Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

Low temperature-mediated repression and far-red light-mediated induction determine morning FLOWERING LOCUS T expression levels.

In order to flower in the appropriate season, plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT). In Arabidopsis, FT messenger RNA levels peak in the morning and evening under natural long-day conditions (LDs). However, the regulatory mechanisms governing morning FT induction remain poorly understood. The morning FT peak is absent in typical laboratory LDs characterized by high red:far-red light (R:FR) ratios and constant temperatures. Here, we demonstrate that ZEITLUPE (ZTL) interacts with the FT repressors TARGET OF EATs (TOEs), thereby repressing morning FT expression in natural environments. Under LDs with simulated sunlight (R:FR = 1.0) and daily temperature cycles, which are natural LD-mimicking environmental conditions, FT transcript levels in the ztl mutant were high specifically in the morning, a pattern that was mirrored in the toe1 toe2 double mutant. Low night-to-morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning. Far-red light counteracted ZTL activity by decreasing its abundance (possibly via phytochrome A (phyA)) while increasing GIGANTEA (GI) levels and negatively affecting the formation of the ZTL-GI complex in the morning. Therefore, the phyA-mediated high-irradiance response and GI play pivotal roles in morning FT induction. Our findings suggest that the delicate balance between low temperature-mediated ZTL activity and the far-red light-mediated functions of phyA and GI offers plants flexibility in fine-tuning their flowering time by controlling FT expression in the morning.

Chemical Systems with Limit Cycles.

The dynamics of a chemical reaction network (CRN) is often modeled under the assumption of mass action kinetics by a system of ordinary differential equations (ODEs) with polynomial right-hand sides that describe the time evolution of concentrations of chemical species involved. Given an arbitrarily large integer [Formula: see text], we show that there exists a CRN such that its ODE model has at least K stable limit cycles. Such a CRN can be constructed with reactions of at most second-order provided that the number of chemical species grows linearly with K. Bounds on the minimal number of chemical species and the minimal number of chemical reactions are presented for CRNs with K stable limit cycles and at most second order or seventh-order kinetics. We also show that CRNs with only two chemical species can have K stable limit cycles, when the order of chemical reactions grows linearly with K.

The effects of brewers' spent grain on high-fat diet-induced fatty liver.

Obesity drives nonalcoholic fatty liver disease (NAFLD). This study investigated the effects of dietary brewers' spent grain (BSG) supplementation on obesity-induced NAFLD. Mice fed a high-fat diet supplemented with 30% BSG (HFD30) had reduced body weight and decreased plasma total cholesterol (TC) concentrations compared with HFD-fed mice. Retroperitoneal white adipose tissue (RWAT) and liver weights were reduced. Consistent with reduced hepatic triacylglycerol, TC, and non-esterified fatty acid concentrations, HFD30-fed mice showed reduced hepatic steatosis. 3-hydroxy-3-methylglutaryl-CoA reductase and low-density lipoprotein receptor genes were increased, whereas carnitine palmitoyltransferase 1 alpha, ATP-binding cassette subfamily A member 1 (Abca1), and cholesterol 7 alpha-hydroxylase genes were upregulated in the liver of HFD30-fed mice. Abca1 gene expression was also increased in epididymal WAT and RWAT of HFD30-fed mice. BSG supplementation increased and decreased fecal fat and bile acid concentrations, respectively. Taken together, BSG supplementation reduced HFD-induced hepatic lipid accumulation by increasing fatty acid oxidation and bile acid synthesis in the liver as well as decreasing lipid absorption in the intestine.

Incorporating age and delay into models for biophysical systems.

In many biological systems, chemical reactions or changes in a physical state are assumed to occur instantaneously. For describing the dynamics of those systems, Markov models that require exponentially distributed inter-event times have been used widely. However, some biophysical processes such as gene transcription and translation are known to have a significant gap between the initiation and the completion of the processes, which renders the usual assumption of exponential distribution untenable. In this paper, we consider relaxing this assumption by incorporating age-dependent random time delays (distributed according to a given probability distribution) into the system dynamics. We do so by constructing a measure-valued Markov process on a more abstract state space, which allows us to keep track of the 'ages' of molecules participating in a chemical reaction. We study the large-volume limit of such age-structured systems. We show that, when appropriately scaled, the stochastic system can be approximated by a system of partial differential equations (PDEs) in the large-volume limit, as opposed to ordinary differential equations (ODEs) in the classical theory. We show how the limiting PDE system can be used for the purpose of further model reductions and for devising efficient simulation algorithms. In order to describe the ideas, we use a simple transcription process as a running example. We, however, note that the methods developed in this paper apply to a wide class of biophysical systems.

