X-ray-Sensitive Doped CaF2-Based MRI Contrast Agents for Local Radiation Dose Measurement.

Ionizing radiation has become widely used in medicine, with application in diagnostic techniques, such as computed tomography (CT) and radiation therapy (RT), where X-rays are used to diagnose and treat tumors. The X-rays used in CT and, in particular, in RT can have harmful side effects; hence, an accurate determination of the delivered radiation dose is of utmost importance to minimize any damage to healthy tissues. For this, medical specialists mostly rely on theoretical predictions of the delivered dose or external measurements of the dose. To extend the practical use of ionizing radiation-based medical techniques, such as magnetic resonance imaging (MRI)-guided RT, a more precise measurement of the internal radiation dose internally is required. In this work, a novel approach is presented to measure dose in liquids for potential future in vivo applications. The strategy relies on MRI contrast agents (CAs) that provide a dose-sensitive signal. The demonstrated materials are (citrate-capped) CaF2 nanoparticles (NPs) doped with Eu3+ or Fe2+/Fe3+ ions. Free electrons generated by ionizing radiation allow the reduction of Eu3+, which produces a very small contrast in MRI, to Eu2+, which induces a strong contrast. Oxidative species generated by high-energy X-rays can be measured indirectly using Fe2+ because it oxidizes to Fe3+, increasing the contrast in MRI. Notably, in the results, a strong increase in the proton relaxation rates is observed for the Eu3+-doped NPs at 40 kV. At 6 MV, a significant increase in proton relaxation rates is observed using CaF2 NPs doped with Fe2+/Fe3+ after irradiation. The presented concept shows great promise for use in the clinic to measure in vivo local ionizing radiation dose, as these CAs can be intravenously injected in a saline solution.

Metabolite Biomarkers of Prolonged and Intensified Pain and Distress in Head and Neck Cancer Patients Undergoing Radio- or Chemoradiotherapy by Means of NMR-Based Metabolomics-A Preliminary Study.

Treatment of head and neck squamous cell carcinoma (HNSCC) has a detrimental impact on patient quality of life. The rate of recognized distress/depression among HNSCC patients ranges from 9.8% to 83.8%, and the estimated prevalence of depression among patients receiving radiotherapy is 63%. Shorter overall survival also occurs in preexisting depression or depressive conditions. The present study analyzes the nuclear magnetic resonance (NMR) blood serum metabolic profiles during radio-/chemoradiotherapy and correlates the detected alterations with pain and/or distress accumulated with the disease and its treatment. NMR spectra were acquired on a Bruker 400 MHz spectrometer and analyzed using multivariate methods. The results indicate that distress and/or pain primarily affect the serum lipids and metabolites of energy (glutamine, glucose, lactate, acetate) and one-carbon (glycine, choline, betaine, methanol, threonine, serine, histidine, formate) metabolism. Sparse disturbances in the branched-chain amino acids (BCAA) and in the metabolites involved in protein metabolism (lysine, tyrosine, phenylalanine) are also observed. Depending on the treatment modality-radiotherapy or concurrent chemoradiotherapy-there are some differences in the altered metabolites.

Applicability of Electron-Beam and Hybrid Plasmas for Polyethylene Terephthalate Processing to Obtain Hydrophilic and Biocompatible Surfaces.

The applicability of beam-plasma chemical reactors generating cold hybrid plasma for the production of noncytotoxic polymeric surfaces with high hydrophilicity and good biocompatibility with human fibroblast culture and human red blood cells was studied. Oxygen hybrid plasma was excited by the joint action of a continuous scanning electron beam and a capacity-coupled RF-gas discharge. Experiments showed that hybrid plasma treatment caused polar oxygen-containing functional group formation in the surface layer of poly (ethylene terephthalate) films. No thermal or radiative damage in tested polymer samples was found. The plasma-modified polymers turned out to be noncytotoxic and revealed good biocompatibility with human fibroblasts BJ-5ta as well as lower hemolytic activity than untreated poly (ethylene terephthalate). Experiments also demonstrated that no phenomena caused by the electrostatic charging of polymers occur in hybrid plasma because the electron beam component of hybrid plasma eliminates the item charge when it is treated. The electron beam can effectively control the reaction volume geometry as well as the fluxes of active plasma particles falling on the item surface. This provides new approaches to the production of abruptly structured patterns or smooth gradients of functionalities on a plane and 3D polymeric items of complicated geometry.

