Human serum albumin as a potential drug delivery system for N-methylated hot spot insulin analogs inhibiting hormone aggregation.

The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind N-methylated analogs of hot spots of native insulin. Three N-methylated derivatives of the A13-A19 fragment of native insulin were used: L(N-Me)YQLENY (1), LYQ(N-Me)LENY (2), and L(N-Me)YQ(N-Me)LENY (3). The studied N-methylated insulin fragments possess inhibiting potential against hormone aggregation. A variety of research techniques, including spectroscopic methods and microscopy assays, were used to study the interaction of HSA with the N-methylated insulin fragments. Based on spectroscopic measurements with Congo Red and Thioflavin T, all the analyzed N-methylated peptides were able to interact with the HSA surface. The CD spectrum registered for HSA in the presence of L(N-Me)YQLENY showed the smallest content of α-helix conformation, indicating the most compact HSA structure. Based on the results of MST, the dissociation constants (Kd) for complexes of HSA and peptides 1-3 were 19.2 nM (complex 1), 15.6 nM (complex 2), and 8.07 nM (complex 3). Microscopy assays, dynamic light scattering measurements as well as computer simulation of protein-ligand interaction also confirmed the possibility of docking the N-methylated inhibitors within HSA.

Selection of collagen IV fragments forming the outer sphere of the native protein: Assessment of biological activity for regenerative medicine.

The aim of this research was to select the fragments that make up the outer layer of the collagen IV (COL4A6) protein and to assess their potential usefulness for regenerative medicine. It was expected that because protein-protein interactions take place via contact between external domains, the set of peptides forming the outer sphere of collagen IV will determine its interaction with other proteins. Cellulose-immobilized protein fragment libraries treated with polyclonal anti-collagen IV antibodies were used to select the peptides forming the outer sphere of collagen IV. In the first test, 33 peptides that strongly interacted with the polyclonal anti-collagen IV antibodies were selected from a library of non-overlapping fragments of collagen IV. The selected fragments of collagen IV (cleaved from the cellulose matrix) were tested for their cytotoxicity, their effects on cell viability and proliferation, and their impact on the formation of reactive oxygen species (ROS). The studies used RAW 264.7 mouse macrophage cells and Hs 680.Tr human fibroblasts. PrestoBlue, ToxiLight™, and ToxiLight 100% Lysis Control assays were conducted. The viability of fibroblasts cultured with the addition of increasing concentrations of the peptide mix did not show statistically significant differences from the control. Fragments 161-170, 221-230, 721-730, 1331-1340, 1521-1530, and 1661-1670 of COL4A6 were examined for cytotoxicity against BJ normal human foreskin fibroblasts. None of the collagen fragments were found to be cytotoxic. Further research is underway on the potential uses of collagen IV fragments in regenerative medicine.

The Comparison of Serum Exosome Protein Profile in Diagnosis of NSCLC Patients.

A thorough study of the exosomal proteomic cargo may enable the identification of proteins that play an important role in cancer development. The aim of this study was to compare the protein profiles of the serum exosomes derived from non-small lung cancer (NSCLC) patients and healthy volunteers (control) using the high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to identify potentially new diagnostic and/or prognostic protein biomarkers. Proteins exclusively identified in NSCLC and control groups were analyzed using several bioinformatic tools and platforms (FunRich, Vesiclepedia, STRING, and TIMER2.0) to find key protein hubs involved in NSCLC progression and the acquisition of metastatic potential. This analysis revealed 150 NSCLC proteins, which are significantly involved in osmoregulation, cell-cell adhesion, cell motility, and differentiation. Among them, 3 proteins: Interleukin-34 (IL-34), HLA class II histocompatibility antigen, DM alpha chain (HLA-DMA), and HLA class II histocompatibility antigen, DO beta chain (HLA-DOB) were shown to be significantly involved in the cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) infiltration processes. Additionally, detected proteins were analyzed according to the presence of lymph node metastasis, showing that differences in frequency of detection of protein FAM166B, killer cell immunoglobulin-like receptor 2DL1, and olfactory receptor 52R1 correlate with the N feature according to the TNM Classification of Malignant Tumors. These results prove their involvement in NSCLC lymph node spread and metastasis. However, this study requires further investigation.

Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine.

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO- as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1-33 and 2.1-2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1ß1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.

Synthesis and properties of the anticodon stem-loop of human mitochondrial tRNAMet containing the disease-related G or m1G nucleosides at position 37.

