First report of filarial nematodes in the genus Onchocerca infecting black flies (Diptera: Simuliidae) in Iran.

Black flies are blood-sucking insects of public health importance, and they are effective vectors of pathogens and parasites, such as filarial nematodes of the genus Onchocerca. Our previous surveys have shown that individuals of Simulium turgaicum are annoying pests of humans and livestock in the Aras River Basin of Iran. In the present study, adult black flies of S. turgaicum were trapped from different ecotopes of five villages in Khoda-Afarin County, Iran. By using a sensitive nested PCR assay and targeting the nuclear 18S rDNA-ITS1 marker, filarial infections were found in 38 (1.89%) of 2005 black flies. Homology exploration of 360 bp of the sequences indicated that the filarial worms are members of the family Onchocercidae, with maximum alignment scores of 93-95%. Phylogenetic analysis showed that two Iranian Onchocerca isolates were clustered in the O. fasciata-O. volvulus lineage and were well separated from other filarial nematodes. Both the entomological evidence (empty abdomen of the specimens) and climatologic data (adequate accumulated degree days for development) suggest that the filarial DNA was probably that of infective larvae of vertebrates. This is the first report of an infection by Onchocerca species in S. turgaicum and the first record of onchocercids in black flies in Iran; however, more research is required to demonstrate transmission of these filarial worms by black flies in nature.

Taxonomic status of the black fly Prosimulium italicum Rivosecchi (Diptera: Simuliidae) based on genetic evidence.

The black fly Prosimulium italicum Rivosecchi, distributed in the Apennines and Sicily, was described as a subspecies of Prosimulium hirtipes (Fries), based on a few morphological details. It subsequently was considered conspecific with P. hirtipes and the name was synonymized. Analyses of polytene chromosome banding patterns and sequences of mitochondrial DNA (COI and COII) revealed deep genetic divergence between P. italicum from Italy and P. hirtipes from northern and central Europe and confirmed the species status of P. italicum. Populations of P. italicum either lack chromosomal inversion IS-9 or carry it as an X-chromosome polymorphism, whereas all analyzed populations of P. hirtipes (Slovakia, Sweden, England, and Scotland) are fixed for IS-9. The average K2P genetic distance was 3.7% between P. italicum and P. hirtipes from northern Europe (Sweden) and 4.3 % between P. italicum and P. hirtipes from central Europe (Slovakia). Cytogenetic analysis showed the presence of two cytoforms of P. hirtipes ('A' in Sweden and Slovakia and 'B' in England and Scotland) and two cytoforms of P. italicum ('A' in Sicily and 'B' in Campania and Basilicata), all of which differ in their sex chromosomes and autosomal polymorphisms, suggesting that P. hirtipes and P. italicum might each be a complex of cryptic species.

New genus and species of cavernicolous cockroach (Blattaria, Nocticolidae) from Vietnam.

The new, small cavernicolous species Helmablatta louisrothi gen. et sp. n. (Nocticolidae) from the Tan-Phu cave (Vietnam) is one of the most morphologically interesting cockroaches. The extremely modified upstanding tergal gland composite from three tergites and may serve for gripping the female head during copulation. This presumption is supported by the presence of a central big hook on tergite 8. Furthermore, both wing pairs are uncommonly adapted to help releasing sex pheromones without raising the wings. Histone 3 DNA-based maximum likelihood analyses indicate a recent origin and close phylogenetic relationship between Nocticola spp. and Helmablatta sp.-consistent with the Quaternary age of the source lava tubes.

Blackflies (Diptera: Simuliidae) in Croatia: species richness, distribution and relationship to surrounding countries.

All records of blackflies (Simuliidae) from the territory of Croatia are summarized, including previously unpublished data. The blackfly fauna of Croatia consists of 28 species. Simulium (Nevermannia) angustitarse (Lundström), Simulium (Nevermannia) cryophilum (Rubtsov) complex, Simulium (Nevermannia) lundstromi (Enderlein), Simulium (Nevermannia) vernum Macquart complex, Simulium (Simulium) argyreatum Meigen, Simulium (Simulium) bezzii (Corti) complex, Simulium paraequinum Puri and Simulium pseudequinum Séguy are reported for the first time from Croatia. Information related to the ecoregions, in which species were found and specific species traits are given. Genus Prosimulium Roubaud is represented by one species only. Genus Simulium Latreille is represented by 27 species in six subgenera, with subgenus Simulium Latreille s. str. being most species rich (13 species) and subgenera Boophthora Enderlein and  Trichodagmia Enderlein represented each by only one species. Compared to the neighboring countries, the Croatian species assemblage is most similar to the fauna of Slovenia and least similar to that of Italy. The relatively low number of species, presence of several species complexes and unclear identity of other species show that further research of blackflies in Croatia is needed.

