RESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord, and there are no effective drug treatments. Low-intensity pulsed ultrasound (LIPUS) has garnered attention as a promising noninvasive neuromodulation method. In this study, we investigate its effects on the motor cortex and underlying mechanisms using the SOD1G93A mouse model of ALS. Our results show that LIPUS treatment delays disease onset and prolongs lifespan in ALS mice. LIPUS significantly increases cerebral blood flow in the motor cortex by preserving vascular endothelial cell integrity and increasing microvascular density, which may be mediated via the ion channel TRPV4. RNA sequencing analysis reveals that LIPUS substantially reduces the expression of genes associated with neuroinflammation. These findings suggest that LIPUS applied to the motor cortex may represent a potentially effective therapeutic tool for the treatment of ALS.
RESUMEN
Oxygen levels vary in the environment. Oxygen availability has a major effect on almost all organisms, and oxygen is far more than a substrate for energy production. However, less is known about related biological processes under hypoxic conditions and about the adaptations to changing oxygen concentrations. The yeast Saccharomyces cerevisiae can adapt its metabolism for growth under different oxygen concentrations and can grow even under anaerobic conditions. Therefore, we developed a microfluidic device that can generate serial, accurately controlled oxygen concentrations for single-cell studies of multiple yeast strains. This device can construct a broad range of oxygen concentrations, [O2] through on-chip gas-mixing channels from two gases fed to the inlets. Gas diffusion through thin polydimethylsiloxane (PDMS) can lead to the equilibration of [O2] in the medium in the cell culture layer under gas cover regions within 2 min. Here, we established six different and stable [O2] varying between ~0.1 and 20.9% in the corresponding layers of the device designed for multiple parallel single-cell culture of four different yeast strains. Using this device, the dynamic responses of different yeast transcription factors and metabolism-related proteins were studied when the [O2] decreased from 20.9% to serial hypoxic concentrations. We showed that different hypoxic conditions induced varying degrees of transcription factor responses and changes in respiratory metabolism levels. This device can also be used in studies of the aging and physiology of yeast under different oxygen conditions and can provide new insights into the relationship between oxygen and organisms. Integration, innovation and insight: Most living cells are sensitive to the oxygen concentration because they depend on oxygen for survival and proper cellular functions. Here, a composite microfluidic device was designed for yeast single-cell studies at a series of accurately controlled oxygen concentrations. Using this device, we studied the dynamic responses of various transcription factors and proteins to changes in the oxygen concentration. This study is the first to examine protein dynamics and temporal behaviors under different hypoxic conditions at the single yeast cell level, which may provide insights into the processes involved in yeast and even mammalian cells. This device also provides a base model that can be extended to oxygen-related biology and can acquire more information about the complex networks of organisms.
Asunto(s)
Oxígeno , Saccharomyces cerevisiae , Análisis de la Célula Individual , Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Dimetilpolisiloxanos/química , Dispositivos Laboratorio en un Chip , Proteínas de Saccharomyces cerevisiae/metabolismo , Diseño de Equipo , Técnicas Analíticas Microfluídicas/instrumentación , MicrofluídicaRESUMEN
The shape of a cell as defined by its membrane can be closely associated with its physiological state. For example, the irregular shapes of cancerous cells and elongated shapes of neuron cells often reflect specific functions, such as cell motility and cell communication. However, it remains unclear whether and which cell shape descriptors can characterize different cellular physiological states. In this study, 12 geometric shape descriptors for a three-dimensional (3D) object were collected from the previous literature and tested with a public dataset of ~400,000 independent 3D cell regions segmented based on fluorescent labeling of the cell membranes in Caenorhabditis elegans embryos. It is revealed that those shape descriptors can faithfully characterize cellular physiological states, including (1) cell division (cytokinesis), along with an abrupt increase in the elongation ratio; (2) a negative correlation of cell migration speed with cell sphericity; (3) cell lineage specification with symmetrically patterned cell shape changes; and (4) cell fate specification with differential gene expression and differential cell shapes. The descriptors established may be used to identify and predict the diverse physiological states in numerous cells, which could be used for not only studying developmental morphogenesis but also diagnosing human disease (e.g., the rapid detection of abnormal cells).
