Quantification of macromolecular crowding and ionic strength in living cells

De intracellulaire omgeving verschilt van het extracellulaire milieu door de hoge dichtheid in macromoleculen. Deze hoge dichtheid aan macromoleculen, macromoleculaire crowding genoemd, kan worden gekwantificeerd met intracellulaire eiwit-gebaseerde sensoren. De mate van crowding wordt bepaald door Förster resonance energy transfer (FRET) tussen twee verschillende fluorescente eiwitten aan de sensor. In dit proefschrift helder ik het mechanisme van crowding sensing op, waarbij duidelijk wordt dat de sensoren zich gedragen als polymeren en dat hun compressie gerelateerd is aan hun grootte en de crowderconcentratie. In cellen is de compressie eveneens afhankelijk van de structuur van de linkerregio in de sensor, wat een belangrijk verschil vormt tussen in vitro en in vivo experimenten. Ik heb een afwijking geïdentificeerd vanwege de langzame maturatie van fluorescente eiwitten en dit opgelost door de expressie van de sensor onder een constitutieve promotor te plaatsen. Door crowding te volgen in Escherichia coli hebben we ontdekt hoe crowding verandert in cellen. Eveneens hebben we de eerste sensoren ontwikkeld om de ionische sterkte in levende cellen te bepalen. Deze sensoren om crowding en ionische sterkte te bepalen stellen ons in staat de fysicochemische aspecten in de cel te begrijpen en dienen als eerste stap om de rol van fysisch-chemische parameters op de biochemische organisatie en fysiologie van de levende cel op te helderen

Suavizando los histogramas

Knowledge of the ionic strength in cells is required to understand the in vivo biochemistry of the charged biomacromolecules. Here, we present the first sensors to determine the ionic strength in living cells, by designing protein probes based on Förster resonance energy transfer (FRET). These probes allow observation of spatiotemporal changes in the ionic strength on the single-cell level

Protective effect of Wuling mycelia, alone and in combination with valproic acid, on pentylenetetrazolinduced epilepsy in rats

Purpose: To determine the inhibitory effects of Wuling mycelia, alone and in combination with valproic acid (VPA) on pentylenetetrazol (PTZ)-induced epileptic seizure in rats, and their protective effects on cognitive impairment.Methods: Sprague-Dawley rats were randomly divided into five groups: control (sham), model, Wuling mycelia, VPA and combination groups. Rats in Wuling mycelia group were given oral Wuling mycelia alone at a dose of 594 mg/kg, while those in VPA group were given oral VPA alone at a dose of 189 mg/kg. In the combination group, rats received oral VPA at a dose of 189 mg/kg co-administered with Wuling mycelia at a dose of 594 mg/kg. One hour after the treatments, the control group was injected with physiological saline intraperitoneally, while the other four groups were injected with PTZ solution once a day for 28 days. Subsequently, seizure intensity, cognitive impairment, neuronal loss and hippocampal expressions of IL-1β, NF-ĸB/p65 and TLR4 were determine in all groups.Results: Combined use of Wuling mycelium and VPA significantly reduced the frequency and the grade of seizures (p <0.01), and also decreased the degree of cognitive impairment (p <0.05). There were marked increases in neuronal damage and hippocampal  expression levels of NF-ĸB/p65, TLR4 and IL1β (inflammatory cytokines) in the model group (p < 0.05). However, these changes were  reversed in the combination treatment group (p < 0.05).Conclusion: Wuling mycelia is a potentially effective adjunct drug for the treatment of refractory epilepsy. The underlying mechanism might involve downregulations of NF-ĸB/p65, TLR4 and IL-1β. Keywords: Wuling mycelia, Refractory epilepsy, Seizure, Traditional Chinese medicine, Hippocampal area, HMGB1/TLR4/NF-κB  signalling pathway, IL-1β, NF-ĸB/p65, TLR

Fabrication and properties of PLA/β-TCP scaffolds using liquid crystal display (LCD) photocuring 3D printing for bone tissue engineering

Introduction: Bone defects remain a thorny challenge that clinicians have to face. At present, scaffolds prepared by 3D printing are increasingly used in the field of bone tissue repair. Polylactic acid (PLA) has good thermoplasticity, processability, biocompatibility, and biodegradability, but the PLA is brittle and has poor osteogenic performance. Beta-tricalcium phosphate (β-TCP) has good mechanical properties and osteogenic induction properties, which can make up for the drawbacks of PLA.Methods: In this study, photocurable biodegradable polylactic acid (bio-PLA) was utilized as the raw material to prepare PLA/β-TCP slurries with varying β-TCP contents (β-TCP dosage at 0%, 10%, 20%, 30%, 35% of the PLA dosage, respectively). The PLA/β-TCP scaffolds were fabricated using liquid crystal display (LCD) light-curing 3D printing technology. The characterization of the scaffolds was assessed, and the biological activity of the scaffold with the optimal compressive strength was evaluated. The biocompatibility of the scaffold was assessed through CCK-8 assays, hemocompatibility assay and live-dead staining experiments. The osteogenic differentiation capacity of the scaffold on MC3T3-E1 cells was evaluated through alizarin red staining, alkaline phosphatase (ALP) detection, immunofluorescence experiments, and RT-qPCR assays.Results: The prepared scaffold possesses a three-dimensional network structure, and with an increase in the quantity of β-TCP, more β-TCP particles adhere to the scaffold surface. The compressive strength of PLA/β-TCP scaffolds exhibits a trend of initial increase followed by decrease with an increasing amount of β-TCP, reaching a maximum value of 52.1 MPa at a 10% β-TCP content. Degradation rate curve results indicate that with the passage of time, the degradation rate of the scaffold gradually increases, and the pH of the scaffold during degradation shows an alkaline tendency. Additionally, Live/dead staining and blood compatibility experiments suggest that the prepared PLA/β-TCP scaffold demonstrates excellent biocompatibility. CCK-8 experiments indicate that the PLA/β-TCP group promotes cell proliferation, and the prepared PLA/β-TCP scaffold exhibits a significant ability to enhance the osteogenic differentiation of MC3T3-E1 cells in vitro.Discussion: 3D printed LCD photocuring PLA/β-TCP scaffolds could improve surface bioactivity and lead to better osteogenesis, which may provide a unique strategy for developing bioactive implants in orthopedic applications

Decreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress

Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that GFP diffusion in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with FRET-based sensors. We apply an osmotic upshift and find that, after an initial increase, the apparent crowding decreases over the course of hours, to arrive at a value lower than before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle-size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm

Erratum

We acknowledge cofunding of the research from the EU ITN project SynCrop (project number 764591), appointment of Luca Mantovanelli