Diosmin Attenuates Methotrexate-Induced Hepatic, Renal, and Cardiac Injury: A Biochemical and Histopathological Study in Mice.

The current study was designed to investigate the beneficial role of diosmin, a biologically active flavonoid, against methotrexate- (MTX-) induced hepatic, renal, and cardiac injuries in mice. Male Swiss albino mice received a single intraperitoneal injection of MTX (at 20 mg/kg, body weight) either alone or in combination with oral diosmin (at 50 or 100 mg/kg body weight, for 10 days). Serum was used to evaluate tissue injury markers, while hepatic, renal, and cardiac tissue samples were obtained for determination of antioxidant activity as well as histopathological examination. Diosmin treatment ameliorated the MTX-induced elevation of serum alkaline phosphatase, aminotransferases, urea, creatinine, lactate dehydrogenase, and creatine kinases as well as plasma proinflammatory cytokines (interleukin-1-beta, interleukin-6, and tumor necrosis factor-alpha). Additionally, both diosmin doses significantly reduced tissue levels of malondialdehyde and nitric oxide and increased those of glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase, and catalase, compared to the MTX-intoxicated group. Histopathological examination showed that diosmin significantly minimized the MTX-induced histological alterations and nearly restored the normal architecture of hepatic, renal, and cardiac tissues. Based on these findings, diosmin may be a promising agent for protection against MTX-induced cytotoxicity in patients with cancer and autoimmune diseases.

Protective Vaccination against Blood-Stage Malaria of Plasmodium chabaudi: Differential Gene Expression in the Liver of Balb/c Mice toward the End of Crisis Phase.

Protective vaccination induces self-healing of otherwise fatal blood-stage malaria of Plasmodium chabaudi in female Balb/c mice. To trace processes critically involved in self-healing, the liver, an effector against blood-stage malaria, is analyzed for possible changes of its transcriptome in vaccination-protected in comparison to non-protected mice toward the end of the crisis phase. Gene expression microarray analyses reveal that vaccination does not affect constitutive expression of mRNA and lincRNA. However, malaria induces significant (p < 0.01) differences in hepatic gene and lincRNA expression in vaccination-protected vs. non-vaccinated mice toward the end of crisis phase. In vaccination-protected mice, infections induce up-regulations of 276 genes and 40 lincRNAs and down-regulations of 200 genes and 43 lincRNAs, respectively, by >3-fold as compared to the corresponding constitutive expressions. Massive up-regulations, partly by >100-fold, are found for genes as RhD, Add2, Ank1, Ermap, and Slc4a, which encode proteins of erythrocytic surface membranes, and as Gata1 and Gfi1b, which encode transcription factors involved in erythrocytic development. Also, Cldn13 previously predicted to be expressed on erythroblast surfaces is up-regulated by >200-fold, though claudins are known as main constituents of tight junctions acting as paracellular barriers between epithelial cells. Other genes are up-regulated by <100- and >10-fold, which can be subgrouped in genes encoding proteins known to be involved in mitosis, in cell cycle regulation, and in DNA repair. Our data suggest that protective vaccination enables the liver to respond to P. chabaudi infections with accelerated regeneration and extramedullary erythropoiesis during crisis, which contributes to survival of otherwise lethal blood-stage malaria.

Differential miRNA Expression in the Liver of Balb/c Mice Protected by Vaccination during Crisis of Plasmodium chabaudi Blood-Stage Malaria.

MicroRNAs are increasingly recognized as epigenetic regulators for outcome of diverse infectious diseases and vaccination efficacy, but little information referring to this exists for malaria. This study investigates possible effects of both protective vaccination and P. chabaudi malaria on the miRNome of the liver as an effector against blood-stage malaria using miRNA microarrays and quantitative PCR. Plasmodium chabaudi blood-stage malaria takes a lethal outcome in female Balb/c mice, but a self-healing course after immunization with a non-infectious blood-stage vaccine. The liver robustly expresses 71 miRNA species at varying levels, among which 65 miRNA species respond to malaria evidenced as steadily increasing or decreasing expressions reaching highest or lowest levels toward the end of the crisis phase on day 11 p.i. in lethal malaria. Protective vaccination does not affect constitutive miRNA expression, but leads to significant (p < 0.05) changes in the expression of 41 miRNA species, however evidenced only during crisis. In vaccination-induced self-healing infections, 18 miRNA-species are up- and 14 miRNA-species are down-regulated by more than 50% during crisis in relation to non-vaccinated mice. Vaccination-induced self-healing and survival of otherwise lethal infections of P. chabaudi activate epigenetic miRNA-regulated remodeling processes in the liver manifesting themselves during crisis. Especially, liver regeneration is accelerated as suggested by upregulation of let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-26a, miR-122-5p, miR30a, miR27a, and mir-29a, whereas the up-regulated expression of miR-142-3p by more than 100% is compatible with the view of enhanced hepatic erythropoiesis, possibly at expense of megakaryopoiesis, during crisis of P. chabaudi blood-stage malaria.