RESUMEN
BACKGROUND: Buccal mucosa squamous cell carcinoma (BMSCC) is an aggressive disease. This study investigated the clinicopathological significance of tumor budding (TB), depth of invasion (DOI), and mode of invasion (MOI) on occult cervical metastasis (CM) of BMSCC. METHODS: Seventy-one cT1-2N0 BMSCC patients were included in this retrospective study. TB, DOI, MOI, and other clinicopathological features were reviewed. Risk factors for occult CM, locoregional recurrence-free survival (LRRFS), and overall survival (OS) were analyzed using logistic regression and Cox's proportional hazard models, respectively. RESULTS: Multivariate analysis with the logistic regression model revealed that MOI, DOI, and TB were significantly associated with occult CM in early-stage BMSCC after adjusting for variates. However, multivariate analysis with the Cox's proportional hazard model found only TB to be a prognostic factor for LRRFS (hazard ratio 15.03, 95% confidence interval [CI] 1.94-116.66; p = 0.01; trend test p = 0.03). No significant association was found between MOI, DOI, or TB and OS. CONCLUSIONS: The optimal predictor of occult CM and prognosis of early-stage BMSCC is TB, which may assist clinicians in identifying patients at high risk of cervical metastasis.
Asunto(s)
Carcinoma de Células Escamosas , Mucosa Bucal , Invasividad Neoplásica , Humanos , Femenino , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Carcinoma de Células Escamosas/secundario , Carcinoma de Células Escamosas/patología , Anciano , Mucosa Bucal/patología , Adulto , Neoplasias de la Boca/patología , Estadificación de Neoplasias , Anciano de 80 o más Años , Factores de Riesgo , Modelos de Riesgos Proporcionales , Pronóstico , Metástasis Linfática/patologíaRESUMEN
Nearly all circadian clocks maintain a period that is insensitive to temperature changes, a phenomenon known as temperature compensation (TC). Yet, it is unclear whether there is any common feature among different systems that exhibit TC. From a general timescale invariance, we show that TC relies on the existence of certain period-lengthening reactions wherein the period of the system increases strongly with the rates in these reactions. By studying several generic oscillator models, we show that this counterintuitive dependence is nonetheless a common feature of oscillators in the nonlinear (far-from-onset) regime where the oscillation can be separated into fast and slow phases. The increase of the period with the period-lengthening reaction rates occurs when the amplitude of the slow phase in the oscillation increases with these rates while the progression speed in the slow phase is controlled by other rates of the system. The positive dependence of the period on the period-lengthening rates balances its inverse dependence on other kinetic rates in the system, which gives rise to robust TC in a wide range of parameters. We demonstrate the existence of such period-lengthening reactions and their relevance for TC in all four model systems we considered. Theoretical results for a model of the Kai system are supported by experimental data. A study of the energy dissipation also shows that better TC performance requires higher energy consumption. Our study unveils a general mechanism by which a biochemical oscillator achieves TC by operating in parameter regimes far from the onset where period-lengthening reactions exist.
RESUMEN
Time of day affects how well the immune system responds to viral or bacterial infections. While it is well known that the immune system is regulated by the circadian clock, the dynamic origin of time-of-day-dependent immunity remains unclear. In this paper, we studied the circadian control of immune response upon infection of influenza A virus through mathematical modeling. Dynamic simulation analyses revealed that the time-of-day-dependent immunity was rooted in the relative phase between the circadian clock and the pulse of viral infection. The relative phase, which depends on the time the infection occurs, plays a crucial role in the immune response. It can drive the immune system to one of two distinct bistable states, a high inflammatory state with a higher mortality rate or a safe state characterized by low inflammation. The mechanism we found here also explained why the same species infected by different viruses has different time-of-day-dependent immunities. Further, the time-of-day-dependent immunity was found to be abolished when the immune system was regulated by an impaired circadian clock with decreased oscillation amplitude or without oscillations.