Pharmaceutical Approach to Develop Novel Photosensitizer Nanoformulation: An Example of Design and Characterization Rationale of Chlorophyll α Derivative.

In this study, we described physico-chemical properties of novel nanoformulation of photosensitizer-pyropheophorbide α 17-diethylene glycol ester (XL) (chlorophyll α derivative), revealing insights into antitumor activity and maintaining quality, meeting the pharmaceutical approach of new nanoformulation design. Our formulation, based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles, increased XL solubility and selective tumor-targeted accumulation. In our research, we revealed, for the first time, that XL binding to polyvinyl alcohol (PVA) enhances XL photophysical activity, providing the rationale for PVA application as a stabilizer for nanoformulations. Results of FTIR, DSC, and XRD revealed the physical interactions between XL and excipients, including PVA, indicating that the encapsulation maintained XL binding to PVA. The encapsulated XL exhibited higher photophysical activity compared to non-encapsulated substance, which can be attributed to the influence of residual PVA. Gamma-irradiation led to degradation of XL; however, successful sterilization of the samples was achieved through the filtration. Importantly, the encapsulated and sterilized XL retained cytotoxicity against both 2D and 3D tumor cell models, demonstrating the potential of the formulated NP-XL for photodynamic therapy applications, but lacked the ability to reactivate epigenetically silenced genes. These findings provide valuable insights into the design and characterization of PLGA-based nanoparticles for the encapsulation of photosensitizers.

Electrospinning of biomimetic materials with fibrinogen for effective early-stage wound healing.

Electrospun biomimetic materials based on polyester of natural origin poly-3-hudroxybutyrate (PHB) modified with hemin (Hmi) and fibrinogen (Fbg) represent a great interest and are potentially applicable in various fields. Here, we describe formulation of the new fibrous PHB-Fbg and PHB-Hmi-Fbg materials with complex structure for biomedical application. The average diameter of the fibers was 3.5 µm and 1.8 µm respectively. Hmi presence increased porosity from 80 % to 94 %, significantly reduced the number of defects, ensured the formation of a larger number of open pores, and improved mechanical properties. Hmi presence significantly improved the molding properties of the material. Hmi facilitated effective Fbg adsorption on the of the PHB wound-healing material, ensuring uniform localization of the protein on the surface of the fibers. Next, we evaluated cytocompatibility, cell behavior, and open wound healing in mice. The results demonstrated that PHB-Fbg and PHB-Hmi-Fbg electrospun materials had pronounced properties and may be promising for early-stage wound healing - the PHB-Hmi-Fbg sample accelerated wound closure by 35 % on the 3rd day, and PHB-Hmi showed 45 % more effective wound closure on the 15th day.

Synthesis and activation of pH-sensitive metal-organic framework Sr(BDC)∞ for oral drug delivery.

Metal-organic frameworks (MOFs) are widely used in the biomedical industry. In this study, we developed a new method for obtaining a metal-organic structure of strontium and terephthalic acid, Sr(BDC), and an alternative activation method for removing DMF from the pores. Sr(BDC) MOFs were successfully prepared and characterized by XRD, FTIR, TGA, and SEM. The importance of the activation steps was confirmed by TGA, which showed that the Sr(BDC)(DMF) sample can contain up to a quarter of the solvent (DMF) before activation. In our study, IR spectroscopy confirmed the possibility of removing DMF by ethanol treatment from the Sr-BDC crystals. A comparative analysis of the effect of the activation method on the specific surface and pore size of Sr-BDC and its sorption properties using the model drug doxorubicin showed that due to the undeveloped surface of the Sr-(BDC)(DMF) sample, it is not possible to obtain an adsorption isotherm and determine the pore size distribution, thus showing the importance of the activation step. Cytotoxicity and apoptosis assays were carried out to study the biological activity of MOFs, and we observed relatively low toxicity in the tested concentration range after 48 h, with over 92% cell survival for Sr(BDC)(DMF) and Sr(BDC)(260 °C), with a decrease only in the highest concentration (800 mg L-1). Similar results were observed in our apoptosis assays, as they revealed low apoptotic population generation of 2.52%, 3.23%, and 2.77% for Sr(BDC)(DMF), Sr(BDC) and Sr(BDC)(260 °C), respectively. Overall, the findings indicate that ethanol-activated Sr(BDC) shows potential as a safe and effective material for drug delivery.

