Lifting the curtain on the freshwater mussel diversity of the Italian Peninsula and Croatian Adriatic coast

Freshwater mussels of the order Unionida have been dramatically declining globally. Despite their ecological importance, conservation of these animals has been hindered by unresolved taxonomy and a lack of data on the distribution and status of populations, especially in southern Europe. Although the Italian Peninsula has been noted as a centre of endemism and one of the major refugia of the glacial ages for several taxa, few studies have been performed on the genetic diversity of Unionida. Most importantly, the taxonomic status of several freshwater mussel populations of the Italian Peninsula is still unresolved. Here we present the first comprehensive dataset for the Unionida of the region spanning Italy and the coastal Croatian region (west of the Dinaric Alps). In total, 191 specimens were collected (85 Anodonta, 64 Unio, 17 Microcondylaea bonellii and 25 Sinanodonta woodiana) from 34 sites across the Italian Peninsula and coastal Croatian river basins for molecular identification (COI, 16S and 28S). Genetic analyses were performed to understand major phylogenetic and phylogeographic patterns. Seven species were detected: three Anodonta species (A. anatina, A. cygnea and A. exulcerata), two Unio species (U. mancus and U. elongatulus), Microcondylaea bonellii, and the invasive Sinanodonta woodiana. The presence of three endemic species (A. exulcerata, U. elongatulus and M. bonellii) confirms the importance of the region as a centre of endemism for freshwater mussels. The Apennine Mountains act as an important biogeographic barrier.     

Toxicity of dodecylpyridinium and cetylpyridinium clorides against phosphate-accumulating bacterium

The antibacterial effect of cationic surfactants against the pure culture of phosphate (P)-accumulating bacterium Acinetobacter junii was investigated. The estimated EC₅₀ values of the N-dodecylpyridinium chloride (DPC) for growth inhibition was 1.4±0.5 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 7.3±2.6 × 10⁻⁵ mol L⁻¹. The estimated EC₅₀ values of the N-cetylpyridinium chloride (CPC) for growth inhibition was 4.9±1.3 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 7.7±2.9 × 10⁻⁶ mol L⁻¹. This suggests the importance of controlling the amounts of cationic surfactants in influent of the wastewater treatment systems in order to avoid the possible failure of the biological P removal from wastewaters.     

Human papilloma virus associated with genital infection

Genital human papillomavirus (HPV) infections are among the most common sexually transmitted diseases. HPV is associated with a spectrum of diseases ranging from benign vulgar verrucae and condylomata accuminata to malignant cancers of the cervix, vulva, anus and penis. Genital HPV is in most cases transmitted sexually, but non-sexual routes of transmission, such as perinatal and autoinoculation, are possible. Men can be a reservoir of the virus that lives in latent or subclinical form on genital mucosa. Such an asymptomatic infection may be an oncogenic factor in the development of cervical cancer Colposcopic examination of the genitalia after the application of 3-5% acetic acid is a reliable method for the identification of subclinical HPV infection. Successful therapy of anogenital warts is characterized by their complete clearance, as well as by the lack of recurrence. Current treatments do not reliably eradicate HPV infections. The diagnosis and therapy of HPV infection in men is potentially beneficial because the eradication of penile HPV infection may decrease the reservoir of the virus.     

Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions

We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.     

Characterization of the three selected probiotic strains for the application in food industry

Previously selected bacterial probiotic strains Enterococcus faecium L3, Lactobacillus plantarum L4 and Lactobacillus acidophilus M92 have shown their potential as functional starter cultures in silage, white cabbage and milk fermentation. Therefore, the phenotypic and genotypic characteristics important for their application in food industry were investigated. Pulsed-field gel electrophoresis (PFGE) of NotI digested genomic DNA, in combination with physiological traits determined by API tests, made a useful tool for identification of these probiotic strains and differentiation among them. Lyophilized probiotic cells remained viable during 75 days of storage at −20, +4 and +15°C, while fresh concentrated cells remained viable only at −20°C with addition of glycerol as cryoprotectant. After the lyophilization with addition of skim milk as lyoprotectant, the viability of L. acidophilus M92, L. plantarum L4 and E. faecium L3 was reduced by only 0.37, 0.44 and 0.50 log, respectively. Furthermore, probiotic strains L. acidophilus M92, L. plantarum L4, and E. faecium L3, demonstrated anti-Salmonella activity, and L. acidophilus M92 having also antilisterial activity demonstrated by in vitro competition test. Overnight cultures and cell-free supernatants of the three probiotic strains exerted also an antagonistic effect against the Gram-positive and Gram-negative test microorganisms examined, demonstrated by the agar-well diffusion test. The inhibition of Listeria monocytogenes, Salmonella typhimurium, Yersinia enterocolitica, and Acinetobacter calcoaceticus obtained, achieved by the neutralized, 5-fold concentrated supernatant of L. plantarum L4, may be the result of its bacteriocinogenic activity. On the basis of these results, the application of the three examined probiotic strains may become a point of great importance in respect of food safety.     