A novel primer set for improved direct gene sequencing of human bocavirus genotype-1 from clinical samples.

Human bocavirus genotype (HBoV-1) is a parvovirus associated with respiratory tract infections in children with different degrees of severity. The current study intended to improve the direct gene sequencing of the HBoV-1 using a newly developed primer set. Screening the presence of human bocavirus infection among in-patients children suffering from lower respiratory tract infections was another aim of the current study. Nasopharyngeal swab samples from in-patients children suffering from lower respiratory tract infections were examined. The real-time polymerase chain reaction was used for the initial screening as a highly sensitive method to detect the HBoV. Genotyping of real-time positive samples was attempted by direct sequencing of PCR amplicons using NP, VP1/2 and the newly developed VP/NC primers. HBoV-1 was present in 56.8% of the examined children. The newly developed primer set successfully amplified all real-time PCR positive samples, however, the other primer pairs did not reliably detect real-time PCR positive samples. The gene sequences of the detected HBoV-1 showed conserved sequences to each other with a low rate of discrepancies. The high rate of infection and the similarity between the detected strains strongly suggest nosocomial infections.

Development of species-specific primers for identification of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia.

Schistosoma mansoni is mediated through the intermediate host Biomphalaria arabica which lives in Saudi Arabia. Molecular characterization and identification of this intermediate host are important for epidemiological studies of schistosomiasis. The present work aimed to determine the molecular variations among the populations of B. arabica found in Southern part of Saudi Arabia, and to develop species-specific primers for identification of these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Five populations of Saudi B. arabica snails were collected from freshwater bodies. Three populations were collected from Asser and two populations were collected from AL-Baha. Genomic DNA was extracted from snails and was amplified using five different RAPD-PCR primers. The banding patterns of amplified materials by primers P1 and P5 were identical in all populations. However, the rest primers displayed intra-specific differences among populations with variable degrees. Largest sizes of RAPD-PCR products were cloned into TA cloning vector as a preparatory step for DNA sequence analysis. After sequencing, similarity searches of obtained DNA sequences revealed that there are no similar sequences submitted to genebank data bases and its associated banks. The results obtained will be helpful in the development of simultaneous identification of B. arabica snails and diagnosis of S. mansoni infection within it in a single step by an implementation of multiplex PCR.

Developing species-specific primers to identify Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia.

This work aimed to determine the inter- and intra-specific variations in populations of Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia, and to develop species-specific primers to identify these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Two populations of B. truncatus were collected from Asser and Bisha (A and B), and two B. beccari populations were collected from Mahial Asser and Merba (C and D). The snails' genomic DNA was extracted and amplified using 5 different primers. The primers displayed variable intra- and inter-specific differences across the populations. The largest RAPD-PCR fragments were cloned into a vector as a preparatory step for sequencing. Similarity searches for the sequenced cloned inserts revealed no similar sequences in the GenBank database or its associated databases. Specific primers used to target the B. truncatus and B. beccari genomes were designed using the Gene Runner program and based on the DNA sequences obtained from RAPD fragment sequence analyses. Using these primers for specific PCRs resulted in expected single-band PCR products of 536 bp for B. beccari and 478 bp for B. truncatus. These results will be helpful for simultaneously identifying B. truncatus and B. beccari snails and diagnosing S. haematobium infections within the snails using single step multiplex PCR.

Light and ultrastructural description of Meglitschia mylei n. sp. (myxozoa) from Myleus rubripinnis (Teleostei: Serrasalmidae) in the Amazon River system.

