Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC(50) of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC(50) = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.     

Depolymerization of insulin amyloid fibrils by albumin-modified magnetic fluid

Pathogenesis of amyloid-related diseases is associated with the presence of protein amyloid deposits. Insulin amyloids have been reported in a patient with diabetes undergoing treatment by injection of insulin and causes problems in the production and storage of this drug and in pplication of insulin pumps. We have studied the interference of insulin amyloid fibrils with a series of 18 albumin magnetic fluids (MFBSAs) consisting of magnetite nanoparticles modified by different amounts of bovine serum albumin (w/w BSA/Fe?O? from 0.005 up to 15). We have found that MFBSAs are able to destroy amyloid fibrils in vitro. The extent of fibril depolymerization was affected by nanoparticle physical-chemical properties (hydrodynamic diameter, zeta potential and isoelectric point) determined by the BSA amount present in MFBSAs. The most effective were MFBSAs with lower BSA/Fe?O? ratios (from 0.005 to 0.1) characteristic of about 90% depolymerizing activity. For the most active magnetic fluids (ratios 0.01 and 0.02) the DC50 values were determined in the range of low concentrations, indicating their ability to interfere with insulin fibrils at stoichiometric concentrations. We assume that the present findings represent a starting point for the application of the active MFBSAs as therapeutic agents targeting insulin amyloidosis.     

Immunomodulatory efficiency of poly(2-oxazolines)

Poly(2-oxazolines) represent promising polymer materials for biomedical applications. The activation of mouse lymphoid macrophage line P388.D1 (clone 3124) by two selected representatives of poly(2-oxazolines), namely poly(2-ethyl-2-oxazoline) (PETOX100) and poly[2-(4-aminophenyl)-2-oxazoline-co-2-ethyl-2-oxazoline] (AEOX10), was assessed in vitro. The immunomodulatory efficacy of both polymers was evaluated via the induced release of pro-inflammatory cytokines (TNF-α, IL-1α and IL-6) and the acceleration of reactive free radicals. The present study revealed effective structure-immunomodulating associations of AEOX10 and PETOX100, which are desirable in biomedical and pharmaceutical applications of aliphatic and aromatic poly (2-oxazolines) in vivo.     

The influence of zeolite catalysts on the products of rapeseed oil cracking

The article is devoted to the study of rapeseed oil cracking in the presence of zeolite catalysts in order to prepare the liquid fraction with the properties close to fossil diesel.To evaluate the cracking products, the chromatographic methods GL and GC/MS were used, together with the determination of viscosities, acid values and densities. The cracking of rapeseed oil in the presence of zeolite-type catalysts in batch arrangement at temperatures between 350 and 440 °C gave a yield of liquid condensate 85 to 90 wt.%. After removal of volatile components 3 to 9 wt.% by distillation at the temperatures up to 190 °C, the GL chromatogram of treated condensate was similar to that of fossil diesel fuel. The paper deals with the influence of the kind and amount of catalyst on composition and properties of treated products with the use of GL and GC/MS chromatography and with the tests of blends of fossil diesel fuel with cracking products at contents up to 7 vol.%. The blended fuels meet all parameters specified by standard EN 590 for diesel fuel.     

Dependence of carbon dioxide sorption on the petrographic composition of bituminous coals from the Czech part of the Upper Silesian Basin, Czech Republic

The effect of the rank and of the maceral composition of bituminous coal on carbon dioxide sorption capacity was studied on the basis of samples from two coal mines (Darkov, ?SM) from the Czech part of the Upper Silesian Basin. The samples from the two mines cover a small but very significant section of coalification within the transition zone between high-volatile bituminous A coal and medium-volatile bituminous coal, where porosity and sorption properties pass through their minima. The coal porous system was characterized by the micropore volume evaluated using the sorption isotherm of carbon dioxide and the volumes of meso-, macro- and coarse pores were determined by high-pressure mercury porosimetry. The micropore fraction in the coal porous system ranged between 53% and 75%. It was particularly high in coals with high vitrinite content, namely collotelinite, and also in coals with high inertinite content. The carbon dioxide sorption capacity was determined from the carbon dioxide sorption isotherms measured using a gravimetric sorption analyzer at 298 K until a relative pressure of 0.015 p/p_s, and was interpreted by characteristic parameters of the Dubinin and Langmuir equations. It was found that the adsorbed amount of CO₂ in the ?SM coal increases with the content of vitrinite and collotelinite, whereas no increase or only a slight increase was observed for the Darkov coal. The tendency of adsorption capacity to depend on maceral composition, and also to some extent on coalification, observed for the ?SM coal, may be related to higher microporosity due to the coalification process or oxidative processes leading to the formation of pseudovitrinite.     

