Omics Analysis of Blood-Responsive Regulon in Bordetella pertussis Identifies a Novel Essential T3SS Substrate

Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC–MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.     

Carbon nanotubes prepared from three-layered copolymer microspheres of acrylonitrile and methylmethacrylate

Carbon nanotubes (CNTs) were synthesized from fine three-layered copolymer microspheres using the polymer blend technique. Diameter of PMMA core/Poly(AN-co-MMA) shell-1/PMMA shell-2 microspheres, prepared by a radical soap-free emulsion polymerization of methylmethacrylate (MMA) and acrylonitrile (AN), was between 400 nm and 500 nm. Microspheres were subjected to melt-spinning at 305 °C, stabilizing in oxygen at 220 °C for 4 h, and finally carbonizing at 1000 °C for 30 min. FE-SEM study of carbonized sample revealed the presence of CNTs arrays on carbon blocks. Similar arrays were observed in a comparative CNTs sample prepared from three-layered microspheres with the pure PAN shells-1 layers. HRTEM showed that the CNTs derived from copolymer microspheres had different structure when compared to the control sample, i.e. CNTs often adhered to each other and contained the internal compartments. The insufficient PMMA shell-2 coating of copolymer microspheres is believed to be a reason for CNTs adhesion. The possible mechanisms of the carbon block formation and the adhesion of CNTs are introduced.     

Cardiovascular,Metabolic and Inflammatory Changes after Ovariectomy and Estradiol Substitution in Hereditary Hypertriglyceridemic Rats

Background: If menopause is really independent risk factor for cardiovascular disease is still under debate. We studied if ovariectomy in the model of insulin resistance causes cardiovascular changes, to what extent are these changes reversible by estradiol substitution and if they are accompanied by changes in other organs and tissues. Methods: Hereditary hypertriglyceridemic female rats were divided into three groups: ovariectomized at 8th week (n = 6), ovariectomized with 17-β estradiol substitution (n = 6), and the sham group (n = 5). The strain of abdominal aorta measured by ultrasound, expression of vascular genes, weight and content of myocardium and also non-cardiac parameters were analyzed. Results: After ovariectomy, the strain of abdominal aorta, expression of nitric oxide synthase in abdominal aorta, relative weight of myocardium and of the left ventricle and circulating interleukin-6 decreased; these changes were reversed by estradiol substitution. Interestingly, the content of triglycerides in myocardium did not change after ovariectomy, but significantly increased after estradiol substitution while adiposity index did not change after ovariectomy, but significantly decreased after estradiol substitution. Conclusion: Vascular and cardiac parameters under study differed in their response to ovariectomy and estradiol substitution. This indicates different effects of ovariectomy and estradiol on different cardiovascular but also extracardiac structures.     

An adsorptive transfer technique coupled with brdicka reaction to reveal the importance of metallothionein in chemotherapy with platinum based cytostatics

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug.     

Synthesis and characterization of collagen/hydroxyapatite: magnetite composite material for bone cancer treatment

Our purpose was obtaining and characterizing a complex composite system with multifunctional role: bone graft material and hyperthermia generator necessary for bone cancer therapy. The designed system was a magnetite enriched collagen/hydroxyapatite composite material, obtained by a co-precipitation method. Due to the applied electromagnetic field the magnetite will induce hyperthermia and cause tumoral cell apoptosis. The complex bone graft system was characterised by XRD, FTIR and SEM, while the hyperthermia was quantify by measuring the temperature increase due to the applied alternative electromagnetical field.     

A protocol for processing the delicate larval and prepupal salivary glands of Drosophila for scanning electron microscopy

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton‐derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four‐step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate‐based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline‐based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point‐dried, mounted on aluminum stubs, sputter coated with gold–palladium alloy and examined in the SEM.     