A single point mutation (A4435G) in the human mitochondrial tRNAMet (hmt-tRNAMet) gene causes severe mitochondrial disorders associated with hypertension, type 2 diabetes and LHON. This mutation leads to the exchange of A37 in the anticodon loop of hmt-tRNAMet for G37 and 1-methylguanosine (m1G37). Here we present the first synthesis and structural/biophysical studies of the anticodon stem and loop of pathogenic hmt-tRNAsMet.

Non-Aggregating Amylin Fragments as an Inhibitors of the Aggregation Process of Susceptible to Aggregation Fragments 18-22, 23-27, and 33-37 of Hormone.

Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1-7, 8-12, 13-17 and 28-32 of amylin would inhibit the aggregation of the amyloidogenic cores 18-22, 23-27, 33-37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1-7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18-22, 23-27, 33-37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1-7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.

Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation.

The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind native human insulin and its A13-A19 and B12-B17 fragments, which are responsible for the aggregation of the whole hormone. To label the hormone and both hot spots, so that their binding positions within the HSA could be identified, 4-(1-pyrenyl)butyric acid was used as a fluorophore. Triazine coupling reagent was used to attach the 4-(1-pyrenyl)butyric acid to the N-terminus of the peptides. When attached to the peptides, the fluorophore showed extended fluorescence lifetimes in the excited state in the presence of HSA, compared to the samples in buffer solution. We also analyzed the interactions of unlabeled native insulin and its hot spots with HSA, using circular dichroism (CD), the microscale thermophoresis technique (MST), and three independent methods recommended for aggregating peptides. The CD spectra indicated increased amounts of the α-helical secondary structure in all analyzed samples after incubation. Moreover, for each of the two unlabeled hot spots, it was possible to determine the dissociation constant in the presence of HSA, as 14.4 µM (A13-A19) and 246 nM (B12-B17). Congo Red, Thioflavin T, and microscopy assays revealed significant differences between typical amyloids formed by the native hormone or its hot-spots and the secondary structures formed by the complexes of HSA with insulin and A13-A19 and B12-B17 fragments. All results show that the tested peptide-probe conjugates and their unlabeled analogues interact with HSA, which inhibits their aggregation.

Conjugates of Chitosan and Calcium Alginate with Oligoproline and Oligohydroxyproline Derivatives for Potential Use in Regenerative Medicine.

New materials that are as similar as possible in terms of structure and biology to the extracellular matrix (external environment) of cells are of great interest for regenerative medicine. Oligoproline and oligohydroxyproline derivatives (peptides 2-5) are potential mimetics of collagen fragments. Peptides 2-5 have been shown to be similar to the model collagen fragment (H-Gly-Hyp-Pro-Ala-Hyp-Pro-OH, 1) in terms of both their spatial structure and biological activity. In this study, peptides 2-5 were covalently bound to nonwovens based on chitosan and calcium alginate. Incorporation of the peptides was confirmed by Fourier transform -infrared (FT-IR) and zeta potential measurements. Biological studies (cell metabolic activity by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and Live/Dead assay) proved that the obtained peptide-polysaccharide conjugates were not toxic to the endothelial cell line EA.hy 926. In many cases, the conjugates had a highly affirmative influence on cell proliferation. The results of this study show that conjugates of chitosan and calcium alginate with oligoproline and oligohydroxyproline derivatives have potential for use in regenerative medicine.

Conjugates of Copper Alginate with Arginine-Glycine-Aspartic Acid (RGD) for Potential Use in Regenerative Medicine.

Current restrictions on the use of antibiotics, associated with increases in bacterial resistance, require new solutions, including materials with antibacterial properties. In this study, copper alginate fibers obtained using the classic wet method were used to make nonwovens which were modified with arginine-glycine-aspartic acid (RGD) derivatives. Stable polysaccharide-peptide conjugates formed by coupling with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-), and materials with physically embedded RGD derivatives, were obtained. The materials were found to be characterized by very high antibacterial activity against S. aureus and K. pneumoniae. Cytotoxicity studies confirmed that the materials are not cytotoxic. Copper alginate conjugates with RGD peptides have strong potential for use in regenerative medicine, due to their biocompatibility and innate antibacterial activity.

Insulin Hot-Spot Analogs Formed with N-Methylated Amino Acid Residues Inhibit Aggregation of Native Hormone.

In this study, N-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin "amyloidogenic" analogs containing N-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3-7). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation.

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues.

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C-terminal, N-terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H-PheCysTrp-OH, H-PheCysTyr-OH, H-PheTyrMet-OH, and H-TrpTyr-OH.