Cryptic Biodiversity and the Origins of Pest Status Revealed in the Macrogenome of Simulium colombaschense (Diptera: Simuliidae), History's Most Destructive Black Fly.

The European black fly Simulium (Simulium) colombaschense (Scopoli), once responsible for as many as 22,000 livestock deaths per year, is chromosomally mapped, permitting its evolutionary relationships and pest drivers to be inferred. The species is 12 fixed inversions removed from the standard sequence of the subgenus Simulium. Three of these fixed inversions, 38 autosomal polymorphisms, and a complex set of 12 X and 6 Y chromosomes in 29 zygotic combinations uniquely characterize S. colombaschense and reveal 5 cytoforms: 'A' in the Danube watershed, 'B' in Italy's Adige River, 'C' in the Aliakmonas River of Greece, 'D' in the Aoös drainage in Greece, and 'E' in the Belá River of Slovakia. 'C' and 'D' are reproductively isolated from one another, and 'B' is considered a cytotype of 'A,' the probable name bearer of colombaschense. The species status of 'E' cannot be determined without additional collections. Three derived polytene sequences, based on outgroup comparisons, place S. colombaschense in a clade of species composed of the S. jenningsi, S. malyschevi, and S. reptans species groups. Only cytoforms 'A' and 'B' are pests. Within the Simuliidae, pest status is reached through one of two principal pathways, both of which promote the production of large populations of blood-seeking flies: (1) colonization of the world's largest rivers (habitat specialization) or (2) colonization of multiple habitat types (habitat generalization). Evolutionary acquisition of the ability to colonize large rivers by an ancestor of the S. jenningsi-malyschevi-reptans clade set the scene for the pest status of S. colombaschense and other big-river members of the clade. In an ironic twist, the macrogenome of S. colombaschense reveals that the name associated with history's worst simuliid pest represents a complex of species, two or more of which are nonpests potentially vulnerable to loss of their limited habitat.

The identity and genetic characterization of Simulium reptans (Diptera: Simuliidae) from central and northern Europe.

Although Simulium reptans Linnaeus is one of the first two blackfly species ever described its identity and taxonomy are still not precisely defined. S. reptans and closely related species from central and northern Europe were characterized based on genetic variability, haplotype number and haplotype distribution. S. galeratum can be considered a synonym of S. reptans, but despite this, two distinct species are present in Great Britain and central Europe. The available name S. reptantoides Carlsson can be used for the second species; earlier reported as S. reptans from Great Britain and central Europe. A total of 80 mitochondrial cytochrome c oxidase I gene sequences were analyzed: 44 S. reptans, 36 S. reptantoides; 38 from Great Britain, 19 from Slovakia, 8 from Lithuania, 3 from Latvia and 12 from Sweden. In 73 individuals a 606 bp section (long sequences), and in all 80 individuals a fully overlapping 453 bp section (short sequences) were analyzed. Results confirmed that S. reptans and S. reptantoides are two genetically isolated species. The variation between these species is 80.01% of total variation; approximately six times higher than the variation among the populations within species. The genetic divergence between species is 7.02% in long sequences and 7.46% in short ones. The genetic divergence within species is 1.18% in S. reptans and 0.83% in S. reptantoides in long sequences, and 1.38% and 1.05% in short sequences. Maximum likelihood trees, maximum parsimony trees and the haplotype network constructed using TCS showed that each species consists of two units, labelled as A and B forms. The distribution of the S. reptans forms is not identical-in Slovakia and the Baltic area only S. reptans B was found, meanwhile both A and B forms were present in Great Britain and Sweden; with the A form clearly dominant. In contrast, both forms of S. reptantoides were present in Great Britain and Slovakia, and absent in Scandinavia and the Baltic area. Additional studies comprising more individuals from larger areas of Europe are required to verify the taxonomic position of these species' forms.

Salivary markers of oxidative stress are related to age and oral health in adult non-smokers.