Multiscale Stochastic Reaction-Diffusion Algorithms Combining Markov Chain Models with Stochastic Partial Differential Equations.

Two multiscale algorithms for stochastic simulations of reaction-diffusion processes are analysed. They are applicable to systems which include regions with significantly different concentrations of molecules. In both methods, a domain of interest is divided into two subsets where continuous-time Markov chain models and stochastic partial differential equations (SPDEs) are used, respectively. In the first algorithm, Markov chain (compartment-based) models are coupled with reaction-diffusion SPDEs by considering a pseudo-compartment (also called an overlap or handshaking region) in the SPDE part of the computational domain right next to the interface. In the second algorithm, no overlap region is used. Further extensions of both schemes are presented, including the case of an adaptively chosen boundary between different modelling approaches.

Comparison of Deterministic and Stochastic Regime in a Model for Cdc42 Oscillations in Fission Yeast.

Oscillations occur in a wide variety of essential cellular processes, such as cell cycle progression, circadian clocks and calcium signaling in response to stimuli. It remains unclear how intrinsic stochasticity can influence these oscillatory systems. Here, we focus on oscillations of Cdc42 GTPase in fission yeast. We extend our previous deterministic model by Xu and Jilkine to construct a stochastic model, focusing on the fast diffusion case. We use SSA (Gillespie's algorithm) to numerically explore the low copy number regime in this model, and use analytical techniques to study the long-time behavior of the stochastic model and compare it to the equilibria of its deterministic counterpart. Numerical solutions suggest noisy limit cycles exist in the parameter regime in which the deterministic system converges to a stable limit cycle, and quasi-cycles exist in the parameter regime where the deterministic model has a damped oscillation. Near an infinite period bifurcation point, the deterministic model has a sustained oscillation, while stochastic trajectories start with an oscillatory mode and tend to approach deterministic steady states. In the low copy number regime, metastable transitions from oscillatory to steady behavior occur in the stochastic model. Our work contributes to the understanding of how stochastic chemical kinetics can affect a finite-dimensional dynamical system, and destabilize a deterministic steady state leading to oscillations.

Quasi-Steady-State Approximations Derived from the Stochastic Model of Enzyme Kinetics.

The paper outlines a general approach to deriving quasi-steady-state approximations (QSSAs) of the stochastic reaction networks describing the Michaelis-Menten enzyme kinetics. In particular, it explains how different sets of assumptions about chemical species abundance and reaction rates lead to the standard QSSA, the total QSSA, and the reverse QSSA. These three QSSAs have been widely studied in the literature in deterministic ordinary differential equation settings, and several sets of conditions for their validity have been proposed. With the help of the multiscaling techniques introduced in Ball et al. (Ann Appl Probab 16(4):1925-1961, 2006), Kang and Kurtz (Ann Appl Probab 23(2):529-583, 2013), it is seen that the conditions for deterministic QSSAs largely agree (with some exceptions) with the ones for stochastic QSSAs in the large-volume limits. The paper also illustrates how the stochastic QSSA approach may be extended to more complex stochastic kinetic networks like, for instance, the enzyme-substrate-inhibitor system.

Germinated Waxy Black Rice Ameliorates Hyperglycemia and Dyslipidemia in Streptozotocin-Induced Diabetic Rats.

This study aimed to examine the anti-diabetic effect of germinated waxy black rice (GWBR) using streptozotocin (STZ)-induced diabetic rats. In the diabetic rats, GWBR supplementation for 8 weeks reduced plasma blood glucose concentrations, improved glucose clearance and prevented diabetes-induced weight loss. Rats with STZ-induced diabetes who received GWBR supplementation exhibited decreased expression of sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter (GLUT) 2 genes and proteins in the small intestine via decreases in hepatocyte nuclear factor (HNF)-1α, HNF-1ß, and HNF-4α, transcriptional factors that are involved in the regulation of SGLT1 and GLUT2, compared with the rats with STZ-induced diabetes that did not receive GWBR supplements. GWBR supplementation also enhanced the expression of GLUT4 and the genes and proteins involved in GLUT4 translocation, such as insulin receptor (IR) and insulin receptor substrate 1 (IRS1), and increased the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB, Akt) proteins in skeletal muscle. GWBR further increased glycogen synthase (GS) 1 by decreasing glycogen synthase kinase (GSK)-3ß in skeletal muscle. Interestingly, GWBR recovered STZ-impaired pancreatic ß-cells, resulting in increased insulin synthesis and secretion. In addition, GWBR reduced serum triglyceride, total cholesterol, low-density lipoprotein cholesterol, aspartate transferase and alanine transferase concentrations and increased high-density lipoprotein cholesterol concentrations. Taken together, these findings suggest that GWBR could be a candidate for improving the diabetic condition by regulating glucose uptake in the intestine and muscle and regulating the secretion of insulin from the pancreas.