The Relationship between Histological Composition and Metabolic Profile in Breast Tumors and Peritumoral Tissue Determined with 1H HR-MAS NMR Spectroscopy.

Breast tumors constitute the complex entities composed of cancer cells and stromal components. The compositional heterogeneity should be taken into account in bulk tissue metabolomics studies. The aim of this work was to find the relation between the histological content and 1H HR-MAS (high-resolution magic angle spinning nuclear magnetic resonance) metabolic profiles of the tissue samples excised from the breast tumors and the peritumoral areas in 39 patients diagnosed with invasive breast carcinoma. The total number of the histologically verified specimens was 140. The classification accuracy of the OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) model differentiating the cancerous from non-involved samples was 87% (sensitivity of 72.2%, specificity of 92.3%). The metabolic contents of the epithelial and stromal compartments were determined from a linear regression analysis of the levels of the evaluated compounds against the cancer cell fraction in 39 samples composed mainly of cancer cells and intratumoral fibrosis. The correlation coefficients between the levels of several metabolites and a tumor purity were found to be dependent on the tumor grade (I vs II/III). The comparison of the levels of the metabolites in the intratumoral fibrosis (obtained from the extrapolation of the regression lines to 0% cancer content) to those levels in the fibrous connective tissue beyond the tumors revealed a profound metabolic reprogramming in the former tissue. The joint analysis of the metabolic profiles of the stromal and epithelial compartments in the breast tumors contributes to the increased understanding of breast cancer biology.

Biocompatibility and Antimicrobial Activity of Electrospun Fibrous Materials Based on PHB and Modified with Hemin.

The effect of the hemin (Hmi) on the structure and properties of nanocomposite electrospun materials based on poly-3-hydroxybutyrate (PHB) is discussed in the article. The additive significantly affected the morphology of fibers allowed to produce more elastic material and provided high antimicrobial activity. The article considers also the impact of the hemin on the biocompatibility of the nonwoven material based on PHB and the prospects for wound healing.

Box-Behnken assisted development and validation of high-performance liquid chromatography method for the simultaneous determination of doxorubicin and vorinostat in polymeric nanoparticles.

While histone deacetylase inhibitors, such as vorinostat, demonstrate a significant effect against hematological cancers, their application for solid tumor treatment is limited. However, there is strong evidence that combinatorial administration of vorinostat and genotoxic agents (e.g., doxorubicin) enhances the antitumoral action of both drugs against tumors. We developed a high-performance liquid chromatography method for the simultaneous determination of doxorubicin and vorinostat in polymeric nanoparticles designed to provide the parenteral administration of both drugs and increase their safety profile. We performed separation on Nucleodur C-18 Gravity column with a mixture of 10 mM potassium dihydrogen phosphate buffer pH 3.9:ACN (90:10 v/v) as mobile phase at 240 nm. The method was linear within the concentration range of 4.2-52.0 µg/ml for both drugs with limits of detection and quantification of 3.5 and 10.7 µg/ml for doxorubicin and 2.5 and 7.7 µg/ml for vorinostat, respectively. The method was precise and accurate over the concentration range of analysis. Drug loading was 5.4% for doxorubicin and 0.8% for vorinostat. Degradation of doxorubicin after irradiation was less than 5%, while the amount of vorinostat decreased at 88% under the same conditions. Thus, the validated method could be adopted for routine simultaneous analysis of doxorubicin and vorinostat in polymeric nanoparticles.

Thymol-Modified Oleic and Linoleic Acids Encapsulated in Polymeric Nanoparticles: Enhanced Bioactivity, Stability, and Biomedical Potential.

