A multi-state dynamical process underpins mechano-adaptation in the bacterial flagellar motor

Adaptation is a defining feature of living systems. The bacterial flagellar motor adapts to changes in external mechanical environment by adding or removing torque-generating stator units. However, the molecular mechanism for mechanosensitive motor remodeling remains unclear. Here, we induced stator disassembly using electrorotation, followed by the time-dependent assembly of the individual stator units into the motor. From these experiments, we extracted detailed statistics of the dwell times underlying the stochastic dynamics of stator unit binding and unbinding. The dwell time distribution contains multiple timescales, indicating the existence of multiple stator unit states. Based on these results, we propose a minimal model with four stator unit states – two bound states with different unbinding rates, a diffusive unbound state, and a recently described transiently detached state. Our minimal model quantitatively explains multiple features of the experimental data and allows us to determine the transition rates between all four states. Our experiments and modeling point towards an emergent picture for mechano-adaptive remodeling of the bacterial flagellar motor in which torque generated by bound stator units controls their effective unbinding rate by modulating the transition between the two bound states. Furthermore, the binding rate of stator units with the motor has a non-monotonic dependence on the number of bound units, likely due to two counter-acting effects of motor’s rotation on the binding process.

The Binding of Aripiprazole to Plasma Proteins in Chronic Renal Failure Patients

The binding of drugs to plasma protein is frequently altered in certain types of renal diseases. We recently reported on the effects of oxidation and uremic toxins on the binding of aripiprazole (ARP) to human serum albumin. In our continuing investigations, we examined the binding of ARP to plasma pooled from patients with chronic renal dysfunction. We examined the issue of the molecular basis for which factors affect the changes in drug binding that accompany renal failure. The study was based on the statistical relationships between ARP albumin binding and biochemical parameters such as the concentrations of oxidized albumin and uremic toxins. The binding of ARP to plasma from chronic renal patients was significantly lower than healthy volunteers. A rational relationship between the ARP binding rate and the concentration of toxins, including indoxyl sulphate (IS) and p-cresyl sulphate (PCS), was found, particularly for IS. Moreover, multiple regression analyses that involved taking other parameters such as PCS or oxidized albumin ratio to IS into account supports the above hypothesis. In conclusion, the limited data reported in this present study indicates that monitoring IS in the blood is a very important determinant in the dosage plan for the administration of site II drugs such as ARP, if the efficacy of the drug in renal disease is to be considered.

Specific features of uranyl ions extraction by interpolymer system based on polyacrylic acid and polyethyleneimine hydrogels

Uranyl ions sorption of by interpolymer system consisting of polyacrylic acid hydrogel (hPAA) and polyethyleneimine hydrogel (hPEI) has been studied. Rate of uranyl ions extraction by the initial polymers and interpolymer system hPAA-hPEI, polymeric chain binding rate and dynamic exchange capacity of initial polymers and interpolymer system hPAA-hPEI were calculated. Based on obtained outcomes it was found that area of maximum rate of uranyl ions extraction is within the ratios of 67%hPAA:33%hPEI and 33%hPAA:67%hPEI. Maximum uranyl ions extraction rate after 48 hours of hydrogels remote interaction was 90.0 %, when polymeric chain binding rate was 9.1 % and dynamic exchange capacity was 1.14 mmol/g. Rate of uranyl ions extraction by the initial polymer hydrogels 100 % hPAA and 100 % hPEI was 68.0 % and 52.0%. Obtained outcomes showed changes of initial polymeric hydrogels sorption properties in intergel system leading to functional groups obtaining higher reactive ability, which makes it possible to use them for further development of highly efficient uranyl ions extraction sorption technology.

Exploring the Application of Bifunctional Metal Chelators in Treating Triple-Negative Breast Cancer

Purpose: In this study, we independently synthesised and labelled a novel bidentate bifunctional chelating agent, 177Lu-3,4-HOPO-Cetuximab, that achieved tight binding between targeting and radioactivity, and evaluated its targeted killing ability of cells in vitro and in vivo.Method: 3,4-HOPO was successfully synthesised through a series of chemical steps using malt phenol as the raw material, which was then coupled with Cetuximab labelled with 177Lu. 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab was tested for its cell viability and cell-binding rate after different times and at different doses by CCK-8 and cell-binding experiments. 177Lu-3,4-HOPO-Cetuximab (~500 μCi) and 177Lu-DOTA-Cetuximab (~500 μCi) were injected into the tail vein of a subcutaneous metastasis mouse model of triple-negative breast cancer with a single injection, and tumour volume growth and body weight changes were regularly monitored for 20 days. The radioactivity distribution in nude mice was analysed after sacrifice, and the treated and untreated tumour tissues were analysed by HE staining.Result: The cell viability of 177Lu-3,4-HOPO-Cetuximab declined exponentially after treatment for 48 h at 50 Bq/mL to 500 kBq/mL, respectively; the cell activity was slowed down from 8 to 96 h at a dose of 500 kBq; while the binding rate of 4T1 cells in 177Lu-3,4-HOPO-Cetuximab from 1 to 24 h, respectively, increased logarithmically, which was similar with 177Lu-DOTA-Cetuximab. After 20 days of treatment, the body weight of nude mice with 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab were hardly changed, while the body weight with physiological saline decreased significantly. The tumour inhibition rate of the 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab were (37.03 ± 11.16)% and (38.7 ± 5.1)%; HE staining showed that tumour cells were affected by the action of 177Lu causing necrosis.Conclusion: The experiments showed that 177Lu-3,4-HOPO-Cetuximab has a certain targeted therapeutic ability for triple-negative breast cancer, and it is expected to become a potential targeted nuclear medicine treatment for triple-negative breast cancer.