The effect of selected hydrocolloids on the rheological properties of processed cheese analogues made with vegetable fats during the cooling phase

The aim of this work was to observe the influence of different hydrocolloids on changes in the rheological properties of processed cheese and their analogues within the process of cooling depending on the type of the fat used (butter, coconut fat and palm oil). κ‐carrageenan, ι‐carrageenan, λ‐carrageenan, arabic gum and locust bean gum were used at a concentration of 1.0% w/w. With the decreasing temperature during the cooling period, an increase in the complex modulus (G*) was observed in all samples tested. Within the cooling period, the sample with the addition of κ‐carrageenan showed both the most significant increase in G* in comparison with the control and the highest values of hardness after a 7‐day storage period regardless of the type of the fat used. The samples with coconut fat were assessed as the hardest. On the other hand, the samples with palm oil showed the lowest hardness (with the same hydrocolloid used). In the gelling hydrocolloids (κ‐ and ι‐carrageenan), only small changes in temperature of coil‐to‐helix transition were observed (in range of 2–7 °C) as a result of the addition of different types of fat.     

Artificial Dim Light at Night during Pregnancy Can Affect Hormonal and Metabolic Rhythms in Rat Offspring

Artificial light at night (ALAN) is considered an environmental risk factor that can interfere with the circadian control of the endocrine system and metabolism. We studied the impact of ALAN during pregnancy on the hormonal and biochemical parameters in rat pups at postnatal (P) days P3, P10, and P20. Control dams (CTRL) were kept in a standard light-dark regime, and ALAN dams were exposed to dim ALAN (<2 lx) during the whole pregnancy. A plasma melatonin rhythm was found in all CTRL groups, whereas in ALAN pups, melatonin was not rhythmic at P3, and its amplitude was lowered at P10; no differences were found between groups at P20. Plasma corticosterone was rhythmic at P20 in both groups, with decreased mesor in ALAN pups. Plasma thyroid hormones exhibited an inconsistent developmental pattern, and vasopressin levels were suppressed at the beginning of the dark phase at P20 in ALAN compared to CTRL. Glucose and cholesterol showed significant daily rhythms in CTRL but not in ALAN offspring at P3. Exposure to ALAN during pregnancy disturbed the development of daily rhythms in measured hormones and metabolites, suggesting that ALAN during pregnancy can act as an endocrine disruptor that can interfere with the normal development of the progeny.     

Effects of Sulforaphane-Induced Cell Death upon Repeated Passage of Either P-Glycoprotein-Negative or P-Glycoprotein-Positive L1210 Cell Variants

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.     

Biomarkers of Bladder Cancer: Cell-Free DNA,Epigenetic Modifications and Non-Coding RNAs

Bladder cancer (BC) is the 10th most frequent cancer in the world. The initial diagnosis and surveillance of BC require a combination of invasive and non-invasive methods, which are costly and suffer from several limitations. Cystoscopy with urine cytology and histological examination presents the standard diagnostic approach. Various biomarkers (e.g., proteins, genes, and RNAs) have been extensively studied in relation to BC. However, the new trend of liquid biopsy slowly proves to be almost equally effective. Cell-free DNA, non-coding RNA, and other subcellular structures are now being tested for the best predictive and diagnostic value. In this review, we focused on published gene mutations, especially in DNA fragments, but also epigenetic modifications, and non-coding RNA (ncRNA) molecules acquired by liquid biopsy. We performed an online search in PubMed/Medline, Scopus, and Web of Science databases using the terms “bladder cancer”, in combination with “markers” or “biomarkers” published until August 2022. If applicable, we set the sensitivity and specificity threshold to 80%. In the era of precision medicine, the development of complex laboratory techniques fuels the search and development of more sensitive and specific biomarkers for diagnosis, follow-up, and screening of BC. Future efforts will be focused on the validation of their sensitivity, specificity, predictive value, and their utility in everyday clinical practice.     

Synthetic and Continuous Jets Impinging on a Circular Cylinder

Synthetic and continuous water jets impinging onto an electrically heated circular cylinder were experimentally investigated. The slot nozzle width was 0.36 mm, the cylinder diameter was 1.2 mm, and the cylinder-to-nozzle spacing related to the slot width was 5–21. Two optical methods were used: qualitative laser-induced fluorescence (LIF) visualization and laser Doppler vibrometry (LDV) measurements. Simultaneously with the optical experiments, the overall convective heat transfer from the circular cylinder was evaluated. The LDV quantified the velocity of the oscillating piezo-driven diaphragm at frequencies from 30 to 68 Hz. A majority of the study was performed at the near-resonant frequencies from 46 to 49 Hz. For all investigated jets, the Reynolds numbers based on the nozzle width ranged from 36 to 171. The LIF visualization revealed a dominant flow separation occurring on the windward cylinder side. This result is attributed to the effect of the miniscales, a relatively small ratio of the nozzle width to the cylinder diameter, and low Reynolds numbers. An increase in the Reynolds number changes the flow pattern from a steady jet-flow separation to a vortex shedding wake-flow regime. The heat transfer experiments were validated in a natural convection regime. An enhancement of the average Nusselt numbers by 4.2–6.2 times by means of the synthetic jets was quantified by comparison with the natural convection regime. A correlation for the average Nusselt number was proposed for both the continuous and synthetic jets.