Unsaturated fatty acids, such as oleic acid (OA) and linoleic acid (LA), are promising antimicrobial and cytostatic agents. We modified OA and LA with thymol (TOA and TLA, respectively) to expand their bioavailability, stability, and possible applications, and encapsulated these derivatives in polymeric nanoparticles (TOA-NPs and TLA-NPs, respectively). Prior to synthesis, we performed mathematical simulations with PASS and ADMETlab 2.0 to predict the biological activity and pharmacokinetics of TOA and TLA. TOA and TLA were synthesized via esterification in the presence of catalysts. Next, we formulated nanoparticles using the single-emulsion solvent evaporation technique. We applied dynamic light scattering, Uv-vis spectroscopy, release studies under gastrointestinal (pH 1.2-6.8) and blood environment simulation conditions (pH 7.4), and in vitro biological activity testing to characterize the nanoparticles. PASS revealed that TOA and TLA have antimicrobial and anticancer therapeutic potential. ADMETlab 2.0 provided a rationale for TOA and TLA encapsulation. The nanoparticles had an average size of 212-227 nm, with a high encapsulation efficiency (71-93%), and released TOA and TLA in a gradual and prolonged mode. TLA-NPs possessed higher antibacterial activity against B. cereus and S. aureus and pronounced cytotoxic activity against MCF-7, K562, and A549 cell lines compared to TOA-NPs. Our findings expand the biomedical application of fatty acids and provide a basis for further in vivo evaluation of designed derivatives and formulations.

Metabolomic Insight into Implications of Induction Chemotherapy Followed by Concomitant Chemoradiotherapy in Locally Advanced Head and Neck Cancer.

The present study compares two groups of locally advanced patients with head and neck squamous cell carcinoma (LA-HNSCC) undergoing concurrent chemoradiotherapy (cCHRT), specifically those for whom it is a first-line treatment and those who have previously received induction chemotherapy (iCHT). The crucial question is whether iCHT is a serious burden during subsequent treatment for LA-HNSCC and how iCHT affects the tolerance to cCHRT. Of the 107 LA-HNSCC patients, 54 received cisplatin-based iCHT prior to cCHRT. The patients were clinically monitored at weekly intervals from the day before until the completion of the cCHRT. The 843 blood samples were collected and divided into two aliquots: for laboratory blood tests and for nuclear magnetic resonance (NMR) spectroscopy (a Bruker 400 MHz spectrometer). The NMR metabolites and the clinical parameters from the laboratory blood tests were analyzed using orthogonal partial least squares analysis (OPLS) and the Mann-Whitney U test (MWU). After iCHT, the patients begin cCHRT with significantly (MWU p-value < 0.05) elevated blood serum lipids, betaine, glycine, phosphocholine, and reticulocyte count, as well as significantly lowered NMR inflammatory markers, serine, hematocrit, neutrophile, monocyte, red blood cells, hemoglobin, and CRP. During cCHRT, a significant increase in albumin and psychological distress was observed, as well as a significant decrease in platelet, N-acetyl-cysteine, tyrosine, and phenylalanine, in patients who received iCHT. Importantly, all clinical symptoms (except the decreased platelets) and most metabolic alterations (except for betaine, serine, tyrosine, glucose, and phosphocholine) resolve until the completion of cCHRT. In conclusion, iCHT results in hematological toxicity, altered lipids, and one-carbon metabolism, as well as downregulated inflammation, as observed at the beginning and during cCHRT. However, these complications are temporary, and most of them resolve at the end of the treatment. This suggests that iCHT prior to cCHRT does not pose a significant burden and should be considered as a safe treatment option for LA-HNSCC.

Structural Optimization of Platinum Drugs to Improve the Drug-Loading and Antitumor Efficacy of PLGA Nanoparticles.

