Defence response of Galleria mellonella larvae to oral and intrahemocelic infection with Pseudomonas entomophila

We report differences in the course of infection of G. mellonella larvae with P. entomophila via intrahemocelic and oral routes. Survival curves, larval morphology, histology, and induction of defence response were investigated. Larvae injected with 10 and 50 cells of P. entomophila activated a dose-dependent immune response, which was manifested by induction of immune-related genes and dose-dependent defence activity in larval hemolymph. In contrast, after the oral application of the pathogen, antimicrobial activity was detected in whole hemolymph of larvae infected with the $10^{3}$ but not $10^{5}$ dose in spite of the induction of immune response manifested as immune-relevant gene expression and defence activity of electrophoretically separated low-molecular hemolymph components. Among known proteins induced after the P. entomophila infection, we identified proline-rich peptide 1 and 2, cecropin D-like peptide, galiomycin, lysozyme, anionic peptide 1, defensin-like peptide, and a 27 kDa hemolymph protein. The expression of the lysozyme gene and the amount of protein in the hemolymph were correlated with inactivity of hemolymph in insects orally infected with a higher dose of P. entomophila, pointing to its role in the host-pathogen interaction

Isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from immunized hemolymph of red palm weevil Rhynchophorus ferrugineus

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation

Right ventricular morphology and function is not related with microRNAs and fibrosis markers in dilated cardiomyopathy

Background: The relationship between right ventricle (RV), extracellular matrix (ECM) fibrosis and fibrosis-linked, circulating microRNAs in dilated cardiomyopathy (DCM) is unknown. Aim: The aim of the study was to uncover the associations between serum markers of ECM metabolism and circulating microRNAs with RV morphological and functional parameters. Methods: The study population consisted of 70 consecutive DCM patients (ejection fraction 24.4 ± ± 7.4%). Based on detailed echocardiographic assessment — 15 patients had normal RV, whereas 55 patients had RV dilatation (RVD) and/or RV systolic dysfunction (RVSD). Procollagens type I and III carboxy- and amino-terminal peptides, osteopontin (OPN), TGF1-b1, connective tissue growth factor (CTGF), MMP-2, MMP-9 and TIMP-1 were measured in serum as well as expression of miR-21, miR-26, miR-29, miR-30 and miR-133a. All patients underwent endomyocardial biopsy. Results: Biopsy-proven fibrosis was evenly distributed in two groups. Serum levels of fibrosis markers did not differ between groups. OPN, CTGF, MMP-2, and TIMP-1 correlated with RV parameters. Only miR-133 a was differently expressed in both groups. MiR-21, miR-26, miR-30, and miR-133a cor­related with RV morphological but without functional parameters. Not a single marker of fibrosis was independently associated with RV. MiR-30 was associated with RV impairment in the logistic regression model adjusted for age, sex, body mass index, and disease duration; however, lost its significance in the more comprehensive model. Conclusions: Right ventricle structural and functional abnormalities are common in DCM. ECM fibrosis and serum markers are not associated with RV impairment. The prognostic value of studied microRNAs on RV is limited in DCM

The relationship between myocardial fibrosis and myocardial microRNAs in dilated cardiomyopathy : a link between mir-133a and cardiovascular events

It is unknown whether fibrosis‐associated microRNAs: miR‐21, miR‐26, miR‐29, miR‐30 and miR‐133a are linked to cardiovascular (CV) outcome. The study evaluated the levels of extracellular matrix (ECM) fibrosis and the prevalence of particular microRNAs in patients with dilated cardiomyopathy (DCM) to investigate any correlation with CV events. Methods: Seventy DCM patients (48 ± 12 years, EF 24.4 ± 7.4%) underwent right ventricular biopsy. The control group was comprised of 7 patients with CAD who underwent CABG and intraoperative biopsy. MicroRNAs were measured in blood and myocardial tissue via qPCR. The end‐point was a combination of CV death and urgent HF hospitalization at the end of 12 months. There were differential levels of circulating and myocardial miR‐26 and miR‐29 as well as myocardial miR‐133a when the DCM and CABG groups were compared. Corresponding circulating and myocardial microRNAs did not correlate with one another. There was no correlation between microRNA and ECM fibrosis. By the end of the 12‐month period of the study, CV death had occurred in 6 patients, and a further 19 patients required urgent HF hospitalization. None of the circulating microRNAs was a predictor of the combined end‐point; however, myocardial miR‐133a was an independent predictor in unadjusted models (HR 1.53; 95% CI 1.14‐2.05; P < .004) and adjusted models (HR 1.57; 95% CI 1.14‐2.17; P < .005). The best cut‐off value for the miR‐133a level for the prediction of the combined end‐point was 0.74 ΔCq, with an AUC of 0.67. The absence of a correlation between the corresponding circulating and myocardial microRNAs calls into question their cellular source. This study sheds new light on the role of microRNAs in ECM fibrosis in DCM, which warrants further exploration

Development of an experimental model for analysis TNF production by human monocytic cell lines stimulated with the peptide BacSp222 from Staphylococcus pseudintermedius