Kefir prevented excess fat accumulation in diet-induced obese mice.

Excessive body fat accumulation can result in obesity, which is a serious health concern. Kefir, a probiotic, has recently shown possible health benefits in fighting obesity. This study investigated the inhibitory effects of 0.1 and 0.2% kefir powder on fat accumulation in adipose and liver tissues of high-fat diet (HFD)-induced obese mice. Kefir reduced body weight and epididymal fat pad weight and decreased adipocyte diameters in HFD-induced obese mice. This was supported by decreased expression of genes related to adipogenesis and lipogenesis as well as reduced proinflammatory marker levels in epididymal fat. Along with reduced hepatic triacylglycerol concentrations and serum alanine transaminase and aspartate transaminase activities, genes related to lipogenesis and fatty acid oxidation were downregulated and upregulated, respectively, in liver tissue. Kefir also decreased serum triacylglycerol, total cholesterol, and low-density lipoprotein-cholesterol concentrations. Overall, kefir has the potential to prevent obesity.

Using a guide template with a handpiece sleeve to locate the abutment screw position of a cement-retained implant restoration.

The existing techniques for drilling a screw access hole in cement-retained restorations are limited by inaccurate drill guidance and ineffective cooling of the drilling area. An approach for fabricating a guide template to provide screw retrievability using computer-aided design and computer-aided manufacturing (CAD/CAM) is described. A handpiece sleeve was made by 3-dimensional printing and incorporating it into a vacuum-formed template. The handpiece sleeve not only guides the head of the handpiece accurately but also enables the cooling water to reach the area of drilling directly.

Thioesterase superfamily member 2/Acyl-CoA thioesterase 13 (Them2/Acot13) regulates adaptive thermogenesis in mice.

Members of the acyl-CoA thioesterase (Acot) gene family hydrolyze fatty acyl-CoAs, but their biological functions remain incompletely understood. Thioesterase superfamily member 2 (Them2; synonym Acot13) is enriched in oxidative tissues, associated with mitochondria, and relatively specific for long chain fatty acyl-CoA substrates. Using Them2(-/-) mice, we have demonstrated key roles for Them2 in regulating hepatic glucose and lipid metabolism. However, reduced body weights and decreased adiposity in Them2(-/-) mice observed despite increased food consumption were not well explained. To explore a role in thermogenesis, mice were exposed to ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C). In response to short term (24-h) exposures to decreasing ambient temperatures, Them2(-/-) mice exhibited increased adaptive responses in physical activity, food consumption, and energy expenditure when compared with Them2(+/+) mice. By contrast, genotype-dependent differences were not observed in mice that were equilibrated (96 h) at each ambient temperature. In brown adipose tissue, the absence of Them2 was associated with reduced lipid droplets, alterations in the ultrastructure of mitochondria, and increased expression of thermogenic genes. Indicative of a direct regulatory role for Them2 in heat production, cultured primary brown adipocytes from Them2(-/-) mice exhibited increased norepinephrine-mediated triglyceride hydrolysis and increased rates of O2 consumption, together with elevated expression of thermogenic genes. At least in part by regulating intracellular fatty acid channeling, Them2 functions in brown adipose tissue to suppress adaptive increases in energy expenditure.

Stochastic analysis of reaction-diffusion processes.

Reaction and diffusion processes are used to model chemical and biological processes over a wide range of spatial and temporal scales. Several routes to the diffusion process at various levels of description in time and space are discussed and the master equation for spatially discretized systems involving reaction and diffusion is developed. We discuss an estimator for the appropriate compartment size for simulating reaction-diffusion systems and introduce a measure of fluctuations in a discretized system. We then describe a new computational algorithm for implementing a modified Gillespie method for compartmental systems in which reactions are aggregated into equivalence classes and computational cells are searched via an optimized tree structure. Finally, we discuss several examples that illustrate the issues that have to be addressed in general systems.

Mechanistic insights into condensate formation of human liver-type phosphofructokinase by stochastic modeling approaches.