Search for New Aggregable Fragments of Human Insulin.

In this study, three independent methods were used to identify short fragment of both chains of human insulin which are prone for aggregation. In addition, circular dichroism (CD) research was conducted to understand the progress of aggregation over time. The insulin fragments (deca- and pepta-peptides) were obtained by solid-phase synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) as a coupling reagent. Systematic studies allowed identification of the new fragments, expected to be engaged in triggering aggregation of the entire structure of human insulin under physiological conditions. It was found that the aggregation process occurs through various structural conformers and may favor the formation of a fibrous structure of aggregate.

Study on the Materials Formed by Self-Assembling Hydrophobic, Aromatic Peptides Dedicated to Be Used for Regenerative Medicine.

The aims of this study were to identify the short aromatic peptides which are able to form highly ordered amyloid-like structures in self-assembling processes, to test the influence of length of hydrophobic peptides on tendency to aggregation, and to check if aggregated peptides fulfill requirements expected for materials useful for scaffolding. All tested hydrophobic peptides were prepared on solid phase by using DMT/NMM/TsO- as a coupling reagent. The progress of aggregation was studied by set of independent tests. All aggregated peptides were found stable under in vitro conditions. All fibrous material formed by self-assembling of peptides does not show any cytotoxic effects on L929 fibroblast cells. Peptides containing tyrosine and tryptophan residues even effectively accelerated the proliferation and stimulated the activity of L929 fibroblasts.

Search for Fibrous Aggregates Potentially Useful in Regenerative Medicine Formed under Physiological Conditions by Self-Assembling Short Peptides Containing Two Identical Aromatic Amino Acid Residues.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H-TyrMetTyr-OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

Mechanical thrombectomy in acute stroke - Five years of experience in Poland.

OBJECTIVES: Mechanical thrombectomy (MT) is not reimbursed by the Polish public health system. We present a description of 5 years of experience with MT in acute stroke in Comprehensive Stroke Centers (CSCs) in Poland. METHODS AND RESULTS: We retrospectively analyzed the results of a structured questionnaire from 23 out of 25 identified CSCs and 22 data sets that include 61 clinical, radiological and outcome measures. RESULTS: Most of the CSCs (74%) were founded at University Hospitals and most (65.2%) work round the clock. In 78.3% of them, the working teams are composed of neurologists and neuro-radiologists. All CSCs perform CT and angio-CT before MT. In total 586 patients were subjected to MT and data from 531 of them were analyzed. Mean time laps from stroke onset to groin puncture was 250±99min. 90.3% of the studied patients had MT within 6h from stroke onset; 59.3% of them were treated with IV rt-PA prior to MT; 15.1% had IA rt-PA during MT and 4.7% - emergent stenting of a large vessel. M1 of MCA was occluded in 47.8% of cases. The Solitaire device was used in 53% of cases. Successful recanalization (TICI2b-TICI3) was achieved in 64.6% of cases and 53.4% of patients did not experience hemorrhagic transformation. Clinical improvement on discharge was noticed in 53.7% of cases, futile recanalization - in 30.7%, mRS of 0-2 - in 31.4% and mRS of 6 in 22% of cases. CONCLUSION: Our results can help harmonize standards for MT in Poland according to international guidelines.

Plasma endotoxin activity rises during ischemic stroke and is associated with worse short-term outcome.

BACKGROUND: Activation of Toll-like receptor 4 (TLR4) contributes to brain injury and poor outcome after cerebral ischemia. The expression of this receptor on monocytes is increased in patients with acute ischemic stroke. Endotoxin is an endogenous ligand for TLR4. The aim of our study was to determine if plasma endotoxin activity is increased in stroke patients and correlates with functional outcome. METHODS: We included 88 patients with ischemic stroke (median age: 71, 56.8% men) and 59 age-matched controls. Plasma endotoxin activity and level of proteins regulating endotoxin interaction with TLR4 (LPS binding protein - LBP and sCD14) were measured in blood samples taken at day 1 (within 24h after stroke symptoms onset), 3 and 6. Short-term functional outcome was assessed at day 14 using modified Rankin Scale. Unfavourable outcome was defined as modified Rankin Scale score>2. RESULTS: Compared to controls, stroke patients had higher plasma endotoxin activity on day 1 (median: 0.39 vs 0.32EU/mL, P=0.03) as well as higher LBP (median: 18.7 vs 11.5µg/mL, P<0.01) and sCD14 level (median: 1330 vs 1070ng/mL, P<0.01). Plasma LPS activity and levels of LBP and sCD14 significantly rose during stroke. Higher LPS activity measured on day 6 was associated with unfavourable outcome (OR: 3.94, 95%CI: 1.03-15.02, P=0.04, adjusted for age and stroke severity). CONCLUSIONS: Plasma endotoxin activity rises during ischemic stroke and is associated with worse short-term outcome.

