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1.
Methods Mol Biol ; 2303: 209-226, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626381

RESUMO

Aggrecan, the most abundant extracellular proteoglycan in cartilage (~35% by dry weight), plays a key role in the biophysical and biomechanical properties of cartilage. Here, we review several approaches based on atomic force microscopy (AFM) to probe the physical, mechanical, and structural properties of aggrecan at the molecular level. These approaches probe the response of aggrecan over a wide time (frequency) scale, ranging from equilibrium to impact dynamic loading. Experimental and theoretical methods are described for the investigation of electrostatic and fluid-solid interactions that are key mechanisms underlying the biomechanical and physicochemical functions of aggrecan. Using AFM-based imaging and nanoindentation, ultrastructural features of aggrecan are related to its mechanical properties, based on aggrecans harvested from human vs bovine, immature vs mature, and healthy vs osteoarthritic cartilage.


Assuntos
Fenômenos Biomecânicos , Fenômenos Biofísicos , Agrecanas , Animais , Cartilagem , Cartilagem Articular , Proteínas da Matriz Extracelular , Humanos , Microscopia de Força Atômica , Proteoglicanas
2.
Biochim Biophys Acta Biomembr ; 1864(1): 183810, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34699769

RESUMO

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, HII). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.


Assuntos
Anexina A2/genética , Membrana Celular/genética , Lipídeos de Membrana/química , Fosfolipídeos/genética , Anexina A2/química , Fenômenos Biofísicos , Cálcio/química , Proteínas de Transporte/química , Proteínas de Transporte/genética , Comunicação Celular/genética , Membrana Celular/química , Citoesqueleto/química , Citoesqueleto/genética , Humanos , Fosfatidilserinas/química , Fosfatidilserinas/genética , Fosfolipídeos/química
3.
Biochim Biophys Acta Biomembr ; 1864(1): 183815, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34748744

RESUMO

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (QII) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and QII phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and QII phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between QII phase stabilization and AMP activity doesn't show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize QII phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize QII phases.


Assuntos
/química , Proteínas de Fusão de Membrana/química , Fusão de Membrana/genética , Lipídeos de Membrana/química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Fenômenos Biofísicos , Metabolismo Energético/genética , Proteínas de Fusão de Membrana/genética , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo
4.
Methods Mol Biol ; 2402: 163-177, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34854044

RESUMO

Lipid domains in model membranes are routinely studied to provide insight into the physical interactions that drive raft formation in cellular membranes. Using small angle neutron scattering, contrast-matching techniques enable the detection of lipid domains ranging from tens to hundreds of nanometers which are not accessible to other techniques without the use of extrinsic probes. Here, we describe a probe-free experimental approach and model-free analysis to identify lipid domains in freely floating vesicles of ternary phase separating lipid mixtures.


Assuntos
Lipídeos , Espalhamento a Baixo Ângulo , Fenômenos Biofísicos , Bicamadas Lipídicas , Microdomínios da Membrana , Difração de Nêutrons , Nêutrons
5.
Methods Mol Biol ; 2393: 315-328, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34837187

RESUMO

Charge sensitive optical detection (CSOD) technique is a label-free method for real-time measurement of molecular interactions. Traditional label-free optical detection techniques mostly measure the mass of a molecule, and they are less sensitive to small molecules. In contrast, CSOD detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, CSOD is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. In CSOD, an optical fiber functionalized with the probe molecule is dipped into a well of a microplate where an alternate perpendicular electrical field is applied to the fiber, which drives the fiber into oscillation because of the presence of surface charge on the fiber. The binding of the target molecules changes the charge of the fiber, and thus the amplitude and phase of the oscillating fiber, which are precisely measured through tracking of the optical images of the fiber tip.


Assuntos
Fenômenos Biofísicos , Cinética , Fibras Ópticas , Física
6.
J Colloid Interface Sci ; 608(Pt 1): 644-651, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34628323

RESUMO

HYPOTHESIS: Mixtures of colloids and supramolecular polymers may exhibit stimuli-responsive phase behaviour. However, in theoretical descriptions of such systems, the polymers are commonly described either as flexible chains or as rigid rods, while in experimental systems supramolecular polymers usually fall in between these two limits. We expect the flexibility of the polymers to have a profound effect on the stimuli-responsive phase behaviour. THEORY: We propose a general approach to predict the phase behaviour of colloidal hard spheres mixed with covalent or supramolecular polymers of arbitrary persistence length using free volume theory and an interpolation between flexible and rigid chains. FINDINGS: The binodals are predicted to shift to lower monomer concentrations as the persistence length is increased, making the polymers more efficient depletants. The persistence length is therefore an additional degree of freedom for manipulating the phase behaviour of colloid-polymer mixtures. We show that by manipulating the persistence length of temperature responsive supramolecular polymers, a wide range of phase diagrams with various topologies can be obtained. For example, we find phase diagrams with a critical point but no triple point or displaying two triple points for temperature-sensitive supramolecular polymers mixed with hard spheres.