Asunto(s)
Relojes Circadianos , Relojes Circadianos/inmunología , Virosis/inmunología , Virosis/virología , Humanos , Virus de la Influenza A/inmunología , Modelos Biológicos , AnimalesRESUMEN
The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been accompanied by the emergence of viral mutations that pose a great challenge to existing vaccine strategies. It is not fully understood with regard to the role of mutations on the SARS-CoV-2 spike protein from emerging viral variants in T cell immunity. In the current study, recombinant eukaryotic plasmids were constructed as DNA vaccines to express the spike protein from multiple SARS-CoV-2 strains. These DNA vaccines were used to immunize BALB/c mice, and cross-T cell responses to the spike protein from these viral strains were quantitated using interferon-γ (IFN-γ) Elispot. Peptides covering the full-length spike protein from different viral strains were used to detect epitope-specific IFN-γ+ CD4+ and CD8+ T cell responses by fluorescence-activated cell sorting. SARS-CoV-2 Delta and Omicron BA.1 strains were found to have broad T cell cross-reactivity, followed by the Beta strain. The landscapes of T cell epitopes on the spike protein demonstrated that at least 30 mutations emerging from Alpha to Omicron BA.5 can mediate the escape of T cell immunity. Omicron and its sublineages have 19 out of these 30 mutations, most of which are new, and a few are inherited from ancient circulating variants of concerns. The cross-T cell immunity between SARS-CoV-2 prototype strain and Omicron strains can be attributed to the T cell epitopes located in the N-terminal domain (181-246 aa [amino acids], 271-318 aa) and C-terminal domain (1171-1273 aa) of the spike protein. These findings provide in vivo evidence for optimizing vaccine manufacturing and immunization strategies for current or future viral variants.
Asunto(s)
COVID-19 , Vacunas de ADN , Animales , Ratones , Humanos , Epítopos de Linfocito T/genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Inmunidad Celular , Mutación , Interferón gamma , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
Differential equation models are widely used to describe genetic regulations, predict multicomponent regulatory circuits, and provide quantitative insights. However, it is still challenging to quantitatively link the dynamic behaviors with measured parameters in synthetic circuits. Here, we propose a dynamic delay model (DDM) which includes two simple parts: the dynamic determining part and the doses-related steady-state-determining part. The dynamic determining part is usually supposed as the delay time but without a clear formula. For the first time, we give the detail formula of the dynamic determining function and provide a method for measuring all parameters of synthetic elements (include 8 activators and 5 repressors) by microfluidic system. Three synthetic circuits were built to show that the DDM can notably improve the prediction accuracy and can be used in various synthetic biology applications.
RESUMEN
Thallium is a rare metal known for its highly toxic nature. Recent research has indicated that the precise determination of Tl isotopic compositions using Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP MS) provides new opportunities for understanding Tl geochemical behavior. While isotopic fractionation of Tl derived from anthropogenic activities (e.g., mining, smelting) have been reported, there is limited information regarding Tl influenced by both natural weathering processes and anthropogenic origins. Herein, we investigated, for the first time, the Tl isotopic compositions in soils across a representative Tl-rich depth profile from the Lanmuchang (LMC) quicksilver mine (southwest China) in the low-temperature metallogenesis zone. The results showed significant variations in Tl isotope signatures (ε205Tl) among different soil layers, ranging from -0.23 to 3.79, with heavier isotope-205Tl enrichment observed in the bottom layers of the profile (ε205Tl = 2.18-3.79). This enrichment of 205Tl was not solely correlated with the degree of soil weathering but was also partially associated with oxidation of Tl(I) by Fe (hydr)oxide minerals. Quantitative calculation using ε205Tl vs. 1/Tl data further indicated that the Tl enrichment across the soil depth profile was predominantly derived from anthropogenic origins. All these findings highlight that the robustness and reliability of Tl isotopes as a proxy for identifying both anthropogenic and geogenic sources, as well as tracing chemical alterations and redox-controlled mineralogical processes of Tl in soils. The nascent application of Tl isotopes herein not only offers valuable insights into the behavior of Tl in surface environments, but also establishes a framework for source apportionment in soils under similar circumstances.
RESUMEN
Biological processes are typically actuated by dynamic multi-subunit molecular complexes. However, interactions between subunits, which govern the functions of these complexes, are hard to measure directly. Here, we develop a general approach combining cryo-EM imaging technology and statistical modeling and apply it to study the hexameric clock protein KaiC in Cyanobacteria. By clustering millions of KaiC monomer images, we identify two major conformational states of KaiC monomers. We then classify the conformational states of (>160,000) KaiC hexamers by the thirteen distinct spatial arrangements of these two subunit states in the hexamer ring. We find that distributions of the thirteen hexamer conformational patterns for two KaiC phosphorylation mutants can be fitted quantitatively by an Ising model, which reveals a significant cooperativity between neighboring subunits with phosphorylation shifting the probability of subunit conformation. Our results show that a KaiC hexamer can respond in a switch-like manner to changes in its phosphorylation level.