Left ventricular geometry and white matter lesions in ischemic stroke patients.

Abnormal left ventricular (LV) geometry is associated with extracardiac organ damage in patients with hypertension. The aim of this study was to determine the relationship between LV geometry and white matter lesions (WMLs) in ischemic stroke patients. We retrospectively analyzed data from 155 patients (median age 62; 49.8% male) with mild ischemic stroke (median National Institutes of Health Stroke Scale score 4) who underwent brain magnetic resonance imaging and echocardiography. Patients were categorized into four groups: normal LV geometry, concentric remodeling, eccentric left ventricular hypertrophy (LVH) and concentric LVH. WMLs were graded using the Fazekas scale on fluid-attenuated inversion recovery images. Extensive WMLs were defined as a Fazekas score > 2. Extensive WMLs were more prevalent in patients with concentric LVH, eccentric LVH and concentric remodeling than in those with normal LV geometry. After adjusting for hypertension, age, diabetes mellitus, hypercholesterolemia, glomerular filtration rate and ischemic heart disease, patients with concentric remodeling [odds ratio (OR) 3.94, 95% confidence interval (CI) 1.26-12.31, p = 0.02] and those with concentric LVH (OR 3.69, 95% CI 1.24-10.95, p = 0.02), but not patients with eccentric LVH (OR 2.44, 95% CI 0.72-8.29, p = 0.15), had higher risk of extensive WMLs than patients with normal LV geometry.

The ß-fibrinogen -455G/A gene polymorphism and the risk of ischaemic stroke in a Polish population.

BACKGROUND AND PURPOSE: Ischaemic stroke is considered to be multifactorial and interactions between environmental and genetic factors play an important role. Although vascular risk factors are well known, the genetic ones are still undiscovered. In the present study, we assessed the significance of the ß-fibrinogen -455G/A gene polymorphism and the risk of ischaemic stroke in a Polish population. MATERIAL AND METHODS: 426 ischaemic stroke patients classified according to stroke aetiologies (small vessel disease, large vessel disease or cardioembolic stroke) and 234 controls were included in the study. The association of the ß-fibrinogen genotypes with ischaemic stroke was tested using logistic regression analysis under dominant, recessive or additive models of inheritance. RESULTS: The allele and genotype distributions of the ß-fibrinogen -455G/A gene polymorphism did not differ significantly between patients and controls (patients: G - 75%, GG - 56.6%, GA - 36.8%, AA - 6.6%; controls: G - 73.7%, GG - 57.3%, GA - 32.9%, AA - 9.8%; p > 0.05, χ2). In addition, logistic regression analysis adjusted for the known risk factors, i.e. hypertension, ischaemic heart disease, myocardial infarction, hypercholesterolaemia, diabetes mellitus and smoking, did not show a role of the studied polymorphism in ischaemic stroke. CONCLUSIONS: The ß-fibrinogen -455G/A gene polymorphism is not a risk factor for ischaemic stroke in a Polish population.

The rs2200733 variant on chromosome 4q25 is a risk factor for cardioembolic stroke related to atrial fibrillation in Polish patients.

BACKGROUND AND PURPOSE: A few single nucleotide polymorphisms (SNPs) on chromosome 4q25, associated with atrial fibrillation (AF), are risk factors for ischaemic stroke. We studied the significance of the SNP rs2200733 on chromosome 4q25 in different types of cardioembolic (CE) stroke. MATERIAL AND METHODS: We genotyped 428 controls and 301 CE stroke patients, among whom 197 (65.4%) presented with high risk sources of embolism (CE stroke related to AF) and 104 with medium risk sources (CE stroke unrelated to AF). The SNP rs2200733 was analysed using real-time polymorphism chain reaction. RESULTS: Both univariate and multivariate regression analyses showed that the studied variant affected risk of all CE strokes or CE strokes related to AF in recessive and additive mo-dels. The two types of CE stroke differed significantly in demographics and distribution of vascular risk factors. CONCLUSIONS: The SNP rs2200733 on chromosome 4q25 is a risk factor for CE stroke related to AF only.