Currently, molecular dynamics simulation is being widely applied to predict drug-polymer interaction, and to optimize drug delivery systems. Our study describes a combination of in silico and in vitro approaches aimed at improvement in polymer-based nanoparticle design for cancer treatment. We applied the PASS service to predict the biological activity of novel carboplatin derivatives. Subsequent molecular dynamics simulations revealed the dependence between the drug-polymer binding energy along with encapsulation efficacy, drug release profile, and the derivatives' chemical structure. We applied ICP-MS analysis, the MTT test, and hemolytic activity assay to evaluate drug loading, antitumor activity, and hemocompatibility of the formulated nanoparticles. The drug encapsulation efficacy varied from 0.2% to 1% and correlated with in silico modelling results. The PLGA nanoparticles revealed higher antitumor activity against A549 human non-small-cell lung carcinoma cells compared to non-encapsulated carboplatin derivatives with IC50 values of 1.40-23.20 µM and 7.32-79.30 µM, respectively; the similar cytotoxicity profiles were observed against H69 and MCF-7 cells. The nanoparticles efficiently induced apoptosis in A549 cells. Thus, nanoparticles loaded with novel carboplatin derivatives demonstrated high application potential for anticancer therapy due to their efficacy and high hemocompatibility. Our results demonstrated the combination of in silico and in vitro methods applicability for the optimization of encapsulation and antitumor efficacy in novel drug delivery systems design.

Efficiency of 124I radioisotope production from natural and enriched tellurium dioxide using 124Te(p,xn)124I reaction.

BACKGROUND: 124I Iodine (T[Formula: see text] = 4.18 d) is the only long-life positron emitter radioisotope of iodine that may be used for both imaging and therapy as well as for 131I dosimetry. Its physical characteristics permits taking advantages of the higher Positron Emission Tomography (PET) image quality, whereas the availability of new molecules to be targeted with 124I makes it a novel innovative radiotracer probe for a specific molecular targeting. RESULTS: In this study Monte Carlo and SRIM/TRIM modelling was applied to predict the nuclear parameters of the 124I production process in a small medical cyclotron IBA 18/9 Cyclone. The simulation production yields for 124I and the polluting radioisotopes were  calculated for the natural and enriched 124TeO2  +  Al2O3  solid targets irradiated with 14.8 MeV protons. The proton beam was degraded energetically from 18 MeV with 0.2 mm Havar foil. The 124Te(p,xn)124I reactions were taken into account in the simulations. The optimal thickness of the target material was calculated using the SRIM/TRIM and Geant4 codes. The results of the simulations were compared with the experimental data obtained for the natural TeO2 +Al2O3 target. The dry distillation technique of the 124-iodine was applied. CONCLUSIONS: The experimental efficiency for the natural Te target was better than 41% with an average thick target (>0.8 mm) yield of 1.32 MBq/µAh. Joining the Monte Carlo and experimental approaches makes it possible to optimize the methodology for the 124I production from the expensive Te enriched targets.

Synergetic Enhancement of Tumor Double-Targeted MRI Nano-Probe.

The conventional targeted delivery of chemotherapeutic and diagnostic agents utilizing nanocarriers is a promising approach for cancer theranostics. Unfortunately, this approach often faces hindered tumor access that decreases the therapeutic index and limits the further clinical translation of a developing drug. Here, we demonstrated a strategy of simultaneously double-targeting the drug to two distinct cites of tumor tissue: the tumor endothelium and cell surface receptors. We used fourth-generation polyamideamine dendrimers modified with a chelated Gd and functionalized with selectin ligand and alpha-fetoprotein receptor-binding peptide. According to the proposed strategy, IELLQAR peptide promotes the conjugate recruitment to the tumor inflammatory microenvironment and enhances extravasation through the interaction of nanodevice with P- and E-selectins expressed by endothelial cells. The second target moiety-alpha-fetoprotein receptor-binding peptide-enhances drug internalization into cancer cells and the intratumoral retention of the conjugate. The final conjugate contained 18 chelated Gd ions per dendrimer, characterized with a 32 nm size and a negative surface charge of around 18 mV. In vitro contrasting properties were comparable with commercially available Gd-chelate: r1 relaxivity was 3.39 for Magnevist and 3.11 for conjugate; r2 relaxivity was 5.12 for Magnevist and 4.81 for conjugate. By utilizing this dual targeting strategy, we demonstrated the increment of intratumoral accumulation, and a remarkable enhancement of antitumor effect, resulting in high-level synergy compared to monotargeted conjugates. In summary, the proposed strategy utilizing tumor tissue double-targeting may contribute to an enhancement in drug and diagnostic accumulation in aggressive tumors.