Human liver-type phosphofructokinase 1 (PFKL) has been shown to regulate glucose flux as a scaffolder arranging glycolytic and gluconeogenic enzymes into a multienzyme metabolic condensate, the glucosome. However, it has remained elusive of how phase separation of PFKL is governed and initiates glucosome formation in living cells, thus hampering to understand a mechanism of glucosome formation and its functional contribution to human cells. In this work, we developed a stochastic model in silico using the principle of Langevin dynamics to investigate how biological properties of PFKL contribute to the condensate formation. The significance of an intermolecular interaction between PFKLs, an effective concentration of PFKL at a region of interest, and its own self-assembled filaments in formation of PFKL condensates and control of their sizes were demonstrated by molecular dynamics simulation using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS). Such biological properties that define intracellular dynamics of PFKL appear to be essential for phase separation of PFKL, which may represent an initiation step for the formation of glucosome condensates. Collectively, our computational study provides mechanistic insights of glucosome formation, particularly an initial stage through the formation of PFKL condensates in living human cells.

Thioesterase superfamily member 2/acyl-CoA thioesterase 13 (Them2/Acot13) regulates hepatic lipid and glucose metabolism.

Members of the acyl-CoA thioesterase (Acot) gene family catalyze the hydrolysis of fatty acyl-CoAs, but their biological functions remain unknown. Thioesterase superfamily member 2 (Them2; synonym Acot13) is a broadly expressed mitochondria-associated Acot. Them2 was previously identified as an interacting protein of phosphatidylcholine transfer protein (PC-TP). Pctp(-/-) mice exhibit altered fatty acid metabolism that is accompanied by reduced hepatic glucose production. To examine the role of Them2 in regulating hepatic lipid and glucose homeostasis, we generated Them2(-/-) mice. In livers of Them2(-/-) mice compared with Them2(+/+) controls, a 1.9-fold increase in the K(m) of mitochondrial thioesterase activity was accompanied by a 28% increase in fatty acyl-CoA concentration. A reciprocal 23% decrease in free fatty acid concentration was associated with reduced activation of peroxisome proliferator-activated receptor α. However, fatty acid oxidation rates were preserved in livers of Them2(-/-) mice, suggesting that Them2 functions to limit ß-oxidation. Hepatic glucose production was also decreased by 45% in the setting of reduced hepatocyte nuclear factor 4α (HNF4α) expression. When fed a high-fat diet, Them2(-/-) mice were resistant to increases in hepatic glucose production and steatosis. These findings reveal key roles for Them2 in the regulation of hepatic metabolism, which are potentially mediated by PC-TP-Them2 interactions.

Continuous production of lipid nanoparticles by multiple-splitting in microfluidic devices with chaotic microfibrous channels.

Polydimethylsiloxane (PDMS) microfluidic devices with chaotic microfibrous channels were fabricated for the continuous production of lipid nanoparticles (LNPs). Electrospun poly(ε-caprolactone) (PCL) microfibrous matrices with different diameters (3.6 ± 0.3, 6.3 ± 0.4, and 12.2 ± 0.8 µm) were used as a template to develop microfibrous channels. The lipid solution (in ethanol) and water phase were introduced into the microfluidic device as the discontinuous and continuous phases, respectively. The smaller diameter of microfibrous channels and the higher flow rate of the continuous phase resulted in the smaller LNPs with a narrower size distribution. The multiple-splitting of the discontinuous phase and the microscale contact between the two phases in the microfibrous channels were the key features of the LNP production in our approach. The LNPs containing doxorubicin with different average sizes (89.7 ± 35.1 and 190.4 ± 66.4 nm) were prepared using the microfluidic devices for the potential application in tumor therapy. In vitro study revealed higher cellular uptake efficiency and cytotoxicity of the smaller LNPs, especially in the HepG2 cells. The microfluidic devices with microfibrous channels can be widely used as a continuous and high-throughput platform for the production of LNPs containing various active agents.

A new method for choosing the computational cell in stochastic reaction-diffusion systems.

