Encapsulation of doxorubicin and ellipticine into apoferritin nanocarriers

Tumor disease is the second most common cause of death in the Czech Republic, right after cardiovascular diseases. The relatively new and growing science field of nanomedicine brought a new insight into the possibilities of studying the tumor diseases with nanotransporters. The encapsulation of the drug into the nanotransponers can improve the properties and distribution of the cytostatics, in particular to reduce the side effects of the cytostatics on the surrounding healthy tissue. This work studied nanotransporter apoferritin (apo-form of ferritin) as a very promising candidate for the clinical use. Optimization of the preparation of apoferritin nanotransporters with two different cytostatics - doxorubicin and ellipticine was studied. Furthermore, apoellipticine was characterized in more detail in terms of its physicochemical properties (stability and size). The results obtained show promising potential of these nanocarriers for the clinical use. Key words anticancer drugs, doxorubicin, ellipticine, nanomedicine, nanocarriers, apoferritinNádorové onemocnění je druhou nejčastější příčinou úmrtí v ČR, hned po kardiovaskulárních chorobách. Relativně nový a velice se rozrůstající vědní obor nanomedicína přinesla nový náhled na možnosti studia národového onemocnění pomocí nanotransportérů. Uložení léčiva do nanotranspotéru může zlepšit vlastnosti a distribuci cytostatika, zejména snížit vedlejší účinky daného cytostatika na okolní zdravou tkáň. Tato práce studovala nanotransportér apoferritin (apo- forma ferritinu) jako velice slibného kandidáta pro klinické využití. Studována byla optimalizace přípravy apoferritinových nanotransportérů se dvěma různými cytostatiky - doxorubicinu a ellipticinu. Dále byl podrobněji charakterizován apoellipticin z hlediska jeho fyzikálně - chemických vlastností (stabilita a velikost). Získané výsledky ukazují slibný potenciál pro klinické použití apodoxorubicinu i apoellipticinu. Klíčová slova protinádorová léčiva, doxorubicin, ellipticin, nanomedicína, nanočástice, apoferritinKatedra biochemieDepartment of BiochemistryFaculty of SciencePřírodovědecká fakult

Effect of cytochromes P450 on metabolism of anticancer drugs bound into apoferritin nanoparticle

Tumour-related diseases are the second most common cause of death in the Czech Republic, right after cardiovascular diseases. Nanomedicine - a novel scientific discipline - shows captivating potential in anticancer treatment with help of so called nanotranporters - nanoparticles capable of transporting other molecules. Encapsulation of a cytostatic drug into a nanoparticle improves its pharmacokinetical and pharmacodynamical properties which helps to reduce adverse side effects on non-tumour healthy tissue. In the scope of this diploma thesis apoferritin - apo-form of ferritin - was studied, since this nanotransporter shows promise for clinical use in anticancer treatment. Effect of hepatic microsomes from premedicated and control rats on biotransformation of doxorubicin cytostatic (Dox) in free and apoferritin nanoparticle-bound forms was investigated at pH 7,4. Over the course of biotransformation two types of metabolites - M1 and M2 - were observed. Regardless of the employed inductor all studied microsomes have exhibited similar metabolism of free doxorubicin and its apoferritin encapsulated form (ApoDox). Our results also imply that doxorubicin can be metabolically processed by rat hepatic microsomes in both free and ApoDox form with similar efficiency. We have also studied biotransformation...Nádorové onemocnění je druhou nejčastější příčinou úmrtí v České republice hned po kardiovaskulárních chorobách. Nový vědní obor - nanomedicína, ukazuje slibný potenciál v protinádorové léčbě pomocí tzv. nanotransportérů - nanočástice schopných přepravovat jiné molekuly. Cytostatika enkapsulovaná do nanotrasportérů zlepšují farmakokinetické i farmakodynamické vlastnosti léčiva. Tím mohou snížit vedlejší účinky cytostatika na zdravou tkáň. V rámci této diplomové práce byl studován apoferritin - apo-forma ferritinu, jenž se ukazuje jako slibný adept pro klinické využití v protinádorové léčbě. Studován byl vliv jaterních mikrosomů kontrolních a premedikovaných potkanů na biotransformaci cytostatika doxorubicinu ve volné formě i ve formě vázané v apoferritinové nanočástici při pH 7,4. Během biotransformace se tvořili dva různé metabolity - M1 a M2. Všechny použité mikrosomy bez ohledu na použitý induktor se podílí na metabolismus doxorubicinu volného i doxorubicinu vázaného v apoferritinu (ApoDox) podobným způsobem. Výsledky zároveň naznačují, že bez ohledu na formu použitého Dox (volný či vázaný v ApoDox) jsou jaterní mikrosomy potkana schopny jeho metabolické přeměny, a to s prakticky stejnou efektivitou. Rovněž byl studován vliv jaterních mikrosomů, cytochromů P450 rodiny 3A4 v Supersomech™ a...Department of BiochemistryKatedra biochemiePřírodovědecká fakultaFaculty of Scienc