The polymer blend technique as a method for designing fine carbon materials

The polymer blend technique is proposed as a method for designing fine carbon materials. In principle a blend consisting of polymers with and without carbon residue after heating is subjected to melt-spinning, stabilization and finally carbonization. A combination of two polymers and control of the blend texture are important for using the technique successfully. In this paper, three prepared fine carbon materials are introduced, i.e. carbon fibers including thin and long pores aligned parallel to the fiber, thin carbon fibers 200-300 nm in diameter and carbon nanotubes with 10-20-nm outer diameter. In particular the carbon nanotubes are described in detail to emphasize the great potential of the polymer blend technique. Further possibilities of the technique are also discussed briefly.     

Intrinsic affinity of acid-activated bentonite towards hydrogen and carbon dioxide

The effect of acid activation on bentonite affinity toward carbon dioxide (CO₂) and hydrogen (H₂) was investigated at ambient conditions. Characterization through X-ray diffraction and fluorescence, thermal gravimetric analysis, nitrogen adsorption-desorption isotherms, Fourier Transform Infrared spectroscopy and differential scanning calorimetry allowed correlating newly induced textural and structural features with adsorptive properties. Optimum acid treatment improved the specific surface area and porosity. The resulting decrease in dehydration temperature indicates decay in hydrophilic character. The affinity improvement towards hydrogen was due to Brønsted acidity suppression and surface basicity attenuation, which are essential requirements for adsorption on aluminosilicates (AS) via weak Lewis Acid-Base interactions, but excessive acid attack was detrimental. Low Si/Al surfaces should be suitable for CO₂ capture, while moderately acid-treated clays should be interesting candidates as hydrogen adsorbents. This allows envisaging promising prospects for low-cost AS-based materials intended for selective CO₂-free capture and storage of hydrogen without energy and safety constraints.     

Ketoprofen affects the mammary immune response in dairy cows in vivo and in vitro

Nonsteroidal anti-inflammatory drugs are commonly administered parenterally in addition to antimicrobial mastitis therapy to increase the well-being of the diseased animal. As mastitis is usually a localized infection of mammary tissue, we tested the hypothesis that a local administration of nonsteroidal anti-inflammatory drugs through the teat canal could have anti-inflammatory effects on the affected area. We investigated the effects of intramammarily administered ketoprofen (KET) during an LPS-induced immune response on somatic cell count (SCC) and blood–milk barrier integrity. In addition, we investigated the effects of KET on the mRNA abundance of immune factors and their prostaglandin E2 secretion in primary bovine mammary epithelial cells in vitro. Six cows received 0.2 µg of LPS (serotype O26:B6) together with 50 mg of KET into one quarter and LPS only in the opposing quarter. The increase of SCC and of serum albumin (SA) and IgG concentrations and the increase of lactate dehydrogenase (LDH) activity in milk induced by LPS were lower in quarters that received KET in addition. In 3 cows, intramammary KET (50 mg) without additional LPS did not affect SCC, SA, IgG, and LDH in milk. Effects of KET on the immune response of mammary epithelial cells in vitro were investigated in cells from 3 cows challenged with or without LPS (0.2 µg/mL) and with or without additional KET in 2 concentrations (1.25 or 2.5 mg/mL). Ketoprofen reduced the LPS-induced increase of mRNA abundance of tumor necrosis factor α, IL-8, serum amyloid A, and cyclooxygenase-2. The mRNA abundance of cyclooxygenase-1 and prostaglandin E synthase was reduced in cells without LPS challenge by addition of KET at 2.5 mg/mL. Furthermore, the LPS-induced secretion of prostaglandin E2 of mammary epithelial cells into the supernatant could not be detected if KET was added. The results demonstrate that intramammary KET diminishes the increase of SCC and reduces the impairment of the blood–milk barrier (based on SA and LDH in milk), leading to a reduced IgG concentration in milk during LPS-induced mastitis. In mammary epithelial cells, KET limits the expression of several immune factors that are increased during an immune response. In summary, intramammary administration of KET reduces the inflammatory response in the mammary gland. However, it remains unclear whether the inhibited transfer of immune cells and IgG from blood into milk after KET administration would reduce the success of the immune defense in infectious mastitis.