BACKGROUND: Salivary concentrations of thiobarbituric acid-reacting substances (TBARS) are associated with the periodontal status assessed as papillary bleeding index (PBI). Whether this association is age independent is currently unclear. Salivary concentrations of advanced oxidation protein products (AOPPs) and advanced glycation end products (AGEs) have not been assessed in relation to age or oral health yet. The aim of our study was to analyse salivary markers of oxidative stress in dental patients in relation to age, gender and oral health. METHODS: Consecutive adult non-smoking dental patients were enrolled (n = 204; aged 19-83 years). PBI and the caries index (CI) were assessed. Markers of oxidative stress, such as TBARS, AOPPs and AGEs, and the total antioxidant capacity (TAC) were measured in saliva samples taken before clinical examination. RESULTS: Linear regression showed that salivary TBARS, AGEs and TAC significantly increase with age (r squared = 5.3%, 2.1% and 5%, respectively). PBI is an independent predictor of salivary TBARS (r squared = 5.5%), and the CI negatively affected AOPPs (r = 3.2%) and positively affected TBARS (r = 2.5%). Gender did not affect any of the analysed parameters. CONCLUSIONS: Age as a significant contributor to the variance should be taken into account in studies focusing on salivary markers of oxidative stress. The relationship between PBI and salivary TBARS confirms results from previous studies. In addition, our results show that the association is age independent. Negative association between the CI and AOPPs might be related to recent findings that AOPP might be actually a marker of non-enzymatic antioxidant status.

Advanced glycation end products in myocardial reperfusion injury.

Advanced glycation end products (AGEs) are associated with cardiovascular diseases. Whether the AGE levels change during myocardial reperfusion injury is currently unknown. The aim of our study was to investigate the dynamics of AGEs in myocardial reperfusion injury and to discuss potential reasons for these changes. The dynamics of AGEs, pentosidine and neopterin in the plasma of patients with acute myocardial infarction (AMI) treated using thrombolysis (n = 40) were analyzed. In addition, AGEs were measured in patients with open heart surgery (n = 12) and rabbits with induced AMI (n = 9). In all three studies of myocardial reperfusion injury, a significant decrease of AGEs was observed (by 26 ± 19% in patients with AMI, by 23 ± 14% in patients with open heart surgery and by 39 ± 10% in rabbits with AMI within 1 day of reperfusion; p < 0.05 in all studies). In additional studies, an association between lower AGEs and an activated immune system (R (2) = 0.09; p < 0.01) and fasting (decrease by 38%; p < 0.01) was shown. AGEs decrease in reperfusion injury of the heart. Indices pointing towards the involvement of immune system activation and fasting are presented. Further studies focusing on the underlying mechanism and on the clinical value of the observed dynamics of AGEs are needed.

Lack of evidence for meteorological effects on infradian dynamics of testosterone.

Climatic factors are known to influence the endocrine system. Previous studies have shown that circannual seasonal variations of testosterone might be partly explained by changes in air temperature. Whether infradian variations are affected by meteorological factors is unknown. To analyze possible effects of meteorological parameters on infradian variations of salivary testosterone levels in both sexes, daily salivary testosterone levels were measured during 1 month in 14 men and 17 women. A correlation analysis between hormonal levels and selected meteorological parameters was performed. The results indicate that high testosterone levels are loosely associated with cold, sunny and dry weather in both sexes. However, only the correlations between testosterone and air temperature (men) and actual cloudiness (women) were statistically significant (p < 0,05). Although some correlations reached the level of statistical significance, the effects of selected meteorological parameters on salivary testosterone levels remain unclear. Further longer-term studies concentrating on air temperature, cloudiness and average relative humidity in relation to the sex hormone axis are needed.

Salivary sex hormones during the menstrual cycle.

Infradian rhythms of sex hormones in women are important in several physiological and pathophysiological processes. Detailed analyses of these rhythms are difficult due to problems with sampling. Salivary levels of sex steroids are widely used when repeated sampling is needed. However, a description of variation during the menstrual cycle with daily sampling is lacking. In our study salivary levels of testosterone, estradiol and progesterone were measured in samples collected daily by 17 young healthy women (21,2+/-0,7 years) during one menstrual cycle. Sex steroid levels were determined using radioimmunoassay. The dynamics of salivary sex steroids in our study resembles the known dynamics in plasma. Similar patterns for estradiol and testosterone confirm the hypothesis that in women testosterone serves as a precursor for estradiol. The primary (periovulatory) peak and secondary (luteal) peak of testosterone are followed by similar peaks of estradiol. Progesterone reaches maximum concentrations during the luteal phase. This study shows that analysis of salivary levels of sex steroids are informative and can be used in neuroendocrine, chronobiological and other research areas, when repeated sampling is needed.

