Hematoencefálica/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Linhagem Celular , Transporte Biológico , Encéfalo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células CultivadasRESUMO
During the Mesolithic in Europe, there is widespread evidence for an increase in exploitation of aquatic resources. In contrast, the subsequent Neolithic is characterised by the spread of farming, land ownership, and full sedentism, which lead to the perception of marine resources subsequently representing marginal or famine food or being abandoned altogether even at the furthermost coastal limits of Europe. Here, we examine biomarkers extracted from human dental calculus, using sequential thermal desorption- and pyrolysis-GCMS, to report direct evidence for widespread consumption of seaweed and submerged aquatic and freshwater plants across Europe. Notably, evidence of consumption of these resources extends through the Neolithic transition to farming and into the Early Middle Ages, suggesting that these resources, now rarely eaten in Europe, only became marginal much more recently. Understanding ancient foodstuffs is crucial to reconstructing the past, while a better knowledge of local, forgotten resources is likewise important today.
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Alga Marinha , Humanos , Europa (Continente) , Agricultura , VerdurasRESUMO
Small-molecule responsive protein switches are powerful tools for controlling cellular processes. These switches are designed to respond rapidly and specifically to their inducer. They have been used in numerous applications, including the regulation of gene expression, post-translational protein modification, and signal transduction. Typically, small-molecule responsive protein switches consist of two proteins that interact with each other in the presence or absence of a small molecule. Recent advances in computational protein design already contributed to the development of protein switches with an expanded range of small-molecule inducers and increasingly sophisticated switch mechanisms. Further progress in the engineering of small-molecule responsive switches is fueled by cutting-edge computational design approaches, which will enable more complex and precise control over cellular processes and advance synthetic biology applications in biotechnology and medicine. Here, we discuss recent milestones and how technological advances are impacting the development of chemical switches.
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Proteínas , Transdução de Sinais , Proteínas/genética , Transdução de Sinais/genética , Processamento de Proteína Pós-Traducional , Biologia SintéticaRESUMO
Physical interactions between proteins are essential for most biological processes governing life1. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications2-9. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein-protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.
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Simulação por Computador , Aprendizado Profundo , Ligação Proteica , Proteínas , Humanos , Proteínas/química , Proteínas/metabolismo , Proteômica , Mapas de Interação de Proteínas , Sítios de Ligação , Biologia SintéticaRESUMO
The ability of the ancient Egyptians to preserve the human body through embalming has not only fascinated people since antiquity, but also has always raised the question of how this outstanding chemical and ritual process was practically achieved. Here we integrate archaeological, philological and organic residue analyses, shedding new light on the practice and economy of embalming in ancient Egypt. We analysed the organic contents of 31 ceramic vessels recovered from a 26th Dynasty embalming workshop at Saqqara1,2. These vessels were labelled according to their content and/or use, enabling us to correlate organic substances with their Egyptian names and specific embalming practices. We identified specific mixtures of fragrant or antiseptic oils, tars and resins that were used to embalm the head and treat the wrappings using gas chromatography-mass spectrometry analyses. Our study of the Saqqara workshop extends interpretations from a micro-level analysis highlighting the socio-economic status of a tomb owner3-7 to macro-level interpretations of the society. The identification of non-local organic substances enables the reconstruction of trade networks that provided ancient Egyptian embalmers with the substances required for mummification. This extensive demand for foreign products promoted trade both within the Mediterranean8-10 (for example, Pistacia and conifer by-products) and with tropical forest regions (for example, dammar and elemi). Additionally, we show that at Saqqara, antiu and sefet-well known from ancient texts and usually translated as 'myrrh' or 'incense'11-13 and 'a sacred oil'13,14-refer to a coniferous oils-or-tars-based mixture and an unguent with plant additives, respectively.