Child safety on various bicycle-mounted seats during vehicle impact

This research addresses an important gap in the state of the art by investigating the safety of vulnerable road users – children transported on bicycle seats. The article focuses on three forms of bicycle-mounted child seats and their kinematics during an accident scenario involving a motor vehicle. The front, rear-frame and rear-rack mounted child seat mounting configurations were considered in this study. The research covers the impact of a sports sedan vehicle against a bicycle equipped with the child seat. The assessment of the child safety was done through numerical simulations by coupling the codes of MADYMO and LS-DYNA. The after-impact kinematics for various baby carriers is presented with the emphasis on child’s head and neck injuries. The results were compared to the full-scale test available in the literature. The findings prove a low protection level for the child provided by the bicycle carriers in all considered cases. The study is further devoted to directions of increasing child safety in this means of transportation. First published online 18 March 201

Modulation of the Blazhko Cycle in LS Her

We present analysis of the RR Lyrae star, LS Her and confirm the previously reported modulation to its Blazhko cycles. We performed Fourier analysis on two sectors (Sector 24 & 25) of data from the Transiting Exoplanet Survey Satellite (TESS) spanning 53 days. We find LS Her to have a primary pulsation period of 0.2308 d and a Blazhko period of 12.7 d in keeping with previously reported results. We also identified side-band frequencies around the Blazhko multiplets suggesting the Blazhko cycle is modulated on a time scale of 112 days. Analysis of the Blazhko effect using the TESS data clearly shows a changing amplitude and phase throughout the four Blazhko cycles. We compared our modeled results, which were based on our TESS frequency analysis, to TESS data (Sector 51) taken ~700 days later and found our modulation model was not a good representation of the data. We then coupled our TESS analysis with the modulation frequency results from Wils et al. (MNRAS 387 (2008) 783-787) and found excellent agreement with the Sector 51 data. To further test this result we obtained ground-based, V-magnitude observations of LS Her in the summer of 2022. This data also showed excellent agreement with our coupled modulation model. We have verified that LS Her is a Blazhko star with a modulated Blazhko period of 109 days, stability over the 862 days of observations, and possible stability lasting over 15 years. We discuss the ramifications of the modulation for other Blazhko stars that show Blazhko effect changes over time.Comment: 9 pages, 10 figure

Umformen von Karton in innovativer Industrieanlage

Im Rahmen eines Kooperationsprojektes wurde eine Form-, Füll- und Verschließanlage zum effizienten Verpacken von Nutzinsekten entwickelt. Diese Anlage ist die welterste industrielle Anwendung der an der TU Dresden weiterentwickelten Tiefziehtechnologie von Karton. Gegenüber des Laborumformversuchsstandes ist die Anlage mit den Stationen „Füllen“ und „Verschließen“ gekoppelt, sodass sich das Verhalten des Tiefziehverfahrens im Gesamtprozess untersuchen lässt. Weiterhin leistet die Anlage sehr viel höhere Taktzahlen als am Versuchsstand möglich, wodurch sich neue Anforderungen für Material und Prozess ergeben. Eine wesentliche Innovation ist die Umformung direkt aus der Kartonbahn, sodass die befüllten und verschlossenen Verpackungen erst am Ende der Prozesskette aus der Bahn getrennt werden müssen. Dies ist durch das gezielte Einbringen von Entlastungsstanzungen möglich und bedarf einer genauen Kenntnis und Kontrolle des Materialflusses. Vorteile sind das vereinfachte Handling und die so erhöhte Ausbringung. Anhand des Projektes wird zudem die Bedeutung und Innovationskraft der Kooperation von KMUs mit Forschungseinrichtungen dargestellt