Increased one week soybean consumption affects spatial abilities but not sex hormone status in men.

BACKGROUND: Increased soybean intake is often recommended for the prevention of hormone-dependent cancer, cardiovascular diseases and age-related cognitive decline. Although isolated phytoestrogens have shown these positive effects, the evidence for such influence of increased consumption of soybeans is lacking. AIM: To prove the effects of short-term increased soybean intake on sex hormone levels and spatial cognitive parameters in men. SUBJECTS AND METHODS: Seven young healthy men were asked to eat 900 g soybeans during 1 week. Sex hormone levels were determined in saliva and plasma, and mental rotation and spatial visualization were quantified by standard psychometric tests. All parameters were assessed before and after the study. RESULTS: Plasma estradiol, total and free testosterone, as well as salivary testosterone and estradiol remained unchained. Spatial cognitive performance was improved after increased soybean intake when considering spatial visualization (P=0.03). The results for mental rotation showed similar dynamics, but the changes were not significant. CONCLUSION: Short-term increased soybean intake does not affect sex hormone status, but improves spatial cognitive performance in young healthy men.

Short-term soybean intake and its effect on steroid sex hormones and cognitive abilities.

OBJECTIVE: To observe the effects of short-term soybean consumption on cognitive spatial abilities and changes in sex endocrine net in both genders. DESIGN: Short-term prospective intervention study. SETTING: Academic medical center. PATIENT(S): Fifty-four healthy females and 32 healthy males (18 - 25 y of age). INTERVENTION(S): Volunteers were asked to eat 2 g/kg per day of soybeans during 1 week. Saliva and plasma samples were taken, and psychometric tests were performed on the 1st (before soybean intake), 7th (after soybean intake), and 14th days (after washout period) of the study. MAIN OUTCOME MEASURE(S): Salivary T and plasma E(2) were measured. Mental rotation (MR) and spatial visualization (SV) tests were performed on the days of sampling. RESULT(S): Soybean intake did not change salivary T and plasma E(2) levels in men. During the washout period, both parameters have shown a tendency to rise. The effect of soybean intake on hormonal parameters in men was, however, dependent on the basal T levels. In women, salivary T as well as plasma E(2) levels showed a tendency toward a decline after soybean intake (the decrease in E(2) was statistically significant) and to increase back toward basal levels during the washout period. Both males and females statistically significantly improved in MR and SV after soybean intake. During the washout period, both genders further improved their results in MR but not in SV. CONCLUSION(S): Short-term soybean consumption alters the concentrations of salivary T and plasma E(2) in both men and women. Spatial abilities appear to be improved by short-term soybean intake, but the exact mechanisms require further study.

Intelligence and salivary testosterone levels in prepubertal children.

BACKGROUND: Hormones are one of the regulatory systems influencing brain-cognition interactions and subsequent emotions and behavior in humans and animals. Sex hormones have been found to influence brain structures prenatally, so as to prepare targeted neuronal circuits for activation during and after puberty. Testosterone is believed to affect cognition and thinking in humans as well as between-sex differences in cognitive abilities. AIM: The aim of this paper was to investigate associations between testosterone and different levels of intelligence in young prepubertal children of both sexes. METHODS: Two hundred and eighty four prepubertal children of both sexes between 6 and 9 years of age provided saliva samples. Of these, 107 were intellectually gifted (IQ above 130), 100 children of average intelligence--randomly chosen from general population (IQ between 70 and 130), and 77 children mentally challenged (IQ less than 70). RESULTS: Our results have revealed the differences in salivary testosterone levels in boys grouped according to IQ, intellectually gifted and mentally challenged boys having lower salivary testosterone levels than their peers characterized by average intelligence proposing the common biological characteristic of minority IQ groups on both ends of the Gauss curve. In girls, no differences in salivary testosterone levels were found among IQ groups. CONCLUSIONS: Our findings are the first that present the relationship between testosterone and the broad range of general IQ in childhood. The boys of average intelligence had significantly higher testosterone levels than both mentally challenged and intellectually gifted boys, with the latter two groups showing no significant difference between each other. The functional implications of the brain-cognition interactions remain to be fully explored with regard to the internal milieu influencing neural substrate.

Salivary thiobarbituric acid reacting substances and malondialdehyde--their relationship to reported smoking and to parodontal status described by the papillary bleeding index.