Meglitschia mylei n. sp. found in the gall bladder of the teleostean fish Myleus rubripinnis (Serrasalmidae) from the middle Amazonian region of Brazil is described using light and transmission electron microscopy. The spores observed in the bile averaged 24.6±0.8 µm long, 8.7±0.4 µm wide and 5.1±0.3 µm thick and were strongly furcate and arcuate ∩-shaped composed of two symmetric equal-sized valves, up to ∼70 nm thick. Each valve possessed one opposed tapering appendage, 20.1±0.7 µm long, oriented parallel towards the basal tip of the appendages and joined along a right suture line forming a thick strand. The strand goes around the central part of the spore, which in turn surrounds two equal and symmetric spherical polar capsules (PC), 2.1±0.3 µm in diameter, located at the same level. Each capsule contains a polar filament with five (rarely six) coils. The binucleate sporoplasm was irregular in shape, contained several sporoplasmosomes, ∼175 nm in diameter and filled all the space of the two caudal appendages. Based on the arc shape of the spore with two tapering caudal appendages oriented to the basis of spores, on the number and position of the PC and of the polar filament coils and arrangements, and on the host specificity, we propose the name M. mylei n. sp. for this new myxozoan. Accordingly, this is the second described species of this genus.

Fingerprint of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia, using RAPD-PCR.

In the time schistosomisis control programs are implemented in many countries, schistosomiasis continues to spread throughout the world. Among these control strategies is the vector control. Within this context, analysis of the genetic variability of the intermediate host snails is important because it allows identification of specific sequences of the genome of this mollusk related to determine their fingerprint. We investigated Biomphalaria arabica, which is found in Saudi Arabia, the intermediate host of Schistosoma mansoni infection. Genetic fingerprint was studied by RAPD-PCR using our own different random primers as well as published primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers of B. arabica. This information will be helpful in the identification of the snails and demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for establishment of genetic barcode development.

Isoenzyme electrophetic characterization of Leishmania major, the causative agent of zoonotic cutaneous Leishmaniasis in North and West Saudi Arabia.

Several expeditions were carried out to four localities (Al-Madinah Almona-warah, Tabouk region, Al-Jouf and Northern Frontiers regions) in Northern and Western Saudi Arabia for sampling zoonotic cutaneous leishmaniasis (ZCL) cases from patients and rodents. Biopsy samples were collected from 51 patients complaining of skin lesions, most of which (40 or 78.4%) proved to be ZCL. Amastigotes were detected in 33 patients (64.7%), but only 30 (58.9%) gave successful growth of promastigotes in the culture media. The positive cases were Saudis 14(35%) and non-Saudis 26 (65%). Five species of rodents were caught, Meriories libycus, Psammomys obesus, Rattus rattus, jaculus and Hystrix indica. The first species was the most dominant (90%) in which Leishmania parasites were detected. The Leishmania isolates from man and rodents were identified by isoenzyme electrophoresis and proved to be Zymodeme LON-4.

Descriptions of two new marine scuticociliates, Pleuronema sinica n. sp. and P. wilberti n. sp. (Ciliophora: Scuticociliatida), from the Yellow Sea, China.

Two new marine scuticociliates, Pleuronema sinica n. sp. and P. wilberti n. sp., collected from the sand beach of Qingdao, China, were investigated in vivo and following protargol impregnation. Ciliates of the genus Pleuronema are normally recognizable by their large sail-like paroral membrane although one species, P. grolierei, has shorter cilia in the paroral membrane. Neither of the new forms has the conspicuous paroral membrane in vivo so in this respect they are not typical members of this genus. Pleuronema sinica is characterized by its large, conspicuously flattened body, the possession of only one preoral kinety, the irregular-shaped macronucleus and the rather unusual structure of the oral apparatus. By contrast P. wilberti has a medium-size broad-oval body, six to eight preoral kineties and a highly differentiated membranelle 3 that is five- or six-rowed. An identification key is supplied for the 15 species of Pleuronema for which the infraciliature is known.

Systematic position of Discocephalus-like ciliates (Ciliophora: Spirotrichea) inferred from SSU rDNA and ontogenetic information.