The inhibition of FGF receptor 1 activity mediates sorafenib-induced antiproliferative effects in human mesothelioma tumor-initiating cells

Tumor-initiating cells (TICs), the subset of cells within tumors endowed with stem-like features, being highly resistant to conventional cytotoxic drugs, are the major cause of tumor relapse. The identification of molecules able to target TICs remains a significant challenge in cancer therapy. Using TIC-enriched cultures (MM1, MM3 and MM4), from 3 human malignant pleural mesotheliomas (MPM), we tested the effects of sorafenib on cell survival and the intracellular mechanisms involved. Sorafenib inhibited cell-cycle progression in all the TIC cultures, but only in MM3 and MM4 cells this effect was associated with induction of apoptosis via the down-regulation of Mcl-1. Although sorafenib inhibits the activity of several tyrosine kinases, its effects are mainly ascribed to Raf inhibition. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with EGF or bFGF causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt and STAT3 phosphorylation. These effects were significantly reduced by sorafenib in bFGF-treated cells, while a slight inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGFR inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. A different picture was observed in MM1 cells, which, releasing high levels of bFGF, showed an autocrine activation of FGFR1 and a constitutive phosphorylation/activation of MEK-ERK1/2. A powerful inhibitory response to sorafenib was observed in these cells, indirectly confirming the central role of sorafenib as FGFR inhibitor. These results suggest that bFGF signaling may impact antiproliferative response to sorafenib of MPM TICs, which is mainly mediated by a direct FGFR targeting

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities

Sterol biosensor reveals LAM-family Ltc1-dependent sterol flow to endosomes upon Arp2/3 inhibition.

Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell

Effects of Sorafenib on Intra-Tumoral Interstitial Fluid Pressure and Circulating Biomarkers in Patients with Refractory Sarcomas (NCI Protocol 6948)

Purpose: Jain Sorafenib is a multi-targeted tyrosine kinase inhibitor with therapeutic efficacy in several malignancies. Sorafenib may exert its anti-neoplastic effect in part by altering vascular permeability and reducing intra-tumoral interstitial hypertension. As correlative science with a phase II study in patients with advanced soft-tissue sarcomas (STS), we evaluated the impact of this agent on intra-tumor interstitial fluid pressure (IFP), serum circulating biomarkers, and vascular density. Patients and Methods: Patients with advanced STS with measurable disease and at least one superficial lesion amenable to biopsy received sorafenib 400 mg twice daily. Intratumoral IFP and plasma and circulating cell biomarkers were measured before and after 1–2 months of sorafenib administration. Results were analyzed in the context of the primary clinical endpoint of time-to-progression (TTP). Results: In 15 patients accrued, the median TTP was 45 days (range 14–228). Intra-tumoral IFP measurements obtained in 6 patients at baseline showed a direct correlation with tumor size. Two patients with stable disease at two months had post-sorafenib IFP evaluations and demonstrated a decline in IFP and vascular density. Sorafenib significantly increased plasma VEGF, PlGF, and SDF1$\alpha$ and decreased sVEGFR-2 levels. Increased plasma SDF1$\alpha$ and decreased sVEGFR-2 levels on day 28 correlated with disease progression. Conclusions: Pretreatment intra-tumoral IFP correlated with tumor size and decreased in two evaluable patients with SD on sorafenib. Sorafenib also induced changes in circulating biomarkers consistent with expected VEGF pathway blockade, despite the lack of more striking clinical activity in this small series