Asunto(s)
Relojes Circadianos , Microscopía por Crioelectrón , Proteínas CLOCK , Análisis por Conglomerados , Modelos EstadísticosRESUMEN
Once challenged by the SARS-CoV-2 virus, the human host immune system triggers a dynamic process against infection. We constructed a mathematical model to describe host innate and adaptive immune response to viral challenge. Based on the dynamic properties of viral load and immune response, we classified the resulting dynamics into four modes, reflecting increasing severity of COVID-19 disease. We found the numerical product of immune system's ability to clear the virus and to kill the infected cells, namely immune efficacy, to be predictive of disease severity. We also investigated vaccine-induced protection against SARS-CoV-2 infection. Results suggested that immune efficacy based on memory T cells and neutralizing antibody titers could be used to predict population vaccine protection rates. Finally, we analyzed infection dynamics of SARS-CoV-2 variants within the construct of our mathematical model. Overall, our results provide a systematic framework for understanding the dynamics of host response upon challenge by SARS-CoV-2 infection, and this framework can be used to predict vaccine protection and perform clinical diagnosis.
Asunto(s)
COVID-19 , Virosis , Humanos , COVID-19/prevención & control , SARS-CoV-2 , Carga ViralRESUMEN
Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Animales , Ratones , Adenosina Trifosfatasas , Microscopía por Crioelectrón , CitoplasmaRESUMEN
Accurate and robust spatial orders are ubiquitous in living systems. In 1952, Turing proposed a general mechanism for pattern formation exemplified by a reaction-diffusion model with two chemical species in a large system. However, in small biological systems such as a cell, the existence of multiple Turing patterns and strong noise can lower the spatial order. Recently, a modified reaction-diffusion model with an additional chemical species is shown to stabilize the Turing pattern. Here, we study non-equilibrium thermodynamics of this three-species reaction-diffusion model to understand the relationship between energy cost and the performance of self-positioning. By using computational and analytical approaches, we show that beyond the onset of pattern formation the positioning error decreases as energy dissipation increases. In a finite system, we find that a specific Turing pattern exists only within a finite range of total molecule number. Energy dissipation broadens this range, which enhances the robustness of Turing pattern against molecule number fluctuations in living cells. The generality of these results is verified in a realistic model of the Muk system underlying DNA segregation in Escherichia coli, and testable predictions are made for the dependence of the accuracy and robustness of the spatial pattern on the ATP/ADP ratio.
Asunto(s)
Modelos Biológicos , Difusión , TermodinámicaRESUMEN
A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.
RESUMEN
Control of dynamical processes is vital for maintaining correct cell regulation and cell-fate decisions. Numerous regulatory networks show oscillatory behavior; however, our knowledge of how one oscillator behaves when stimulated by two or more external oscillatory signals is still missing. We explore this problem by constructing a synthetic oscillatory system in yeast and stimulate it with two external oscillatory signals. Letting model verification and prediction operate in a tight interplay with experimental observations, we find that stimulation with two external signals expands the plateau of entrainment and reduces the fluctuations of oscillations. Furthermore, by adjusting the phase differences of external signals, one can control the amplitude of oscillations, which is understood through the signal delay of the unperturbed oscillatory network. With this we reveal a direct amplitude dependency of downstream gene transcription. Taken together, these results suggest a new path to control oscillatory systems by coupled oscillator cooperativity.
Asunto(s)
Ciclo Celular , Diferenciación Celular , Fenómenos CronobiológicosRESUMEN
Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated protein can increase, we studied ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn finger interacts with the Rpt5 ATPase and its C-terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Surprisingly, these C-terminal interactions are sufficient to activate proteolysis. With ZFAND5 bound, entry into the proteasome's protein translocation channel is wider, and ZFAND5 dissociation causes opening of the 20S gate for substrate entry. Using single-molecular microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.