Oxidative Damage Induced by Phototoxic Pheophorbide a 17-Diethylene Glycol Ester Encapsulated in PLGA Nanoparticles.

Pheophorbide a 17-diethylene glycol ester (XL-8), is a promising high-active derivative of known photosensitizer chlorin e6 used in photodynamic therapy. However, high lipophilicity and poor tumor accumulation limit XL-8 therapeutic application. We developed a novel XL-8 loaded with poly(D,L-lactide-co-glycolide) nanoparticles using the single emulsion-solvent evaporation method. The nanoparticles possessed high XL-8 loading content (4.6%) and encapsulation efficiency (87.7%) and a small size (182 ± 19 nm), and negative surface charge (-22.2 ± 3.8 mV) contributed to a specific intracellular accumulation. Sustained biphasic XL-8 release from nanoparticles enhanced the photosensitizer photostability upon irradiation that could potentially reduce the quantity of the drug applied. Additionally, the encapsulation of XL-8 in the polymer matrix preserved phototoxic activity of the payload. The nanoparticles displayed enhanced cellular internalization. Flow cytometry and confocal laser-scanning microscopy studies revealed rapid XL-8 loaded nanoparticles distribution throughout the cell and initiation of DNA damage, glutathione depletion, and lipid peroxidation via reactive oxygen species formation. The novel nanoformulated XL-8 simultaneously revealed a significant phototoxicity accompanied with enhanced photostability, in contrast with traditional photosensitizers, and demonstrated a great potential for further in vivo studies.

Optimization, Characterization and Pharmacokinetic Study of Meso-Tetraphenylporphyrin Metal Complex-Loaded PLGA Nanoparticles.

The selection of technological parameters for nanoparticle formulation represents a complicated development phase. Therefore, the statistical analysis based on Box-Behnken methodology is widely used to optimize technological processes, including poly(lactic-co-glycolic acid) nanoparticle formulation. In this study, we applied a two-level three-factor design to optimize the preparation of nanoparticles loaded with cobalt (CoTPP), manganese (MnClTPP), and nickel (NiTPP) metalloporphyrins (MeP). The resulting nanoparticles were examined by dynamic light scattering, X-ray diffraction, Fourier transform infrared spectroscopy, MTT test, and hemolytic activity assay. The optimized model of nanoparticle formulation was validated, and the obtained nanoparticles possessed a spherical shape and physicochemical characteristics enabling them to deliver MeP in cancer cells. In vitro hemolysis assay revealed high safety of the formulated MeP-loaded nanoparticles. The MeP release demonstrated a biphasic profile and release mechanism via Fick diffusion, according to release exponent values. Formulated MeP-loaded nanoparticles revealed significant antitumor activity and ability to generate reactive oxygen species. MnClTPP- and CoTPP-nanoparticles specifically accumulated in tissues, preventing wide tissue distribution caused by long-term circulation of the hydrophobic drug. Our results suggest that MnClTPP- and CoTPP-nanoparticles represent the greatest potential for utilization in in anticancer therapy due to their effectiveness and safety.

Non-parametric MRI Brain Atlas for the Polish Population.