Measurement of kinetic constant of protein binding using microfluidics and particle diffusometry

Protein-protein interaction is widely used in biological science and biomedical engineering research Moreira et al. (2007). Accurate measurement of binding kinetics is essential for understanding protein-protein interactions. Current gold standard assays, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI) and quartz crystal microbalance (QCM), can generate precise and real-time kinetics data. However, these methods usually require expensive instruments housed in core facilities and high-level expertise, which is not convenient for most labs to implement. We developed a new method based on microfluidics and particle diffusometry (PD) to measure protein binding kinetics, which only needs very general lab equipment including a fluorescent microscope to take photos, a syringe pump to inject solutions, capillary tubing, a simple chip made on a glass slide and a computer to process images. To measure the binding rate of a protein pair, both proteins are conjugated with beads of different sizes, respectively. The bead solutions are diluted to appropriate concentrations and injected into a Y-junction channel by a syringe pump. In the microchannel, the two kinds of beads will meet at the interface and bind due to surface protein interactions. Therefore, the size of the beads in solution gradually increases and the Brownian motion will be less and less drastic until the reaction is saturated. Taking photos recording this dynamic process, the apparent change in size of the beads can be measured by particle diffusometry and used for extracting binding kinetics. Particle diffusometry is a correlation-based and non-intrusive optical detection method to analyze properties of fluid and particle such as viscosity, temperature and particle diameter Chamarthy et al. (2009); Clayton et al. (2016, 2017b,a); Hohreiter et al. (2002). It was initially developed to determine errors caused by thermal noise in particle image velocimetry (PIV). PD always analyzes image pairs. A single image of a particle laden flow is first used to do auto-correlation, correlating with itself, which will generate a high and sharp peak. Then it is cross-correlated with a successive image with a known time interval ∆t. Because particles slightly deviate away from the initial positions after time ∆t, due to Brownian motion, the correlation peak is lower and broader than that of auto-correlation. Auto- and cross-correlation peaks are fit into Gaussian function to find peak widths, by which the particle size can be computed as long as the viscosity and temperature do not change. Processing the image sets of the protein-conjugated particles’ binding process, we acquire the relation of particle size and time, which can be used to solve protein binding kinetics. An equation of protein interaction and particle volume is derived to work out association rate from particle diameter data acquired by PD. In this study, we measured streptavidin-biotin binding rate. Streptavidin is conjugated with 20nm beads and biotin is immobilized onto 200nm beads. Proteins on the two kinds of beads bind rapidly after mixing in the main channel. It is necessary to choose a narrow area at the interface of particle streams that diffusion does not limit the reaction. Since the liquid is flowing, there is both Brownian motion and advection in particle images. We used EDPIV, a software package developed by Prof. Steven Wereley’s lab, to measure advection velocity. When doing PD analysis, images are shifted following the PIV data to catch up with the flow. The photos are taken at the center layer in the middle of the channel, where there is no velocity gradient. Measuring a series of photo sets along the main channel at several points with known distances to each other, the relation of complex bead size and time can be acquired. Solving for the association constant, the measured value is 1.74 × 107M−1s−1, which is close to that of current gold standard assays. This novel PD-based method is accurate and requires only general lab facilities, making protein binding kinetics measurements accessible and practical for biological and biomedical labs.

Binding Tax Rate Information (WIS) as an expression of the principle of trust in public authorities and the principle of legal certainty

Binding Rate Information (WIS) is a new tax law system institution, in force since 1st November 2019. It is a regulation introducing the possibility of obtaining a decision of the tax authority in the scope of taxing goods and services for the supply of goods, import of goods, intra-community acquisition of goods or provision of services. The issued decision is binding, and therefore provides protection for the taxpayer. In a way, this regulation is an expression of the principles of trust in public authorities and of legal certainty, which is extremely important in the field of tax law. The main research objectives of the article are the legal analysis of the new regulations regarding Binding Rate Information, and an attempt to show that the new WIS institution fully implements the principles of legal certainty and of trust in public authorities as expressed in art. 121 o.p. The author uses the dogmatic-legal method to analyze the legal texts as well as the views of doctrine and case-law.

Antibody–Drug Conjugate to Treat Meningiomas

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody–drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of <5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of <10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

FEATURES OF SORPTION OF URANYL IONS BY POLYMETHACRYLIC ACID-POLY-4-VINYLPYRIDINE INTERPOLYMER SYSTEM

The sorption extraction of uranyl ions by interpolymer system consisting of polymethacrylic acid hydrogel (hPMAA) and poly-4-vinylpyridine hydrogel (hP4VP) was studied. Sorption and kinetic characteristics of initial polymers and hPMAA-hP4VP intergel system were determined. Maximum uranyl ions extraction rate - 82,8% - is observed in interpolymer system within the ratios of 100% hPMAA and 67% hPMAA:33%hP4VP 56 hours later,when polymeric chain binding rate was 9,97% and effective dynamic exchange capacity was 1,15mmol/g. Obtained outcomes show changes of initial polymeric hydrogels sorption properties ininterpolymer system, which makes it possible to use them for further development of highly efficient uranyl ions extraction sorption technology.