The Prodiscocephalus-like ciliates, or discocephalines, are cephalized organisms that are traditionally considered to be hypotrichs (sensu lato) but whose precise systematic position has long been uncertain. The main reasons for this are that these organisms exhibit several intermediate morphological and morphogenetic features and that hitherto none has been investigated using molecular methods. In the present study, the cortical development of Prodiscocephalus borrori was observed during binary division and this can be summarized as follows: (i) in the parental adoral zone of membranelles, only the posterior end is renewed by dedifferentiation of the old structures; (ii) the oral primordium in the opisthe occurs de novo on the cell surface as seen in other typical stichotrichs; (iii) in both dividers, the undulating membranes anlage does not split longitudinally in the usual way but, instead, divides transversely to form the paroral and endoral membranes; (iv) usually seven frontoventral transverse cirral anlagen are formed in the primary mode which then divide into two sets, one each for the proter and opisthe; (v) both left and right marginal rows divide into two parts, thus giving rise to a post-lateral marginal segment at the posterior end of each; (vi) invariably five caudal cirri are formed at the posterior end of the three rightmost dorsal kinety anlagen. Thus, it was found that, like other related discocephalines, P. borrori exhibits more similarities to stichotrichs than to euplotids. Based on a combination of morphological and morphogenetic data, a phylogenetic tree was constructed which suggests that the discocephalines group within the stichotrichs and separate from the euplotids. In addition, the complete small-subunit rRNA gene (SSU rDNA) of P. borrori was sequenced and analysed. In the resulting SSU rDNA tree, the discocephalines represent an intermediate group between the euplotids and the Stichotrichia-Oligotrichia-Choreotrichia assemblage, albeit with low bootstrap support. From these data, we conclude that the discocephalines might be a divergent, or possibly an ancestral, group within the Stichotrichia. Furthermore, our findings further support the suggestion that these organisms should be considered as a distinct order, i.e. Discocephalida Wicklow, 1982, in the subclass Stichotrichia Small & Lynn, 1985.

Morphology and infraciliature of two new marine urostylid ciliates: Metaurostylopsis struederkypkeae n. sp. and Thigmokeronopsis stoecki n. sp. (Ciliophora, Hypotrichida) from China.

The morphology and infraciliature of two new marine urostylid ciliates, Metaurostylopsis struederkypkeae n. sp. and Thigmokeronopsis stoecki n. sp., collected from the coastal waters off Qingdao (Tsingtao), China, have been investigated. Metaurostylopsis struederkypkeae n. sp. is characterized by the slender body shape, small size, rose-reddish cell colour, and having two kinds of pigment-like granules. The larger pigment-like granules are yellow-green or grass-green in colour, oval in shape, and flattened, whereas the smaller ones are wine-reddish. Infraciliature and nuclear apparatus are similar to the well-known Metaurostylopsis marina. Thigmokeronopsis stoecki n. sp. is characterized by its large size with dark brown cell colour and grass-green cortical granules, which are large, blood-cell shaped, and sparsely distributed. The thigmotactic ciliature is conspicuous: 11-14 rows of densely arranged cirri occupy the most postoral area. Keys are provided for all the known species in both genera.

Identification of three highly confused marine Loxophyllum (Ciliophora: Pleurostomatida) with a key to seven congeners from the China Sea.

Three highly confused Loxophyllum helus-like morphotypes (i.e. Loxophyllum rostratum Cohn, 1866, Loxophyllum sinicum n. sp., and Loxophyllum simplex Kahl, 1933) found in mariculture waters near the coast of Qingdao, China, were investigated with emphasis on their live morphology and infraciliature. Comparative descriptions of these three organisms are presented and synonyms are critically discussed. The validity of L. sinicum n. sp. is confirmed mainly by the combination of the distribution of extrusomes, features of general living morphology, morphometric data, and the characters of the somatic ciliature. Two previously reported organisms under the name of L. helus by Dragesco (1966, variety a) and by Dragesco and Dragesco-Kernéis (1986) are discussed and are believed to be synonyms of L. sinicum. Furthermore, two isolates described by Dragesco (1960) and Ozaki and Yagiu (1943) under the name of L. helus are very likely, in our opinion, misidentifications, and might be two unknown forms. In the light of the current study, a key is presented to the seven clearly defined marine Loxophyllum species found in the coastal areas of north China Sea.

Phylogeny of some systematically uncertain urostyloids--Apokeronopsis, Metaurostylopsis, Thigmokeronopsis (Ciliophora, Stichotrichia) estimated with small subunit rRNA gene sequence information: discrepancies and agreements with morphological data.