Introduction: The application of magnetic resonance imaging (MRI) to acquire detailed descriptions of the brain morphology in vivo is a driving force in brain mapping research. Most atlases are based on parametric statistics, however, the empirical results indicate that the population brain tissue distributions do not exhibit exactly a Gaussian shape. Our aim was to verify the population voxel-wise distribution of three main tissue classes: gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF), and to construct the brain templates for the Polish (Upper Silesian) healthy population with the associated non-parametric tissue probability maps (TPMs) taking into account the sex and age influence. Material and Methods: The voxel-wise distributions of these tissues were analyzed using the Shapiro-Wilk test. The non-parametric atlases were generated from 96 brains of the ethnically homogeneous, neurologically healthy, and radiologically verified group examined in a 3-Tesla MRI system. The standard parametric tissue proportion maps were also calculated for the sake of comparison. The maps were compared using the Wilcoxon signed-rank test and Kolmogorov-Smirnov test. The volumetric results segmented with the parametric and non-parametric templates were also analyzed. Results: The results confirmed that in each brain structure (regardless of the studied sub-population) the data distribution is skewed and apparently not Gaussian. The determined non-parametric and parametric templates were statistically compared, and significant differences were found between the maps obtained using both measures (the maps of GM, WM, and CSF). The impacts of applying the parametric and non-parametric TPMs on the segmentation process were also compared. The GM volumes are significantly greater when using the non-parametric atlas in the segmentation procedure, while the CSF volumes are smaller. Discussion and Conclusion: To determine the population atlases the parametric measures are uncritically and widely used. However, our findings suggest that the mean and parametric measures of such skewed distribution may not be the most appropriate summary statistic to find the best spatial representations of the structures in a standard space. The non-parametric methodology is more relevant and universal than the parametric approach in constructing the MRI brain atlases.

Expression of acid cleavable Asp-Pro linked multimeric AFP peptide in E. coli.

BACKGROUND: Difficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. RESULTS: Multimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. CONCLUSIONS: A recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.

Grading of endometrial cancer using 1H HR-MAS NMR-based metabolomics.

The tissue metabolomic characteristics associated with endometrial cancer (EC) at different grades were studied using high resolution (400 MHz) magic angle spinning (HR-MAS) proton spectroscopy. The metabolic profiles were obtained from 64 patients (14 with grade 1 (G1), 33 with grade 2 (G2) and 17 with grade 3 (G3) tumors) and compared with the profile acquired from 10 patients with the benign disorders. OPLS-DA revealed increased valine, isoleucine, leucine, hypotaurine, serine, lysine, ethanolamine, choline and decreased creatine, creatinine, glutathione, ascorbate, glutamate, phosphoethanolamine and scyllo-inositol in all EC grades in reference to the non-transformed tissue. The increased levels of taurine was additionally detected in the G1 and G2 tumors in comparison to the control tissue, while the elevated glycine, N-acetyl compound and lactate-in the G1 and G3 tumors. The metabolic features typical for the G1 tumors are the increased dimethyl sulfone, phosphocholine, and decreased glycerophosphocholine and glutamine levels, while the decreased myo-inositol level is characteristic for the G2 and G3 tumors. The elevated 3-hydroxybutyrate, alanine and betaine levels were observed in the G3 tumors. The differences between the grade G1 and G3 malignances were mainly related to the perturbations of phosphoethanolamine and phosphocholine biosynthesis, inositol, betaine, serine and glycine metabolism. The statistical significance of the OPLS-DA modeling was also verified by an univariate analysis. HR-MAS NMR based metabolomics provides an useful insight into the metabolic reprogramming in endometrial cancer.

NMR-Based Metabolomics in Investigation of the Radiation Induced Changes in Blood Serum of Head and Neck Cancer Patients and Its Correlation with the Tissue Volumes Exposed to the Particulate Doses.

In the present study, we analyze the nuclear magnetic resonance (NMR) blood serum metabolic profiles of 106 head and neck squamous cell carcinoma (HNSCC) patients during radio (RT) and concurrent radio-chemotherapy (CHRT). Four different fractionation schemes were compared. The blood samples were collected weekly, from the day before the treatment until the last week of CHRT/RT. The NMR spectra were acquired on A Bruker 400 MHz spectrometer at 310 K and analyzed using multivariate methods. Seven metabolites were found significantly to be altered solely by radiotherapy: N-acetyl-glycoprotein (NAG), N-acetylcysteine, glycerol, glycolate and the lipids at 0.9, 1.3 and 3.2 ppm. The NMR results were correlated with the tissue volumes receiving a particular dose of radiation. The influence of the irradiated volume on the metabolic profile is weak and mainly limited to sparse correlations with the inflammatory markers, creatinine and the lymphocyte count in RT and the branched-chain amino-acids in CHRT. This is probably due to the optimal planning and delivery of radiotherapy improving sparing of the surrounding normal tissues and minimizing the differences between the patients (caused by the tumor location and size).