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Embalsamamento , Múmias , Humanos , Antigo Egito , Embalsamamento/economia , Embalsamamento/história , Embalsamamento/métodos , Cromatografia Gasosa-Espectrometria de Massas , História Antiga , Múmias/história , Resinas Vegetais/análise , Resinas Vegetais/história , Cerâmica/química , Cerâmica/história , Alcatrões/análise , Alcatrões/história , Óleos de Plantas/análise , Óleos de Plantas/história , Região do Mediterrâneo , Clima Tropical , Florestas , Traqueófitas/química , Comércio/históriaRESUMO
An in-depth understanding of the properties of gastric fluid(s) prior to an in vivo pharmacokinetic investigation can vastly improve predictions of in vivo performance. Previously, properties of animal and human gastric fluids have been characterized with varying methods. Unfortunately, characterization has often not been thorough, and some properties, such as density and viscosity, have not been reported. Here, human, porcine and canine gastric fluids were harvested and characterized for pH, viscosity, surface tension, density, and osmolarity. We found that the variability of pH and surface tension between dogs was significantly higher than the variability between pigs, and, furthermore, gastric fluids collected from the same canine species (beagles) housed in two different countries (Denmark and China) had surprisingly different pH values. Next, an in vitro dissolution study in diluted gastric fluids from each species was performed using minitablets containing ibuprofen. Human gastric fluids and porcine gastric fluids showed similar dissolution profiles and corroborated well with biorelevant human Fasted State Simulated Gastric Fluid (FaSSGF). In contrast, differences in canine gastric fluids caused highly variable dissolution results. We systematically compared our findings to those in the literature and based on this evaluation, propose obtaining aspirates from the animals used for in vivo studies to ensure knowledge on the fluid properties affecting the performance of the formulated drug in question.
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Estômago , Animais , Cães , Humanos , Suínos , Composição de Medicamentos , Solubilidade , China , Administração OralRESUMO
Peptides play important roles in regulating biological processes and form the basis of a multiplicity of therapeutic drugs. To date, only about 300 peptides in human have confirmed bioactivity, although tens of thousands have been reported in the literature. The majority of these are inactive degradation products of endogenous proteins and peptides, presenting a needle-in-a-haystack problem of identifying the most promising candidate peptides from large-scale peptidomics experiments to test for bioactivity. To address this challenge, we conducted a comprehensive analysis of the mammalian peptidome across seven tissues in four different mouse strains and used the data to train a machine learning model that predicts hundreds of peptide candidates based on patterns in the mass spectrometry data. We provide in silico validation examples and experimental confirmation of bioactivity for two peptides, demonstrating the utility of this resource for discovering lead peptides for further characterization and therapeutic development.
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Aprendizado de Máquina , Peptídeos , Humanos , Camundongos , Animais , Espectrometria de Massas , Peptídeos/química , MamíferosRESUMO
PURPOSE: More than 15 years have passed since the first description of the unbound brain-to-plasma partition coefficient (Kp,uu,brain) by Prof. Margareta Hammarlund-Udenaes, which was enabled by advancements in experimental methodologies including cerebral microdialysis. Since then, growing knowledge and data continue to support the notion that the unbound (free) concentration of a drug at the site of action, such as the brain, is the driving force for pharmacological responses. Towards this end, Kp,uu,brain is the key parameter to obtain unbound brain concentrations from unbound plasma concentrations. METHODS: To understand the importance and impact of the Kp,uu,brain concept in contemporary drug discovery and development, a survey has been conducted amongst major pharmaceutical companies based in Europe and the USA. Here, we present the results from this survey which consisted of 47 questions addressing: 1) Background information of the companies, 2) Implementation, 3) Application areas, 4) Methodology, 5) Impact and 6) Future perspectives. RESULTS AND CONCLUSIONS: From the responses, it is clear that the majority of the companies (93%) has established a common understanding across disciplines of the concept and utility of Kp,uu,brain as compared to other parameters related to brain exposure. Adoption of the Kp,uu,brain concept has been mainly driven by individual scientists advocating its application in the various companies rather than by a top-down approach. Remarkably, 79% of all responders describe the portfolio impact of Kp,uu,brain implementation in their companies as 'game-changing'. Although most companies (74%) consider the current toolbox for Kp,uu,brain assessment and its validation satisfactory for drug discovery and early development, areas of improvement and future research to better understand human brain pharmacokinetics/pharmacodynamics translation have been identified.
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Barreira Hematoencefálica , Fármacos do Sistema Nervoso Central , Descoberta de Drogas , Encéfalo , Descoberta de Drogas/métodos , HumanosRESUMO
Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system.
