A Thermostable Salmonella Phage Endolysin,Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.     

Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological targets with dioxin-like compounds emphasizing the potential for interactions.     

Criteria for Viability Assessment of Discarded Human Donor Livers during Ex Vivo Normothermic Machine Perfusion

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37°C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.     

Somatovariants of Sardinian recruits from the three principal geographic areas of the island

Using the constitutional method based on somatovariants, three samples with a total of 1207 recruits, divided according to the three main geographic areas of Sardinia (North, Center, South) were compared. Individuals from the center of the island are lighter and shorter than those belonging to the other two regions. Since this somatic typology corresponds to expectations based on data analyzed in a previous investigation, the somatotype method can be considered to be a valuable instrument for quick, preliminary constitutional typings.     

A scanning electron microscopic study of the dorsal surface of the human tongue

To study the dorsal surface of the human tongue using a scanning electron microscopy (SEM), tissue specimens were taken from the anterior part of the tongues of 15 individuals aged from 21- to 28-years-old. The formalin-fixed samples were processed routinely for SEM. With SEM the surface of the normal tongue mucosa was shown to be rather evenly covered by filiform papillae, with some fungiform papillae scattered among them. Filiform papillae consisted of two parts: the body and hairs. The mucosal surface of the body was smooth; the squamous epithelial cells were polygonal, and their boundaries were prominent. On the surface of the superficial epithelial cells were parallel or branching microplicae. Each filiform papilla had 6-10 hairs, which were scaled and covered by an extensive plaque of microorganism. The upper surface of the fungiform papillae was smooth; only a few desquamating cells were seen. The superficial cells had a pitted appearance and cell boundaries overlapped. Taste pores, up to 3 pores in a single papilla, were found on the upper surface. Desquamation was more pronounced on the base of the fungiform papillae than on the upper surface. In almost all fungiform papillae some hairs protruded from the base. Parallel microplicae were found on the surface of the superficial cells of the base. The structure and function of the human tongue, as well as the microplicae of its superficial cells, are compared to those of various species of animals.     

In Vivo Administration of Ethanol Enhances the Function of the γ-Aminobutyric Acid-Dependent Chloride Channel in the Rat Cerebral Cortex

The effect of in vivo administration of ethanol on the gamma-aminobutyric acidA (GABAA) receptor-coupled chloride channel was studied by measuring ex vivo t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in the rat cerebral cortex. Intragastric administration of ethanol (0.5-1 g/kg) elicited in 40 min a significant decrease of [35S]TBPS binding to unwashed cortical membrane preparations, an effect mimicked by diazepam (0.5-1 mg/kg, i.p.). However, Scatchard plot analysis indicated that, unlike the case with diazepam, the decrease was entirely due to a reduction in the apparent affinity of [35S]TBPS receptors with no change in the total number of binding sites. Moreover, ethanol, like diazepam, reduced the increase of [35S]TBPS binding elicited by isoniazid (350 mg/kg, s.c.), an inhibitor of the GABAergic transmission. Finally, ethanol markedly potentiated the inhibitory action of diazepam on [35S]TBPS binding. The results suggest that ethanol, like benzodiazepines, enhances the function of the GABAA-coupled chloride channel.     

Comparison of five cladoceran species in short- and long-term copper exposure

The study was carried out to assess the effect of both short-and long-term copper exposure on five cladoceran species differing in body size and habitat, i.e. Daphnia magna, D. pulex, D. galeata, Bosmina longirostris, and Chydorus sphaericus. The species-specific 48 h EC₅₀ values for fed neonates were used to determine the chronic exposure levels (52% and 65% of the EC₅₀ values). The experiment was run at two food levels. Long-term copper exposure retarded growth in each of the species studied. However, the copper treatments did not affect the clutch sizes of the experimental animals. At a low food level, copper exposure increased mortality in every species studied. The intrinsic rate of increase, r, was reduced in the copper-exposed populations. The sensitivity to copper was higher in the small lake species, compared to D. magna and D. pulex that are commonly used in standard toxicity tests. Of the species studied, B. longirostris had the highest sensitivity to both acute and chronic copper stress. The different sensitivities of the species studied are discussed in an ecological context.