Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

High Prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and Other Helminth Infections among People in Khammouane Province, Lao PDR

The prevalence of liver and intestinal helminth infections, including Opisthorchis, Haplorchis, Phaneropsolus, hookworms, Enterobius, and Taenia, was surveyed in Khammouane province, Lao PDR. Fecal specimens were collected from 1,242 people (590 men and 652 women) in 3 Mekong riverside villages and were examined by the Kato-Katz thick smear technique. The overall helminth egg positive rate was 81.1%. The positive rate for small trematode eggs, including Opisthorchis viverrini, heterophyids, and lecithodendriids, was 81.1% and the positive rate for hookworms was 6.7%. To obtain adult worms, 35 people who were positive for small trematode eggs were treated with 20-30 mg/kg praziquantel and 10-15 mg/kg pyrantel pamoate, and then purged. Diarrheic stools were collected from 33 of these people and searched for helminth parasites using a stereomicroscope. Mixed infections with various helminths (Haplorchis taichui, Haplorchis yokogawai, Prosthodendrium molenkampi, Phaneropsolus bonnei, echinostomes, hookworms, Trichostrongylus spp., Trichuris trichiura, Enterobius vermicularis, and/or Taenia saginata) were found. The total number of helminth specimens collected was 20,907 (approximately 634 per person). The most common species was H. taichui, followed by P. molenkampi, O. viverrini, P. bonnei, E. vermicularis, hookworms, and Trichostrongylus spp. These results show that diverse species of intestinal nematodes, trematodes, and cestodes are infecting humans in Khammouane province, Lao PDR.     

Fishborne Trematode Metacercariae in Luang Prabang,Khammouane, and Saravane Province,Lao PDR

Fishborne trematode (FBT) metacercariae were investigated in fish from 3 Provinces of Lao PDR. Total 242 freshwater fish of 40 species were collected in local markets of Luang Prabang (59 fish of 16 species), Khammouane (81 fish of 19 species), and Saravane (97 fish of 14 species), and each of them was examined by artificial digestion method. Four species of metacercariae (Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, and Centrocestus formosanus) were detected. O. viverrini was detected in 35 fish (14.5%), and their density was 252 per infected fish (Luang Prabang, 88 metacercariae in 5 fish; Khammouane, 187 in 6 fish; Saravane, 303 in 24 fish). H. taichui was found in 102 fish (42.1%), and their density was 485 per infected fish (Luang Prabang, 260 metacercariae in 38 fish; Khammouane, 1,084 in 23 fish; Saravane, 359 in 41 fish). H. yokogawai was detected in 92 fish (38.0%), and their density was 222 per infected fish (Luang Prabang, 362 metacercariae in 17 fish; Khammouane, 126 in 20 fish; Saravane, 214 in 55 fish). Metacercariae of C. formosanus were found in 8 fish (3.3%), and their density was 3 per infected fish. In the present study, it has been confirmed that FBT metacercariae, in particular, H. taichui, H. yokogawai, and O. viverrini, are highly prevalent in fish from Luang Prabang, Khammouane, and Saravane Province, Lao PDR.     

Identification of parasite DNA in common bile duct stones by PCR and DNA sequencing

We attempted to identify parasite DNA in the biliary stones of humans via PCR and DNA sequencing. Genomic DNA was isolated from each of 15 common bile duct (CBD) stones and 5 gallbladder (GB) stones. The patients who had the CBD stones suffered from cholangitis, and the patients with GB stones showed acute cholecystitis, respectively. The 28S and 18S rDNA genes were amplified successfully from 3 and/or 1 common bile duct stone samples, and then cloned and sequenced. The 28S and 18S rDNA sequences were highly conserved among isolates. Identity of the obtained 28S D1 rDNA with that of Clonorchis sinensis was higher than 97.6%, and identity of the 18S rDNA with that of other Ascarididae was 97.9%. Almost no intra-specific variations were detected in the 28S and 18S rDNA with the exception of a few nucleotide variations, i.e., substitution and deletion. These findings suggest that C. sinensis and Ascaris lumbricoides may be related with the biliary stone formation and development.     