How to choose the computational compartment or cell size for the stochastic simulation of a reaction-diffusion system is still an open problem, and a number of criteria have been suggested. A generalized measure of the noise for finite-dimensional systems based on the largest eigenvalue of the covariance matrix of the number of molecules of all species has been suggested as a measure of the overall fluctuations in a multivariate system, and we apply it here to a discretized reaction-diffusion system. We show that for a broad class of first-order reaction networks this measure converges to the square root of the reciprocal of the smallest mean species number in a compartment at the steady state. We show that a suitably re-normalized measure stabilizes as the volume of a cell approaches zero, which leads to a criterion for the maximum volume of the compartments in a computational grid. We then derive a new criterion based on the sensitivity of the entire network, not just of the fastest step, that predicts a grid size that assures that the concentrations of all species converge to a spatially-uniform solution. This criterion applies for all orders of reactions and for reaction rate functions derived from singular perturbation or other reduction methods, and encompasses both diffusing and non-diffusing species. We show that this predicts the maximal allowable volume found in a linear problem, and we illustrate our results with an example motivated by anterior-posterior pattern formation in Drosophila, and with several other examples.

Garlic Scape (Allium sativum L.) Extract Decreases Adipogenesis by Activating AMK-Activated Protein Kinase During the Differentiation in 3T3-L1 Adipocytes.

Regulating adipogenesis and lipogenesis in white adipose tissue (WAT) is an efficient strategy to reduce obesity. This study investigates whether garlic scape extract (GSE) has anti-adipogenic and anti-lipogenic effects and which stage of adipogenesis is critical for its effect using 3T3-L1 cells. 3T3-L1 cells that were treated with GSE during adipogenesis and differentiation exhibited reduced peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein a (Cebpa) and Cebpb, acetyl-CoA carboxylase, fatty acid synthase, sterol regulatory element binding protein 1c, diacylglycerol acyltransferase 1, and perilipin 1 genes. When the cells were treated with GSE during postdifferentiation or during preadipocytes, they showed less reduction and no change, respectively. Consistent with this, lipid accumulation was strongly reduced in the cells that were treated during adipogenesis and differentiation and to a lesser extent in the cells that were treated during preadipocytes and postdifferentiation. Phosphorylation on AMP-activated protein kinase (AMPK) and its downstream proteins was increased together with increased carnitine palmitoyl transferase 1α and phosphorylation on hormone-sensitive lipase in the cells that were treated with GSE during differentiation. In summary, GSE reduced intracellular lipid accumulation by suppressing adipogenic and lipogenic genes and proteins by possibly the activation of AMPK signaling pathway during adipocyte differentiation. This result indicates that garlic scape may have the potential to prevent obesity by regulating lipid metabolism in WAT.

Fabrication of Microfiber-Templated Microfluidic Chips with Microfibrous Channels for High Throughput and Continuous Production of Nanoscale Droplets.

A polydimethylsiloxane (PDMS) microfluidic chip with well-interconnected microfibrous channels was fabricated by using an electrospun poly(ε-caprolactone) (PCL) microfibrous matrix and 3D-printed pattern as templates. The microfiber-templated microfluidic chip (MTMC) was used to produce nanoscale emulsions and spheres through multiple emulsification at many small micro-orifice junctions among microfibrous channels. The emulsion formation mechanisms in the MTMC were the cross-junction dripping or Y-junction splitting at the micro-orifice junctions. We demonstrated the high throughput and continuous production of water-in-oil emulsions and polyethylene glycol-diacrylate (PEG-DA) spheres with controlled size ranges from 2.84 µm to 83.6 nm and 1.03 µm to 45.7 nm, respectively. The average size of the water droplets was tuned by changing the micro-orifice diameter of the MTMC and the flow rate of the continuous phase. The MTMC theoretically produced 58 trillion PEG-DA nanospheres per hour without high shear force. In addition, we demonstrated the higher encapsulation efficiency of the PEG-DA microspheres in the MTMC than that of the microspheres fabricated by ultrasonication. The MTMC can be used as a powerful platform for the large-scale and continuous productions of emulsions and spheres.

Regulatory role for phosphatidylcholine transfer protein/StarD2 in the metabolic response to peroxisome proliferator activated receptor alpha (PPARalpha).

Phosphatidylcholine transfer protein (PC-TP, a.k.a. StarD2) is abundantly expressed in liver and is regulated by PPARalpha. When fed the synthetic PPARalpha ligand fenofibrate, Pctp(-/-) mice exhibited altered lipid and glucose metabolism. Microarray profiling of livers from fenofibrate fed wild type and Pctp(-/-) mice revealed differential expression of a broad array of metabolic genes, as well as their regulatory transcription factors. PC-TP expression in cell culture controlled the activities of both PPARalpha and HNF4alpha, suggesting that the mechanism by which it modulates hepatic metabolism is at least in part via activation of transcription factors that govern nutrient homeostasis.