Structural investigation of chondroitin/dermatan sulfate oligosaccharides from human skin fibroblast decorin

Hybrid chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan chains, derived from decorin secreted by human skin fibroblasts, were shown to interact with FGF-2, as did oligosaccharides derived therefrom by chondroitin B lyase digestion. In a first attempt to identify the biologically active sequence, a novel protocol for structural analysis of enzyme-resistant oligosaccharides larger than standard trisulfated hexasaccharides was developed. The method bases on capillary electrophoresis (CE) for separating oversulfated species in offline combination with nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the negative ion mode. Under optimized CE and ESI-MS conditions, up to 12-mer oligosaccharides with different degrees of sulfation were identified. A novel tandem MS protocol (CID-VE) was applied to elucidate the structure of a previously undescribed pentasulfated CS/DS hexasaccharide, Delta-4,5-IdoAGalNAc[GlcAGalNAc]2(5S). In this molecular species, detected as a triply charged ion at m/z 511.38, three sulfates are found in the IdoAGalNAcGlcA moiety offering two structural variants: one containing sulfated IdoA together with a disulfated GalNAc moiety and in the other one both uronic acids, that is, GlcA and IdoA and the amino sugar each carry a sulfate ester group.     

Migration of phthalates from plastic products to model solutions

The aim of this investigation was to determine the level and rate of migration of phthalates, compounds used as plastic softeners, from various plastic products into model solutions and to assess the possible adverse effects of the phthalate amounts released on human health, thus to contribute to harmonization of the opinions on the maximal allowed human exposure to these compounds through environmental factors. Nine specimens of plastic toys, 16 specimens of plastic food containers and 10 specimens of other plastic consumer goods were analyzed. The specimens of plastic products were submitted to 10-day action of model solutions. Three model solutions were used: distilled water, 10% ethyl alcohol, and 3% acetic acid. Identification and quantification of the phthalates released were performed by the method of gas chromatography on days 1, 5 and 10 of exposure, at a detection limit of 0.005 microgram/kg. On day 10, the highest level of released phthalates (54.5 mg/kg) was measured in distilled water, followed by 44.4 mg/kg in 3% acetic acid and 32.3 mg/kg in 10% ethyl alcohol. According to plastic product categories, the highest pooled level of phthalates released to all three solutions was recorded for plastic toys (66.2 mg/kg), followed by food containers (37.6 mg/kg) and other consumer goods (27.4 mg/kg). According to plastic product categories, toys showed the most rapid phthalate release, with 65.4% (43.3 of 66.2 mg/kg) of the pooled level of phthalates released to all three solutions recorded on day 1. As indicated by the study results, the levels of phthalates released would not present a hazard for human health, not even over a prolonged period of time. However, data on the highest and fastest pooled phthalate release from plastic toys, and this especially to distilled water simulating salivary action, point to the need of continuous evaluation and amendments of the legislation on phthalates in consumer goods.     

Encephalitis or encephalopathy during an influenza-A epidemic

Six female patients with encephalitis, mean age 36.5 (17-60) years, were admitted to the hospital during the 2000-2001 influenza A (H1N1) epidemic in the Osijek--Baranja County. In three (50.0%) patients, the manifestation of encephalitis occurred on day 4 or 5, and in two (33.3%) patients within 24-48 hours of the onset of influenza symptoms. The disease manifestations included headache, elevated body temperature, generalized fatigue, and consciousness disturbance through coma. Three (50.0%) patients had grand mal seizures. Pathologic electroencephalography findings were recorded in all six (100%) patients, whereas computed tomography showed cerebral edema in three (50.0%) patients. Elevated levels of hepatic enzymes and peripheral blood leukopenia were found in two (33.3%) patients in whom encephalitis developed early upon the onset of influenza. One (16.6%) of these patients died, whereas permanent sequels remained in the other two (33.3%) patients.     

Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry

Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.     

Selective infection of E. coli as a function of a specific molecular interaction

Selective infection of phage is when the bacterial infection depends on the specific molecular interaction between an antigen and a phage-displayed protein sequence such as an antibody. Engineering of the normal infection into pathways, directed by a specific protein--protein interaction, has raised several mechanistic questions. Here, we address the type of display and the affinity between the interacting pairs. The deleted phage R408d3 was used for the first time in selective infection and was shown to exhibit a superior performance compared to the VCSM13 phage. Furthermore, the affinity between the interacting pairs also affected the selective infection process and a correlation between affinity and infection efficiency was detected, thus implying that selective infection is the method of choice for selection of rare high-affinity interactions in molecular libraries.