Fishborne Trematode Metacercariae Detected in Freshwater Fish from Vientiane Municipality and Savannakhet Province, Lao PDR

Freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR were examined by the muscle compression and artificial digestion methods to know the infection status with trematode metacercariae. In the fish from Savannakhet, 2 species of metacercariae, Opisthorchis viverrini and Haplorchis taichui, were detected. O. viverrini metacercariae were found in 6 species of fish, Puntius brevis, Hampala dispar, Esomus metallicus, Mystacoleucus marginatus, Puntioplites falcifer, and Cyclocheilichthys armatus. H. taichui metacercariae were detected in 3 species of fish, P. brevis, P. falcifer, and M. marginatus. In the fish from Vientiane, 4 species of metacercariae, O. viverrini, H. taichui, Haplorchis yokogawai, and Centrocestus formosanus, were detected. Among them, O. viverrini metacercariae were found in 7 species of fish, Onychostoma elongatum, C. armatus, H. dispar, P. brevis, Cyclocheilichthys repasson, Osteochilus hasseltii, and Hypsibarbus lagleri. The metacercariae of H. taichui were detected in 6 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, and Cirrhinus molitorella. The metacercariae of H. yokogawai were found in 9 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, O. hasseltii. C. molitorella, Hypsibarbus wetmorei, and H. lagleri. The metacercariae of C. formosanus were detected in 4 species of fish, C. repasson, P. brevis, O. hasseltii, and C. molitorella. From these results, it is confirmed that fishborne trematode metacercariae, i.e. O. viverrini, H. taichui, H. yokogawai and C. formosanus, are prevalent in various species of freshwater fish from Savannakhet Province and Vientiane Municipality, Lao PDR.     

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.     

Intestinal Helminth Infections in Feral Cats and a Raccoon Dog on Aphaedo Island, Shinan-gun, with a Special Note on Gymnophalloides seoi Infection in Cats

Four feral cats and a raccoon dog purchased from a local collector on Aphaedo Island, Shinan-gun, where human Gymnophalloides seoi infections are known to be prevalent, were examined for their intestinal helminth parasites. From 2 of 4 cats, a total of 310 adult G. seoi specimens were recovered. Other helminths detected in cats included Heterophyes nocens (1,527 specimens), Pygidiopsis summa (131), Stictodora fuscata (4), Acanthotrema felis (2), Spirometra erinacei (15), toxocarids (4), and a hookworm (1). A raccoon dog was found to be infected with a species of echinostome (55), hookworms (7), toxocarids (3), P. summa (3), and S. erinacei (1). No G. seoi was found in the raccoon dog. The results indicate that feral cats and raccoon dogs on Aphaedo are natural definitive hosts for intestinal trematodes and cestodes, including G. seoi, H. nocens, and S. erinacei. It has been first confirmed that cats, a mammalian species other than humans, play the role of a natural definitive host for G. seoi on Aphaedo Island.     

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.     

Human Infections with Spirometra decipiens Plerocercoids Identified by Morphologic and Genetic Analyses in Korea

Tapeworms of the genus Spirometra are pseudophyllidean cestodes endemic in Korea. At present, it is unclear which Spirometra species are responsible for causing human infections, and little information is available on the epidemiological profiles of Spirometra species infecting humans in Korea. Between 1979 and 2009, a total of 50 spargana from human patients and 2 adult specimens obtained from experimentally infected carnivorous animals were analyzed according to genetic and taxonomic criteria and classified as Spirometra erinaceieuropaei or Spirometra decipiens depending on the morphology. Morphologically, S. erinaceieuropaei and S. decipiens are different in that the spirally coiled uterus in S. erinaceieuropaei has 5-7 complete coils, while in S. decipiens it has only 4.5 coils. In addition, there is a 9.3% (146/1,566) sequence different between S. erinaceieuropaei and S. decipiens in the cox1 gene. Partial cox1 sequences (390 bp) from 35 Korean isolates showed 99.4% (388/390) similarity with the reference sequence of S. erinaceieuropaei from Korea (G1724; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) and an additional 15 Korean isolates revealed 99.2% (387/390) similarity with the reference sequences of S. decipiens from Korea (G1657; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679). Based on morphologic and molecular databases, the estimated population ratio of S. erinaceieuropaei to S. decipiens was 35: 15. Our results indicate that both S. erinaceieuropaei and S. decipiens found in Korea infect humans, with S. erinaceieuropaei being 2 times more prevalent than S. decipiens. This study is the first to report human sparganosis caused by S. decipiens in humans in Korea.     

