Different aggregation approaches in the chironomid community and the threshold of acceptable information loss

Due to the problem of identification, Chironomidae larvae, although very abundant, are often avoided or not properly used in bioassessment programs. The aim of this work was to test how different aggregation processes—taxonomic resolution and the random aggregation approach (best practicable aggregation of species—BestAgg) affect the analysis of chironomid communities regarding any information loss. The self-organizing map method, together with classification strength analysis and Spearman’s rank correlation, revealed that the genus-level and BestAgg-abundance matrix most accurately approximated the species-level community pattern. The subfamily-level dataset was ineffective at presenting the chironomid community structure, with a substantially lower concordance with the species-level dataset. The biologic environmental gradients analyses presented the same set of important environmental variables for the species-level, genus-level, and BestAgg-abundance matrix. The indicator values analysis showed that indicator genera provide information very close to that gained from species indicators. According to our results, the numeric relationship between species and higher taxa influences taxonomic scaling, limiting Chironomidae family aggregation, with acceptable information loss only up to genus level. In addition, the BestAgg approach, with the maximum level of aggregation, properly assesses the community structure and consequently describes environmental conditions.     

Inhibition of mTOR-Dependent Autophagy Sensitizes Leukemic Cells to Cytarabine-Induced Apoptotic Death

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.     

Probucol Release from Novel Multicompartmental Microcapsules for the Oral Targeted Delivery in Type 2 Diabetes

In previous studies, we developed and characterised multicompartmental microcapsules as a platform for the targeted oral delivery of lipophilic drugs in type 2 diabetes (T2D). We also designed a new microencapsulated formulation of probucol-sodium alginate (PB-SA), with good structural properties and excipient compatibility. The aim of this study was to examine the stability and pH-dependent targeted release of the microcapsules at various pH values and different temperatures. Microencapsulation was carried out using a Büchi-based microencapsulating system developed in our laboratory. Using SA polymer, two formulations were prepared: empty SA microcapsules (SA, control) and loaded SA microcapsules (PB-SA, test), at a constant ratio (1:30), respectively. Microcapsules were examined for drug content, zeta potential, size, morphology and swelling characteristics and PB release characteristics at pH 1.5, 3, 6 and 7.8. The production yield and microencapsulation efficiency were also determined. PB-SA microcapsules had 2.6 ± 0.25% PB content, and zeta potential of −66 ± 1.6%, suggesting good stability. They showed spherical and uniform morphology and significantly higher swelling at pH 7.8 at both 25 and 37°C (p < 0.05). The microcapsules showed multiphasic release properties at pH 7.8. The production yield and microencapsulation efficiency were high (85 ± 5 and 92 ± 2%, respectively). The PB-SA microcapsules exhibited distal gastrointestinal tract targeted delivery with a multiphasic release pattern and with good stability and uniformity. However, the release of PB from the microcapsules was not controlled, suggesting uneven distribution of the drug within the microcapsules.KEY WORDS: anti-inflammatory, antioxidant, artificial-cell microencapsulation, diabetes mellitus, probucol, type 2 diabetes     

Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers,Transmission via Cannibalism,and Inhibition of Spore Germination

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.     

Biotransformation of <Emphasis Type="SmallCaps">l</Emphasis>-Selenomethionine and Selenite in Rat Gut Contents

l-Selenomethionine (SeMet) and sodium selenite are widely used selenium nutritional supplements with potential benefit in preventing cancer. However, supplementation is not without risks of toxicity if intake is too high. The aim of the present study was to investigate SeMet and selenite metabolism in the gastrointestinal tract with particular focus on the formation of the volatile selenium excretion products, dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). Adult male Wistar rats (n = 5) were euthanized, their intestinal tracts removed and the contents of jejunum, ileum, caecum and colon used to prepare 10% suspensions in saline. SeMet and selenite (0.5–0.6 mM) were then incubated with these suspensions at 37°C for 3 h. Caecum and colon contents were the most metabolically active towards SeMet with 30% and 15% metabolized over 3 h. DMDSe was the only volatile selenium metabolite detected accounting for 8.7 ± 1.3% of the selenium lost in caecum contents. Selenite was completely metabolized by caecum contents and 73% by colon contents under the same conditions forming DMSe (5.7 ± 0.9% of the selenium lost in caecum) and a precipitate of red amorphous elemental selenium. Based on previous literature and these results, we conclude that the gut microbiota contributes to the excretion of excess selenium through the production of methylated selenium compounds and elemental selenium.     