Assuntos
Coloides , Modelos Químicos , Fenômenos Biofísicos , Polímeros , Temperatura
7.
Cell Mol Life Sci ; 79(1): 27, 2021 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-34971423

RESUMO

The rate-limiting serine biogenesis enzyme PHGDH is overexpressed in cancers. Both serine withdrawal and genetic/pharmacological inhibition of PHGDH have demonstrated promising tumor-suppressing activities. However, the enzyme properties of PHGDH are not well understood and the discovery of PHGDH inhibitors is still in its infancy. Here, oridonin was identified from a natural product library as a new PHGDH inhibitor. The crystal structure of PHGDH in complex with oridonin revealed a new allosteric site. The binding of oridonin to this site reduced the activity of the enzyme by relocating R54, a residue involved in substrate binding. Mutagenesis studies showed that PHGDH activity was very sensitive to cysteine mutations, especially those in the substrate binding domain. Conjugation of oridonin and other reported covalent PHGDH inhibitors to these sites will therefore inhibit PHGDH. In addition to being inhibited enzymatically, PHGDH can also be inhibited by protein aggregation and proteasome-mediated degradation. Several tested PHGDH cancer mutants showed altered enzymatic activity, which can be explained by protein structure and stability. Overall, the above studies present new biophysical and biochemical insights into PHGDH and may facilitate the future design of PHGDH inhibitors.


Assuntos
Fenômenos Biofísicos , Inibidores Enzimáticos/farmacologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/farmacologia , Inibidores Enzimáticos/química , Ácidos Glicéricos/metabolismo , Humanos , Mutação/genética , NAD/metabolismo , Fosfoglicerato Desidrogenase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Proteólise/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34916290

RESUMO

Recent studies have revealed that extensive heterogeneity of biological systems arises through various routes ranging from intracellular chromosome segregation to spatiotemporally varying biochemical stimulations. However, the contribution of physical microenvironments to single-cell heterogeneity remains largely unexplored. Here, we show that a homogeneous population of non-small-cell lung carcinoma develops into heterogeneous subpopulations upon application of a homogeneous physical compression, as shown by single-cell transcriptome profiling. The generated subpopulations stochastically gain the signature genes associated with epithelial-mesenchymal transition (EMT; VIM, CDH1, EPCAM, ZEB1, and ZEB2) and cancer stem cells (MKI67, BIRC5, and KLF4), respectively. Trajectory analysis revealed two bifurcated paths as cells evolving upon the physical compression, along each path the corresponding signature genes (epithelial or mesenchymal) gradually increase. Furthermore, we show that compression increases gene expression noise, which interplays with regulatory network architecture and thus generates differential cell-fate outcomes. The experimental observations of both single-cell sequencing and single-molecule fluorescent in situ hybridization agrees well with our computational modeling of regulatory network in the EMT process. These results demonstrate a paradigm of how mechanical stimulations impact cell-fate determination by altering transcription dynamics; moreover, we show a distinct path that the ecology and evolution of cancer interplay with their physical microenvironments from the view of mechanobiology and systems biology, with insight into the origin of single-cell heterogeneity.


Assuntos
Tamanho Celular , Transição Epitelial-Mesenquimal/genética , Microambiente Tumoral/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fenômenos Biofísicos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/metabolismo , Análise de Célula Única
9.
Biochemistry ; 60(51): 3856-3867, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34910875

RESUMO

The T-cell protein tyrosine phosphatase (TCPTP/PTPN2) targets a broad variety of substrates across different subcellular compartments. In spite of that, the structural basis for the regulation of TCPTP's activity remains elusive. Here, we investigated whether the activity of TCPTP is regulated by a potential allosteric site in a comparable manner to its most similar PTP family member (PTP1B/PTPN1). We determined two crystal structures of TCPTP at 1.7 and 1.9 Å resolutions that include helix α7 at the TCPTP C-terminus. Helix α7 has been functionally characterized in PTP1B and was identified as its allosteric switch. However, its function is unknown in TCPTP. Here, we demonstrate that truncation or deletion of helix α7 reduced the catalytic efficiency of TCPTP by ∼4-fold. Collectively, our data supports an allosteric role of helix α7 in regulation of TCPTP's activity, similar to its function in PTP1B, and highlights that the coordination of helix α7 with the core catalytic domain is essential for the efficient catalytic function of TCPTP.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 2/química , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Regulação Alostérica , Sítio Alostérico/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Fenômenos Biofísicos , Domínio Catalítico/genética , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica em alfa-Hélice , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
10.
Phys Rev Lett ; 127(13): 138102, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34623834