Infection status of pond smelts, Hypomesus olidus, and other freshwater fishes with trematode metacercariae in 6 large lakes

In order to determine the current infection status of pond smelts, Hypomesus olidus, and other freshwater fishes with trematode metacercariae, a total of 4,861 pond smelts and 18 other freshwater fishes, collected from the Soyang and Uiam Lakes in Gangwon-do, the Unam Lake in Jeollabuk-do, the Jangseong Lake in Jeollanam-do, the Uirim-ji (lake) in Chungcheongbuk-do, and the Andong Lake in Gyeongsangbuk-do, were individually digested with 1% pepsin-HCl and examined under a dissecting microscope. In all pond smelts caught from the 6 lakes, we were unable to detect any known human infectious trematode metacercariae in Korea. However, in other freshwater fishes, such as, Squalidus japonicus coreanus (Unam Lake), and Zacco platypus (Jangseong Lake) and Opsariichthys uncirostris amurensis (Jangseong Lake), metacercariae of human-infecting trematodes, i.e., Clonorchis sinensis and Metagonimus sp. were detected, respectively.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics,and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

Gastrointestinal myiasis by larvae of Sarcophaga sp. and Oestrus sp. in Egypt: report of cases, and endoscopical and morphological studies

Four cases of gastric or intestinal myiasis are reported. The cases contain 2 males (1 child 10 years old, and 1 adult 40 years old) and 2 females (1 girl 18 years old, and 1 adult 50 years old) from Minia Governorate, Southern Egypt. Three of them, including cases no. 1, 3, and 4, were gastric myiasis, and complained of offensive hematemesis of bright red blood. Minute moving worms, larvae of the fly, were found in the vomitus. On the other hand, case no. 2 had intestinal myiasis, and complained of abdominal distention, nausea, vomiting, and diarrhea. The stool of case 2 was mixed with blood, and minute moving worms were observed in the stool. Endoscopy was performed to explore any pathological changes in the stomach of the patients. The larvae were collected and studied macroscopically, microscopically, and us-ing a scanning electron microscope (SEM) to identify their species. Three different types of larvae were identified. The larvae isolated from case 1 were diagnosed as the second stage larvae of Sarcophaga species, and the larvae isolated from case 2 were the third stage larvae of Sarcophaga species. On the other hand, the larvae isolated from cases 3 and 4 were diagnosed as the third stage larvae of Oestrus species.     

Mitochondrial DNA variation in the malaria vector Anopheles minimus across China,Thailand and Vietnam: evolutionary hypothesis,population structure and population history

The effects of Pleistocene environmental fluctuations on the distribution and diversity of organisms in Southeast Asia are much less well known than in Europe and North America. In these regions, the combination of palaeoenvironmental reconstruction and inferences about population history from genetic data has been very powerful. In Southeast Asia, mosquitoes are good candidates for the genetic approach, with the added benefit that understanding the relative contributions of historical and current processes to population structure can inform management of vector species. Genetic variation among populations of Anopheles minimus was examined using 144 mtDNA COII sequences from 23 sites in China, Thailand and Vietnam. Haplotype diversity was high, with two distinct lineages that have a sequence divergence of over 2% and exhibit different geographical distributions. We compare alternative hypotheses concerning the origin of this pattern. The observed data deviate from the expectations based on a single-panmictic population with or without growth, or a stable but spatially structured population. However, they can be readily accommodated by a model of past fragmentation into eastern and western refugia, followed by growth and range expansion. This is consistent with the palaeoenvironmental reconstructions currently available for the region.     