The Differences in the Cellular and Plasma Antioxidative Capacity Between Transient and Defined Focal Brain Ischemia: Does it Suggest Supporting Time-Dependent Neuroprotection Therapy?

There are many opened questions about the precocious role of oxidative stress in the physiopathology of the early stage of transitory ischemic attack (TIA) and defined focal brain ischemia, as well as about its correlation with clinical severity, short-lasting and clinical outcome prediction in these conditions. The study evaluates the values of glutathione (GSH), glutathione peroxidase, and superoxide dismutase (SOD) in hemolysates and total thiol content (–SH), advanced oxidation protein products (AOPP), SOD, and malondialdehyde (MDA) in plasma, in TIA and stroke patients in the early stage of their neurological onset. The results are interpreted in view of the potential relationship between tested parameters and clinical severity and clinical outcome prediction. Better hemolysates’ and total antioxidant profile with higher values of AOPP were observed in TIA compared to stroke patients (p < 0.05). The stroke patients with initially better clinical presentation showed better antioxidant profile with lower values of AOPP (p < 0.05). In TIA patients, this was observed for GSH, –SH content, and AOPP (p < 0.05), which correlated with a short risk for stroke occurrence in this group (p < 0.01). Beyond MDA values, all tested parameters showed correlation with clinical outcome in stroke patients (p < 0.05). The measurement of oxidative stress in TIA and stroke patients would be important for identifying patients’ subgroups which might receive supporting therapy providing better neurological recovery and clinical outcome. That approach might give us an additional view of a short-lasting risk of stroke occurrence after TIA, and its clinical outcome and prognosis.     

Inulinase immobilization on polyethylene glycol/polypyrrole multiwall carbon nanotubes producing a catalyst with enhanced thermal and operational stability

This paper describes the development of a simple method for mixed non‐covalent and covalent bonding of partially purified inulinase on functionalized multiwall carbon nanotubes (f‐MWCNTs) with polypyrrole (PPy). The pyrrole (Py) was electrochemically polymerized on MWCNTs in order to fabricate MWCNTs/PPy nanocomposite. Two multiple forms of enzyme were bound to N‐H functional groups from PPy and ‐COO^? from activated MWCNTs to yield a stable MWCNTs/PPy/PEG immobilized preparation with increased thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to confirm functionalization of nanoparticles and immobilization of the enzyme. The immobilization yield of 85% and optimal enzyme load of 345 μg protein onto MWCNTs was obtained. The optimum reaction conditions and kinetic parameters were established using the UV‐Vis analytical assay. The best functional performance for prepared heterogeneous catalyst has been observed at pH 3.6 and 10, and at the temperatures of 60 and 80ºC. The half‐life (t_1/2) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99 min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle.     

Topography of stimulation sites with aversive effects in the rat's medial hypothalamus (author's transl)]

The intensity of the aversive effects induced by an electrical stimulation of various sites in the rat's medial hypothalamus was evaluated through analysing the relation between stimulation intensity and the induced switch-off responses. Series of "hot points" were found in both the ventro-medial and the dorso-medial nuclei. Stimulation sites with less marked aversive effects were found in a periventricular region.     

Light scattering of normal human lens I. Application of random density and orientation fluctuation theory.

Light-scattering intensities in the I parallel and I+ mode were obtained on thin sections of three human lenses. Random density and orientation fluctuation theory, without cross correlation, was employed to evaluate light-scattering parameters. Both the density correlation distances, as well as the orientation correlation distances, were related to structural elements in the lens fiber cell that have been observed by other investigators with different techniques. The magnitude of these fluctuations were evaluated, and it was demonstrated that the density fluctuations are the main contributors to light scattering in normal human lenses. Changes in the light-scattering parameters were evaluated as a function of position within the lens. The changes observed agree with the biochemical data in the literature that reflects that an aging process occurs when one proceeds from the periphery of the lens toward the center.     

No evidence of Neandertal mtDNA contribution to early modern humans

The retrieval of mitochondrial DNA (mtDNA) sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.