RESUMEN
Thallium (Tl) is a highly toxic heavy metal, which is harmful to plants and animals even in trace amounts. Migration behaviors of Tl in paddy soils system remain largely unknown. Herein, Tl isotopic compositions have been employed for the first time to explore Tl transfer and pathway in paddy soil system. The results showed considerably large Tl isotopic variations (ε205Tl = -0.99 ± 0.45 ~ 24.57 ± 0.27), which may result from interconversion between Tl(I) and Tl(III) under alternative redox conditions in the paddy system. Overall higher ε205Tl values of paddy soils in the deeper layers were probably attributed to abundant presence of Fe/Mn (hydr)oxides and occasionally extreme redox conditions during alternative dry-wet process which oxidized Tl(I) to Tl(III). A ternary mixing model using Tl isotopic compositions further disclosed that industrial waste contributed predominantly to Tl contamination in the studied soil, with an average contribution rate of 73.23%. All these findings indicate that Tl isotopes can be used as an efficient tracer for fingerprinting Tl pathway in complicated scenarios even under varied redox conditions, providing significant prospect in diverse environmental applications.
RESUMEN
As an absolute quantification method at the single-molecule level, digital PCR (dPCR) offers the highest accuracy. In this work, we developed a 3D scalable chamber-array chip that multiplied the number of partitions by stacking chamber-array layers and realized digital loop-mediated isothermal amplification to quantify DNA molecules. It greatly increases the number of partitions to improve the performance of dPCR without increasing the chip size, the operation workflow complicity, and operation time. For the three-chamber-array-layer chip which contains 200,000 reactors of a 0.125 nL volume, it has been proved that the reagent filling and partition were finished within 3 min, and the whole detection could be finished within 1 h. The method demonstrated that it could be scalable to a six-chamber-array layer, which contains 400,000 reactors without increasing the size of the chip and the complication of filling/partition workflow but only takes an additional hour for scanning. Due to its potential for high throughput, low cost, and simple operation, our device may significantly expand the clinical application range of dPCR.
Asunto(s)
ADN , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/genética , Nanotecnología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Context-dependency of mammalian transcriptional elements has hindered the quantitative investigation of multigene expression stoichiometry and its biological functions. Here, we describe a host- and local DNA context-independent transcription system to gradually fine-tune single and multiple gene expression with predictable stoichiometries. The mammalian transcription system is composed of a library of modular and programmable promoters from bacteriophage and its cognate RNA polymerase (RNAP) fused to a capping enzyme. The relative expression of single genes is quantitatively determined by the relative binding affinity of the RNAP to the promoters, while multigene expression stoichiometry is predicted by a simple biochemical model with resource competition. We use these programmable and modular promoters to predictably tune the expression of three components of an influenza A virus-like particle (VLP). Optimized stoichiometry leads to a 2-fold yield of intact VLP complexes. The host-independent orthogonal transcription system provides a platform for dose-dependent control of multiple protein expression which may be applied for advanced vaccine engineering, cell-fate programming and other therapeutic applications.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Transcripción Genética , Animales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas/genética , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) has become a big threaten to global health. The current strategy for treatment of MDR-TB and extensive drug resistant tuberculosis (XDR-TB) is with low efficacy and high side effect. While new drug is fundamental for cure MDR-TB, repurposing the Food and Drug Administration (FDA)-approved drugs represents an alternative soluation with less cost. METHODS: The activity of 8 tetracycline-class antibiotics against mycobacterium tuberculosis (M.tb) were determined by Minimum Inhibitory Concentration (MIC) in vitro. A transposon M.smeg libraries was generated by using the Harm phage and then used to isolate the conditional growth mutants in doxycycline containing plate. Eleven mutants were isolated and genomic DNAs were extracted using the cetyltrimethyl ammonium bromide (CTAB) method and analyzed by whole genome sequencing. RESULTS: We found that three of eight drugs efficiently inhibited mycobacteria growth under the peak plasma concentration in the human body. Further tests showed these three tetracycline analogs (demeclocycline, doxycycline and methacycline) had antimicrobial activity against seven clinical isolates, including MDR and XDR strains. Among them, Doxycycline had the lowest MICs in all mycobacteria strains tested in this study. By using a transposon library, we identify the insertion of transposon in two genes, porin and MshA, associatewith the resistant to doxycycline. CONCLUSIONS: Our findings show that tetracycline analogs such as doxycycline, has bactericidal activity against not only drug sensitive M.tb, but also clinical MDR and XDR strains, provided proof of concept to repurpose doxycycline to fight MDR-TB and XDR-TB. Further investigations are warranted to clarify the underlying mechanism and optimize the strategy in combination with other anti-TB drugs.