Ectomyelois Heinrich, 1956 in China,with descriptions of two new species and a key (Lepidoptera,Pyralidae, Phycitinae)

Only three species belonging to the genus Ectomyelois Heinrich, 1956 are recorded from China, of which two species, Ectomyelois bipectinalis sp. n. and Ectomyelois furvivena sp. n. are described as new. We discuss the status of Ectomyelois that has been treated as a junior synonym by previous authors; we treat it as a valid genus, revised status, based on characters of the venation and female genitalia. Photographs of the adults and illustrations of the genitalia are given, along with a key to the three known Chinese species.     

Redescription of Heligmosomoides neopolygyrus,Asakawa and Ohbayashi, 1986 (Nematoda: Heligmosomidae) from a Chinese Rodent,Apodemus peninsulae (Rodentia: Muridae); with comments on Heligmosomoides polygyrus polygyrus (Dujardin, 1845) and related species in China and Japan

Heligmosomoides neopolygyrus, Asakawa and Ohbayashi, 1986 (Nematoda, Heligmosomoidea) is redescribed from Apodemus peninsulae from Rangtang, Sichuan, China. A morphological review of the Heligmosomoides spp. belonging to the “polygyrus line” proposed by Asakawa (1988) is made using new characters. This enabled us to distinguish two subspecies in Mus musculus (Heligmosomoides polygyrus bakeri from Japan and H. p. polygyrus from China) and two valid species in Apodemus spp. (H. neopolygyrus from Japan [in A. peninsulae] and from China [in A. agrarius] and H. asakawae from China [in A. uralensis]). Three parasite species of A. agrarius and A. peninsulae, previously identified by Asakawa et al. (1993) as H. neopolygyrus, are considered to be Heligmosomoides incertae sedis. This is the first report of H. neopolygyrus in A. peninsulae from China.     

Highly organized but pliant active site of DNA polymerase beta: compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses

To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg(258) is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe(272), Asn(279), and Arg(283)) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg(258)) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.     

Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer

Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichiacoli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.     

Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model

Immunization of jirds with Bm-alt-2 elicited partial protection against challenge infection with the filarial parasite Brugia malayi. In this study, we initially compared the protective immune responses elicited following immunization with recombinant Bm-ALT-2 protein regimen and Bm-alt-2 DNA regimen. These studies showed that protein vaccination conferred approximately 75% protection compared to DNA vaccination that conferred only 57% protection. Analysis of the protective immune responses showed that the protein immunization promoted a Th2-biased response with an increase in IL-4, IL-5 and IgG1 responses, whereas, the DNA vaccine promoted a Th1-biased response with profound IFN-gamma and IgG2a responses. Since protein vaccination gave better results than DNA vaccination, we then wanted to evaluate whether a prime-boost vaccination that combined DNA prime and protein boost will significantly increase the protective responses induced by the protein vaccine. Our results suggest that prime-boost vaccination had no added advantage and was comparatively less effective (64% protection) than the Bm-ALT-2 protein alone vaccination. Prime boost vaccination generated mixed Th1/Th2 responses with a slightly diminished Th2 responses compared to protein vaccination. Thus, our results suggest that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the mechanism of protection appears to be largely mediated by a Th2-biased response.     

RNA targeting by DNA binding drugs: Structural,conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H-form of poly(rC)·poly(rG)

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4′,6-diamidino-2-phenylindole (DAPI) with the right handed Watson–Crick base paired A-form and the left-handed Hoogsteen base paired H^L-form of poly(rC)·poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H^L-form of poly(rC)·poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H^L-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H^L-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H^L-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA–ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.     

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults

Background

The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.

Methods

0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.

Results

Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone > 1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone < 1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.

Conclusion

Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.