RESUMO

Because the normal operation of the eye depends on sensitive morphogenetic processes for its eventual shape, developmental flaws can lead to wide-ranging ocular defects. However, the physical processes and mechanisms governing ocular morphogenesis are not well understood. Here, using analytical theory and nonlinear shell finite-element simulations, we show, for optic vesicles experiencing matrix-constrained growth, that elastic instabilities govern the optic cup morphogenesis. By capturing the stress amplification owing to mass increase during growth, we show that the morphogenesis is driven by two elastic instabilities analogous to the snap through in spherical shells, where the second instability is sensitive to the optic cup geometry. In particular, if the optic vesicle is too slender, it will buckle and break axisymmetry, thus, preventing normal development. Our results shed light on the morphogenetic mechanisms governing the formation of a functional biological system and the role of elastic instabilities in the shape selection of soft biological structures.


Assuntos
Olho/crescimento & desenvolvimento , Modelos Biológicos , Fenômenos Biofísicos , Morfogênese , Disco Óptico/crescimento & desenvolvimento
11.
Phys Rev Lett ; 127(13): 138103, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34623840

RESUMO

Nanopores in 2D materials are highly desirable for DNA sequencing, yet achieving single-stranded DNA (ssDNA) transport through them is challenging. Using density functional theory calculations and molecular dynamics simulations we show that ssDNA transport through a pore in monolayer hexagonal boron nitride (h-BN) is marked by a basic nanomechanical conflict. It arises from the notably inhomogeneous flexural rigidity of ssDNA and causes high friction via transient DNA desorption costs exacerbated by solvation effects. For a similarly sized pore in bilayer h-BN, its self-passivated atomically smooth edge enables continuous ssDNA transport. Our findings shed light on the fundamental physics of biopolymer transport through pores in 2D materials.


Assuntos
Compostos de Boro/química , DNA de Cadeia Simples/química , Nanoestruturas/química , Fenômenos Biofísicos , Modelos Químicos , Simulação de Dinâmica Molecular , Nanoporos
12.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638522

RESUMO

We investigated the influence of a solvent's composition on the stability of desorbed and multiply charged RNAse S ions by analyzing the non-covalent complex's gas-phase dissociation processes. RNAse S was dissolved in electrospray ionization-compatible buffers with either increasing organic co-solvent content or different pHs. The direct transition of all the ions and the evaporation of the solvent from all the in-solution components of RNAse S under the respective in-solution conditions by electrospray ionization was followed by a collision-induced dissociation of the surviving non-covalent RNAse S complex ions. Both types of changes of solvent conditions yielded in mass spectrometrically observable differences of the in-solution complexation equilibria. Through quantitative analysis of the dissociation products, i.e., from normalized ion abundances of RNAse S, S-protein, and S-peptide, the apparent kinetic and apparent thermodynamic gas-phase complex properties were deduced. From the experimental data, it is concluded that the stability of RNAse S in the gas phase is independent of its in-solution equilibrium but is sensitive to the complexes' gas-phase charge states. Bio-computational in-silico studies showed that after desolvation and ionization by electrospray, the remaining binding forces kept the S-peptide and S-protein together in the gas phase predominantly by polar interactions, which indirectly stabilized the in-bulk solution predominating non-polar intermolecular interactions. As polar interactions are sensitive to in-solution protonation, bio-computational results provide an explanation of quantitative experimental data with single amino acid residue resolution.


Assuntos
Biologia Computacional/métodos , Ribonucleases/química , Solventes/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Fenômenos Biofísicos/fisiologia , Bovinos , Simulação por Computador , Ribonucleases/análise , Termodinâmica
13.
Molecules ; 26(20)2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34684706

RESUMO

The remarkable lubrication properties of normal articular cartilage play an essential role in daily life, providing almost frictionless movements of joints. Alterations of cartilage surface or degradation of biomacromolecules within synovial fluid increase the wear and tear of the cartilage and hence determining the onset of the most common joint disease, osteoarthritis (OA). The irreversible and progressive degradation of articular cartilage is the hallmark of OA. Considering the absence of effective options to treat OA, the mechanosensitivity of chondrocytes has captured attention. As the only embedded cells in cartilage, the metabolism of chondrocytes is essential in maintaining homeostasis of cartilage, which triggers motivations to understand what is behind the low friction of cartilage and develop biolubrication-based strategies to postpone or even possibly heal OA. This review firstly focuses on the mechanism of cartilage lubrication, particularly on boundary lubrication. Then the mechanotransduction (especially shear stress) of chondrocytes is discussed. The following summarizes the recent development of cartilage-inspired biolubricants to highlight the correlation between cartilage lubrication and OA. One might expect that the restoration of cartilage lubrication at the early stage of OA could potentially promote the regeneration of cartilage and reverse its pathology to cure OA.