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Anticorpos Monoclonais , Antineoplásicos Imunológicos , Administração Oral , Animais , Disponibilidade Biológica , Cápsulas , Imunoterapia , Peptídeos , SuínosRESUMO
This paper presents the earliest evidence for the exploitation of lignite (brown coal) in Europe and sheds new light on the use of combustion fuel sources in the 2nd millennium BCE Eastern Mediterranean. We applied Thermal Desorption/Pyrolysis-Gas Chromatography-Mass Spectrometry and Polarizing Microscopy to the dental calculus of 67 individuals and we identified clear evidence for combustion markers embedded within this calculus. In contrast to the scant evidence for combustion markers within the calculus samples from Egypt, all other individuals show the inhalation of smoke from fires burning wood identified as Pinaceae, in addition to hardwood, such as oak and olive, and/or dung. Importantly, individuals from the Palatial Period at the Mycenaean citadel of Tiryns and the Cretan harbour site of Chania also show the inhalation of fire-smoke from lignite, consistent with the chemical signature of sources in the northwestern Peloponnese and Western Crete respectively. This first evidence for lignite exploitation was likely connected to and at the same time enabled Late Bronze Age Aegean metal and pottery production, significantly by both male and female individuals.
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Diurnal variation in enzymes, hormones, and other biological mediators has long been recognized in mammalian physiology. Developments in pharmacobiology over the past few decades have shown that timing drug delivery can enhance drug efficacy. Here, we report the development of a battery-free, refillable, subcutaneous, and trocar-compatible implantable system that facilitates chronotherapy by enabling tight control over the timing of drug administration in response to external mechanical actuation. The external wearable system is coupled to a mobile app to facilitate control over dosing time. Using this system, we show the efficacy of bromocriptine on glycemic control in a diabetic rat model. We also demonstrate that antihypertensives can be delivered through this device, which could have clinical applications given the recognized diurnal variation of hypertension-related complications. We anticipate that implants capable of chronotherapy will have a substantial impact on our capacity to enhance treatment effectiveness for a broad range of chronic conditions.
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Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.
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Peso Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Peptídeos Semelhantes ao Glucagon/farmacologia , Vias Neurais/efeitos dos fármacos , Animais , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/efeitos dos fármacos , Camundongos , RatosRESUMO
It is evident that the emergence of infectious diseases, which have the potential for spillover from animal reservoirs, pose an ongoing threat to global health. Zoonotic transmission events have increased in frequency in recent decades due to changes in human behavior, including increased international travel, the wildlife trade, deforestation, and the intensification of farming practices to meet demand for meat consumption. Influenza A viruses (IAV) possess a number of features which make them a pandemic threat and a major concern for human health. Their segmented genome and error-prone process of replication can lead to the emergence of novel reassortant viruses, for which the human population are immunologically naïve. In addition, the ability for IAVs to infect aquatic birds and domestic animals, as well as humans, increases the likelihood for reassortment and the subsequent emergence of novel viruses. Sporadic spillover events in the past few decades have resulted in human infections with highly pathogenic avian influenza (HPAI) viruses, with high mortality. The application of conventional vaccine platforms used for the prevention of seasonal influenza viruses, such as inactivated influenza vaccines (IIVs) or live-attenuated influenza vaccines (LAIVs), in the development of vaccines for HPAI viruses is fraught with challenges. These issues are associated with manufacturing under enhanced biosafety containment, and difficulties in propagating HPAI viruses in embryonated eggs, due to their propensity for lethality in eggs. Overcoming manufacturing hurdles through the use of safer backbones, such as low pathogenicity avian influenza viruses (LPAI), can also be a challenge if incompatible with master strain viruses. Non-replicating adenoviral (Ad) vectors offer a number of advantages for the development of vaccines against HPAI viruses. Their genome is stable and permits the insertion of HPAI virus antigens (Ag), which are expressed in vivo following vaccination. Therefore, their manufacture does not require enhanced biosafety facilities or procedures and is egg-independent. Importantly, Ad vaccines have an exemplary safety and immunogenicity profile in numerous human clinical trials, and can be thermostabilized for stockpiling and pandemic preparedness. This review will discuss the status of Ad-based vaccines designed to protect against avian influenza viruses with pandemic potential.