Głównym celem pracy był wybór modelu eksperymentalnego pozwalającego na zbadanie wpływu bakteriocyny BacSp222 na produkcję TNF przez ludzkie linie monocytarne. W pierwszym etapie zoptymalizowano kanapkowy test ELISA do detekcji ludzkiego TNF. Następnie zbadano wpływ LPS na produkcję TNF przez wybrane, ludzkie linie monocytarno-makrofagowe. Ostatecznie, wybrano linię komórkową THP-1, która po różnicowaniu przez PMA i po stymulacji LPS wykazywała ekspresję TNF. W ostatnim etapie badań komórki stymulowano bakteriocyną BacSp222 i zauważono, że niezróżnicowane komórki THP-1 produkowały więcej TNF, niż komórki zróżnicowane za pomocą PMA. Co więcej, bakteriocyna BacSp222 obniżała aktywności metaboliczną zróżnicowanych komórek.The main aim of the work was designed a model for analysis TNF expression after stimulation human monocytes cell lines with bacteriocin BacSp222. In the first stage of research the sandwich ELISA test for detection of cytokine concentrations was optimized. Then studied LPS influence on TNF production by chosen human monocyte-macrophage cell lines. Finally, THP-1 cell line, which after differentiation by PMA and stimulation LPS expressed TNF, was chosen. In the last stage of the work cells stimulated BacSp222 and noted that undifferentiated cells produced more TNF than differentiated by PMA cells. Furthermore, BacSp222 reduced the metabolic activity in differentiated cells

Analysis of the cytotoxicity of micelles of sodium alginate and curcumin and 2-hydroxyoleic acid embedded liposomes

Choroby nowotworowe są jedną z najczęstszych przyczyn zgonów na świecie. Naukowcy od lat próbują znaleźć związki, które pozwolą poprawić skuteczność terapii przeciwnowotworowych. Potencjalnych terapeutyków poszukuje się w nowo projektowanych związkach jak i w substancjach występujących naturalnie w przyrodzie. Niektóre związki ze względu na swój hydrofobowy charakter czy też słabą biodostępność, wymagają czy to modyfikacji chemicznych czy też specjalnej formulacji, celem poprawy ich skuteczności działania. W ostatnich latach nanotechnologia umożliwiła opracowanie wielu terapii opartych na związkach, których wcześniej nie można było podać pacjentowi ze względu na negatywne skutki uboczne, słabą rozpuszczalność lub niską skuteczność in vivo. Celem wykonanych doświadczeń była analiza cytotoksyczności miceli alginianu sodu i kurkuminy (AA-Cur) oraz liposomów z wbudowanym kwasem 2-hydroksyoleinowym (2-OHOA). W tym celu zbadano żywotność komórek mysich linii nowotworowych: B16F10, MC38-CEA i 4T1 poddanych działaniu badanych nanomateriałów. Następnie sprawdzono wpływ miceli i liposomów na indukcję apoptozy oraz progresję cyklu komórkowego. Dla liposomów sprawdzono również aktywność hemolityczną względem ludzkich erytrocytów. Dla każdego z badanych nanomateriałów obserwowana była dawkozależna cytotoksyczność względem mysich komórek nowotworowych. Jednak, zarówno dla miceli AA-Cur jak i liposomów z wbudowanym 2-OHOA nie wykazano znaczącego wpływu na progresję cyklu komórkowego. W badanych komórkach hodowanych z AA-Cur nie była wykrywana fragmentacja DNA genomowego. Liposomy z wbudowanym 2-OHOA powodowały rozpad ludzkich erytrocytów, w związku z czym podanie ich do organizmu jest wykluczone. Badane nanomateriały nie wykazują potencjału do stosowania ich jako samodzielnych chemioterapeutyków.Cancers are one of the most frequent causes of deaths in the world. For years, scientists have been looking for compounds, which could help improve anti-cancers therapies. Potential therapeutics are searched among newly designed compounds as well as substances naturally occurring in nature. Some compounds, due to their hydrophobic character or poor bioavailability, require either chemical modification or a special formulation to improve their effectiveness. In recent years, nanotechnology has made it possible to develop many compound-based therapies that were previously unavailable to the patient due to negative side effects, poor solubility or low in vivo efficacy.The purpose of presented experiments was to analyse the cytotoxicity of micelles of sodium alginate and curcumin (AA-Cur) and 2-hydroxyoleic acid (2-OHOA) embedded liposomes. For this purpose, viability of murine tumour cell lines: B16F10, MC38-CEA and 4T1 treated with the studied nanomaterials was tested. Next, the effect of micelles and liposomes on the induction of apoptosis and progression of the cell cycle were checked. Haemolytic activity on human erythrocytes was also checked for liposomes.For each of the studied nanomaterials, dose-dependent cytotoxicity was observed against murine tumour cells. However, for both AA-Cur micelles and 2-OHOA embedded liposomes no significant influence on the cell cycle progression has been demonstrated. In the examined cells cultured with AA-Cur there were no signs of genomic DNA fragmentation. Liposomes with embedded 2-OHOA caused haemolysis of human erythrocytes, therefore their application to the body is excluded. The studied nanomaterials do not have the potential to be used as independent chemotherapeutics