The small subunit rRNA (SSrRNA) genes of seven species of urostyloids representing four genera were sequenced. These were: Apokeronopsis crassa, A. bergeri, Anteholosticha sp-QD-1, Metaurostylopsis sp-QD-1, M. sp-QD-2, M. sp-QD-3 and Thigmokeronopsis sp-QD-1. Gene trees were constructed in order to investigate their phylogenetic relationships. The results indicate that: (1) Apokeronopsis, Thigmokeronopsis and Metaurostylopsis form a well-supported, clearly isolated, monophyletic group; (2) Metaurostylopsis species analysed consistently group together indicating that it is a well-outlined genus; (3) the validity of the genus Apokeronopsis is supported; (4) the separation of Holosticha and Anteholosticha is supported although Anteholosticha species exhibit a high molecular diversity; (5) Pseudokeronopsis and Thigmokeronopsis, which have been considered closely related, may not share a recent common ancestor, casting doubt on the monophyly of the family Pseudokeronopsidae.

Redescription of a poorly known marine ciliate, Leptoamphisiella vermis Gruber, 1888 n. g., n. comb. (ciliophora, stichotrichia, pseudoamphisiellidae), from the Yellow Sea, China.

Leptoamphisiella vermisGruber, 1888 n. g., n. comb. (basionym Epiclintes vermisGruber, 1888) is an extraordinarily large and worm-like marine stichotrichous ciliate. Based on a population isolated recently from coastal waters of Qingdao, China, the living morphology and infraciliature are redescribed and its taxonomic position is defined. Accordingly, a new diagnosis for this species is suggested: large, marine Leptoamphisiella with a conspicuous layer of pellicular alveoli; 400-1000 microm x 40-70 microm in vivo; body band-like, highly flexible; about 40 membranelles; always three frontal and two buccal cirri; 52-80 transverse cirri (TC) extending to the posterior end of the buccal field; 57-79 left midventral and 44-62 right midventral cirri; 62-102 cirri in left and 63-91 cirri in right marginal rows (MR); 9-13 dorsal kineties that extend the full body length; about 100 macronuclear nodules and 5-13 micronuclei. The diagnosis for the new genus is as follows: vermiform Pseudoamphisiellidae with strongly contractile body, differentiated frontal, buccal, and highly developed TC; two remarkably separated midventral rows; one MR on each side of the body; frontoterminal and caudal cirri absent. Leptoamphisiella vermis n. comb. is fixed as the type species of the new genus.

Morphogenesis in the marine spirotrichous ciliate Apokeronopsis crassa (Claparède & Lachmann, 1858) n. comb. (Ciliophora: Stichotrichia), with the establishment of a new genus, Apokeronopsis n. g., and redefinition of the genus Thigmokeronopsis.

Morphogenetic events during the division of the marine spirotrichous ciliate, Apokeronopsis crassa (Claparède & Lachmann 1858) n. comb. were investigated. Compared with members of the well-known genera Thigmokeronopsis, Uroleptopsis, and Pseudokeronopsis, A. crassa has one row of buccal cirri, high number of transverse cirri, clearly separated midventral rows, lacks thigmotactic cirri and a gap in adoral zone, its undulating membranes (UMs) anlage forms one cirrus and marginal rows and dorsal kineties form apokinetally during division. All these characteristics indicate that this organism represents a new taxon at the generic level, and hence a new genus is suggested, Apokeronopsis n. g. It is defined as thus: Pseudokeronopsidae with Pseudokeronopsis-like bicorona of frontal cirri and one marginal row on each side; one row of two or more buccal cirri in ordinary position; two midventral rows distinctly separated, hence of cirri that are not in a typical zig-zag pattern; high number of transverse cirri, caudal cirri absent, and frontoterminal cirri present; thigmotactic cirri absent, many macronuclear nodules fuse into many masses as well as marginal and dorsal kineties form apokinetally during morphogenesis. At the same time, the genus ThigmokeronopsisWicklow, 1981 is redefined, and one new combination, Apokeronopsis antarctica (Petz, 1995) n. comb. is proposed. The morphogenetic events of A. crassa are characterized as follows: (1) In the proter, the adoral zone of membranelles and UMs are completely renewed by the oral primordium. The UM anlage is formed apokinetally on the dorsal wall of the buccal cavity and is hence clearly separated from the frontoventral-transverse (FVT) cirral anlagen in the proter. (2) Frontoventral-transverse cirral anlagen are generated de novo in the outermost region of the cortex to the right of the old UMs. (3) A row of buccal cirri arises from FVT cirral streak I. (4) The marginal rows and dorsal kineties originate de novo in both dividers; no caudal cirri are formed. (5) The last FVT-streak contributes two frontoterminal cirri. (6) The many macronuclear nodules fuse into many masses (about 50 segments) during division, unlike a singular or branched mass as described in other urostylids.