BACKGROUND: Thiobarbituric reacting substances (TBARS) are markers of lipoperoxidation. The best-known specific TBARS is malondialdehyde (MDA). Results from our previous studies have shown that TBARS can be measured in saliva and are increased in patients with gingivitis. Whether MDA is the main TBARS in saliva from patients with altered parodontal status is unknown. Aim. To observe the relationship between the parodontal status and TBARS, MDA and the number of epithelial cells in saliva. SUBJECTS & METHODS: In Study I saliva and plasma samples of 15 patients (8F, 7M) suffering from inflammatory periodontal diseases were gathered and TBARS levels were measured in these samples. In Study II saliva samples from 217 consecutive stomatologic patients were collected and analysed for TBARS spectrofluorometrically, MDA by high-performance liquid chromatography and epithelial cell count by light microscopy. Papillary bleeding index (PBI) was determined in standard stomatologic examination. RESULTS: In Study I results from our previous studies showing no correlation between salivary and plasma TBARS levels were confirmed. This indicates that the local salivary level of TBARS is unlikely to be directly affected by systemic oxidative stress. In Study II higher PBI was associated independently (adjusted for age and sex) tightly with higher TBARS (p<0.001) and with lower number of epithelial cells in saliva (p<0.05). Smokers had higher salivary MDA levels (p<0.003) and lower number of epithelial cells in saliva (p<0.01). CONCLUSION: Salivary TBARS are a simple parameter that partially reflects the parodontal status with a potential usefulness in the clinical stomatology. We show herein that salivary MDA is dependent on age and smoking, but there is no correlation between MDA and PBI. Further studies should uncover the main salivary TBARS compound in patients with altered parodontal status and trace the origin of these salivary lipoperoxidation markers.

Endocrine and cognitive effects of short-time soybean consumption in women.

BACKGROUND: Soy phytoestrogens are known to influence the hormonal status acting as partial estrogen agonists. Soy-derived food supplements are advised for hormone replacement therapy, prevention of atherosclerosis, age-related cognitive decline and even hormone-dependent cancer, although results from clinical studies are controversial. Whether increased soybean intake can affect the endocrine status and cognitive abilities is largely unknown. AIM: To observe the effects of 1 week of increased soybean intake on sex hormone levels and spatial cognitive abilities in women. SUBJECTS AND METHODS: 16 young healthy female volunteers were asked to eat 900 g of soybeans within 1 week. Salivary testosterone (T), free and total plasma T, salivary and plasma estradiol (E) were measured by radioimmunoassay before and after the study period. Mental rotation (MR) and spatial visualization (SV) psychological tests were done at the days of sampling. RESULTS: Soybean intake increased total plasma T levels (p < 0.02) while decreasing salivary T (p < 0.01) and not altering free plasma T levels. Salivary and plasma E levels were not changed. The results of MR and SV tests were improved after the study period. CONCLUSION: Short-time increased soybean intake alters the level of total plasma and salivary T and improves spatial cognition in women. Whether this effect is mediated by modulation of estrogen receptors, changes in sex hormone-binding globulin production or changes in activity of steroid-competent enzymes needs further study.

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

BACKGROUND: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. AIM: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. METHODS AND RESULTS: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.

Effects of anabolic steroids and antioxidant vitamins on ethanol-induced tissue injury.

Various mechanisms are involved in the process of ethanol-induced tissue impairment. Oxidative stress and its effects are among the most important. We compared the effects of antioxidant vitamins (vitamin C and E in combination) and steroids (testosterone and nandrolone separately) on the toxicity of ethanol in rats. Animals (male Wistar rats, n = 48) were randomised into following groups-Control, Ethanol, Testosterone, Ethanol + Testosterone, Ethanol + Nandrolone, Ethanol + Vitamins. Alcohol was given daily by gavage in a dose of 5 g/kg of body weight. On the 27th day of the study the animals were sacrificed by decapitation and tissue samples were taken. Metabolic status, parameters of the hepatic metabolism, hormone levels (testosterone, ACTH, corticosterone), lipoperoxidation markers (malondialdehyde and conjugated diens in forebrain cortex and in cerebellum) and advanced glycation end-products were analysed. Tissue samples underwent histological examination. Histological outcomes showed a protective effect of antioxidants on hepatic and cerebellar injury caused by chronic ethanol intake. Anabolic steroids protected especially the central nervous tissue against the toxicity of alcohol. Both, antioxidant vitamins and anabolic steroids protect against the ethanol-induced toxicity, however, this effect is tissue specific.