Assuntos
Cartilagem/fisiologia , Osteoartrite/fisiopatologia , Líquido Sinovial/metabolismo , Animais , Fenômenos Biofísicos/fisiologia , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Fricção , Humanos , Ácido Hialurônico/metabolismo , Mecanotransdução Celular , Estresse Mecânico
14.
Nat Methods ; 18(10): 1239-1246, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34608318

RESUMO

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.


Assuntos
Adenosina Trifosfatases/metabolismo , Fenômenos Biofísicos , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/fisiologia , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Membrana Celular/metabolismo , Escherichia coli , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química
15.
Elife ; 102021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34569929

RESUMO

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic Caenorhabditis elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signaling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g., zebrafish).


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Ligação a Calmodulina/química , Contração Muscular/fisiologia , Proteínas Musculares/química , Conformação Proteica , Animais , Animais Geneticamente Modificados , Fenômenos Biofísicos , Caenorhabditis elegans , Conectina , Transferência Ressonante de Energia de Fluorescência/métodos
16.
Curr Protoc ; 1(9): e237, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34499805

RESUMO

Eukaryotic cells contain numerous membrane-bound and membraneless organelles that provide spatiotemporal control for diverse biological processes. The liquid-liquid phase separation of proteins has been proposed as the driving force behind the formation of membraneless organelles. Here, we describe a method to determine the phase separation activities of proteins in plants. This basic method includes protocols for an in vivo fluorescence recovery after photobleaching assay in Nicotiana benthamiana using transient expression, an in vitro liquid droplet reconstitution assay using purified recombinant proteins, and an in vivo fluorescence recovery after photobleaching assay in Arabidopsis thaliana using stable transgenic plants. With these assays, the phase separation characteristics of a protein can be determined. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Detection of protein phase separation activities in N. benthamiana Support Protocol: Fluorescence recovery after photobleaching assay Basic Protocol 2: Detection of protein phase separation in vitro Basic Protocol 3: Detection of protein phase separation in stable transgenic Arabidopsis thaliana plants.


Assuntos
Fracionamento Químico/métodos , Organelas , Proteínas de Plantas , Fenômenos Biofísicos , Membranas
17.
Phys Rev Lett ; 127(9): 094501, 2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34506163

RESUMO

Turbulent puffs are ubiquitous in everyday life phenomena. Understanding their dynamics is important in a variety of situations ranging from industrial processes to pure and applied science. In all these fields, a deep knowledge of the statistical structure of temperature and velocity space-time fluctuations is of paramount importance to construct models of chemical reaction (in chemistry) and of condensation of virus-containing droplets (in virology and/or biophysics) and optimal mixing strategies in industrial applications. As a matter of fact, results of turbulence in a puff are confined to bulk properties (i.e., average puff velocity and typical decay or growth time) and date back to the second half of the 20th century. There is, thus, a huge gap to fill to pass from bulk properties to two-point statistical observables. Here, we fill this gap by exploiting theory and numerics in concert to predict and validate the space-time scaling behaviors of both velocity and temperature structure functions including intermittency corrections. Excellent agreement between theory and simulations is found. Our results are expected to have a profound impact on developing evaporation models for virus-containing droplets carried by a turbulent puff, with benefits to the comprehension of the airborne route of virus contagion.


Assuntos
Modelos Biológicos , Modelos Teóricos , Microbiologia do Ar , Fenômenos Biofísicos , COVID-19/transmissão , COVID-19/virologia
18.
Nature ; 597(7874): 33-34, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471278
19.
Elife ; 102021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34581668

RESUMO

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.


Assuntos
Bioensaio/métodos , Imunoadsorventes/química , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Fenômenos Biofísicos , Magnetismo , Ligação Proteica , Proteômica
20.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576104

RESUMO

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.


Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fenômenos Biofísicos , Sinalização do Cálcio , Citosol/metabolismo , Células HeLa , Humanos , Cinética
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