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Adenoviridae/genética , Vetores Genéticos , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/prevenção & controle , Influenza Humana/prevenção & controle , Orthomyxoviridae/patogenicidade , Zoonoses Virais , Animais , Aves , Interações Hospedeiro-Patógeno , Humanos , Imunidade Celular , Imunidade Humoral , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/imunologia , Influenza Humana/transmissão , Influenza Humana/virologia , Orthomyxoviridae/imunologia , Vacinação , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêuticoRESUMO
Protein engineering and synthetic biology stand to benefit immensely from recent advances in silico tools for structural and functional analyses of proteins. In the context of designing novel proteins, current in silico tools inform the user on individual parameters of a query protein, with output scores/metrics unique to each parameter. In reality, proteins feature multiple "parts"/functions and modification of a protein aimed at altering a given part, typically has collateral impact on other protein parts. A system for prediction of the combined effect of design parameters on the overall performance of the final protein does not exist. Function2Form Bridge (F2F-Bridge) attempts to address this by combining the scores of different design parameters pertaining to the protein being analyzed into a single easily interpreted output describing overall performance. The strategy comprises of (a) a mathematical strategy combining data from a myriad of in silico tools into an OP-score (a singular score informing on a user-defined overall performance) and (b) the F2F Plot, a graphical means of informing the wetlab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F predictive output was compared with wetlab data from a range of synthetic proteins designed, built, and tested for this study. Statistical/machine learning approaches for predicting overall performance, for use alongside the F2F plot, were also examined. Comparisons between wetlab performance and F2F predictions demonstrated close and reliable correlations. This user-friendly strategy represents a pivotal enabler in increasing the accessibility of synthetic protein building and de novo protein design.
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Anticorpos/química , Coagulase/química , Aprendizado de Máquina , Mucina-1/química , Biologia Sintética/métodos , Anticorpos/metabolismo , Coagulase/metabolismo , Humanos , Modelos Estatísticos , Mucina-1/metabolismo , Engenharia de Proteínas/métodos , Staphylococcus aureus/química , Relação Estrutura-AtividadeRESUMO
The rich Mediterranean imports found in Early Celtic princely sites (7th-5th cent. BC) in Southwestern Germany, Switzerland and Eastern France have long been the focus of archaeological and public interest. Consumption practices, particularly in the context of feasting, played a major role in Early Celtic life and imported ceramic vessels have consequently been interpreted as an attempt by the elite to imitate Mediterranean wine feasting. Here we present the first scientific study carried out to elucidate the use of Mediterranean imports in Early Celtic Central Europe and their local ceramic counterparts through organic residue analyses of 99 vessels from Vix-Mont Lassois, a key Early Celtic site. In the Mediterranean imports we identified imported plant oils and grape wine, and evidence points towards appropriation of these foreign vessels. Both Greek and local wares served for drinking grape wine and other plant-based fermented beverage(s). A wide variety of animal and plant by-products (e.g. fats, oils, waxes, resin) were also identified. Using an integrative approach, we show the importance of beehive products, millet and bacteriohopanoid beverage(s) in Early Celtic drinking practices. We highlight activities related to biomaterial transformation and show intra-site and status-related differences in consumption practices and/or beverage processing.
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Cerâmica/análise , Grão Comestível/química , Comportamento Alimentar , Vinho , Animais , Arqueologia , Bebidas/análise , Europa (Continente) , França , Alemanha , Grécia , Humanos , Milhetes/química , Óleos de Plantas/análise , Suíça , Vitis/química , Ceras/químicaRESUMO
Absorption enhancers are often a major component of solid oral peptide formulations as compared to the active pharmaceutical ingredient and excipients. This commonly results in poor tabletability that is hard to mitigate in direct compaction by addition of small amounts of excipients. To improve the tabletability of bulky absorption enhancers, the model absorption enhancers, sodium cholate and deoxycholic acid, were co-spray-dried with hydroxypropyl methylcellulose E5, where the percentage of absorption enhancers was not lower than 90% (w/w). The physicochemical properties of the resulting powders were assessed by laser diffraction, scanning electron microscopy, X-ray powder diffraction, thermogravimetric analysis, and differential scanning calorimetry. The powders were compressed into tablets, and the tabletability was evaluated. Co-spray drying with 10% of hydroxypropyl methylcellulose significantly improved the tabletability of the both absorption enhancers. Moreover, it was demonstrated that small particle size and amorphous state rather than high moisture content contributed to the improved tabletability of the spray-dried powders. The study suggests that spray drying technology can be promising to overcome the poor tabletability of oral peptide formulation consisting of large amounts of absorption enhancers.