[Clinical characteristics of 21 infertile women with non-classic 21-hydroxylase deficiency].

Objective: To investigate the clinical features of infertile women with non-classic 21-hydroxylase deficiency (21-OHD). Methods: The study enrolled 21 infertile women with non-classic 21-OHD in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from January 2009 to December 2018. The clinical presentation, endocrine hormone, glucolipid metabolism and treatment outcome were retrospectively analyzed. The diagnosis of non-classic 21-OHD was comprehensively based on clinical and hormonal characteristics. Results: Among 21 cases, the age was (29.9±2.9) years, the mean age at menarche was (13.6±2.0) years, body mass index was (22.1±2.9) kg/m2, and 38% (8/21) had oligomenorrhea. Hirsutism was diagnosed in 3 cases (14%, 3/21). Clitoromegaly was seen in 14% (3/21) and polycystic ovarian morphology was found in 33% (7/21) of the patients. The mean serum level of basal progesterone was (11.3±21.0) nmol/L, with 48% (10/21) having high basal progesterone level; after therapy by glucocorticoid, the level of progesterone was (1.9±2.0) nmol/L. Serum 17-hydroxyprogesterone concentration was (66.4±123.6) nmol/L; after therapy by glucocorticoid, it was (2.4±1.8) nmol/L. In the study increased testosterone, androstenedione and dehydroepiandrosterone sulfate were present in 62% (13/21), 52% (11/21) and 43% (9/21), respectively; and 52% (11/21) of patients manifested androgen excess and basal progesterone elevation; androgen levels decreased after therapy by glucocorticoid. The pregnancy rate was 76% (16/21). Out of 19 pregnancies, 6/19 ended in spontaneous miscarriages. Conclusions: Infertile women with non-classic 21-OHD are characterized by hyperandrogenism and basal progesterone elevation, whereas gonad axis disorder is not apparent. After no response to conventional therapy, adult infertile women with non-classic 21-OHD could achieve a desirable pregnancy outcome with proper treatment of glucocorticoid.

[Preparation of folate-targeted magnetic nanocomposites loaded with TFPI-2 plasmid and cisplatin and evaluation of its targeting and inhibitory effect on nasopharyngeal carcinoma HNE-1 cells in vitro].

OBJECTIVE: To prepare a novel folate-targeted magnetic nanocomposites loaded with tissue facor pathway inhibitor 2 (TFPI-2) and cisplatin (CDDP) and to investigate its targeting ability and anti-tumor effect on nasopharyngeal carcinoma HNE-1 cells in vitro. METHODS: The copolymer folic acid-polyethylene glycol-polyethyleneimine (FA-PEG-PEI) was synthesized through amidation reaction, and then FA-PEG-PEI/ magnetic nanoparticles-CDDP/TFPI-2 (MNP-CDDP/TFPI-2) nanocomposites was obtained by electrostatic adsorption between TFPI-2 plasmid and magnetic nanoparticles loaded with CDDP (MNP-CDDP) with vortex FA-PEG-PEI. (1)H Nuclear Magnetic Resonance ((1)H NMR ) was used to determine if FA-PEG-PEI was synthesized. The particle size, zeta potential and morphology were detected by dynamic light scattering (DLS) and transmission electron microscope (TEM). The content of Fe and CDDP was measured by phenanthroline and o-phenylenediamine (OPDA) colourimetry. Agarose gel electrophoresis was used to analyze the binding ability of FA-PEG-PEI/MNP-CDDP to TFPI-2 plasmid. Molecular targeted uptake of FA-PEG-PEI/ MNP-CDDP/TFPI-2 coupling with green fluorescent protein (GFP) in NPC cells were observed by Prussian-blue iron staining and fluorescence microscope. The levels of TFPI-2 protein expression after transfection were evaluated by Western blot. The effects of nanocomposites on HNE-1 cells proliferation and apoptosis were measured with Cell Counting Kit-8(CCK-8) and flow cytometry. RESULTS: Special peak value of FA, PEG and PEI were showed on (1)H NMR spectrogram. The mean size and zeta potential of FA-PEG-PEI/MNP-CDDP/TFPI-2 were 141.1 nm and 21.5 mV. The nanocomposites showed a good monodispersity and an insufficient size uniformity under TEM. The content of Fe and CDDP were 116.2 µg/ml and 92.88 µg/ml, respectively. Agarose gel electrophoresis showed TFPI-2 could be encapsulated completely and protected from digestion of DNA enzyme as the mass ratio of FA-PEG-PEI/ MNP-CDDP and TFPI-2 plasmid was equal or higher than 1∶1. More blue-stained magnetic granulars and green fluorescence were seen in folate receptor (FR)-positive HNE-1 cells than in FR-negative CNE-2 (P<0.05) under microscope and fluorescence microscope. The level of TFPI-2 protein expression in HNE-1cells increased significantly after transfection by FA-PEG-PEI/ MNP-CDDP/TFPI-2, compared with other control groups (FA-PEG-PEI/MNP-CDDP group and TFPI-2 group), all P<0.05. The nanocomposites inhibitory effect on HNE-1 including cell growth inhibition rate (64.00%) and apoptosis rate (49.61%) were significantly higher than that in FA-PEG-PEI/MNP group (8.19%, 9.26%), FA-PEG-PEI/TFPI-2 group (40.35%, 19.85%) and FA-PEG-PEI/MNP-CDDP group(56.15%, 36.46%)(P<0.05). CONCLUSION: FA-PEG-PEI/MNP-CDDP/TFPI-2 nanocomposites was successfully synthesized using amidation and electrostatic adsorption technology and has a good molecular targeting and inhibitory effect on FR-positive HNE-1cells in vitro.

Insulin resistance in first-trimester pregnant women with pre-pregnant glucose tolerance and history of recurrent spontaneous abortion.

Insulin resistance (IR) has been reported to play an important role in recurrent spontaneous abortion (RSA) among patients with polycystic ovary syndrome (PCOS). However, scanted materials exist regarding the independent effect of IR on RSA. The aim of this study is to investigate the status of IR in first trimester pregnant patients with normal pre-pregnant glucose tolerance and history of RSA. This two-center case-control study enrolled totally 626 first trimester pregnant women including 161 patients with a history of recurrent spontaneous abortion, who were pre-pregnantly glucose-tolerant according to oral glucose tolerance test (OGTT), and 465 women with no history of abnormal pregnancies of any kind. Clinical, biochemical and hormonal parameters were simultaneously measured in all participants. Serum beta-HCG, estradiol, progesterone, fasting plasma glucose and fasting plasma insulin levels, as well, the calculated homeostasis model assessment of insulin resistance index (HOMA-IR), fasting plasma glucose/insulin ratio(G/I) and pregnancy outcome were analyzed and compared. Serum beta-HCG and progesterone were found to be significantly lower in RSA group compared to controls. Subjects in RSA group were found to have higher HOMA-IR and lower G/I ratio than those in control group. Serum beta-HCG and progesterone were negatively correlated with HOMA-IR, and positively with G/I ratio even after adjustment for BMI. The spontaneous abortion rate within first trimester pregnancy of RSA patients was significantly higher than that in controls. In conclusion, woman with recurrent spontaneous abortion and normal pre-pregnant glucose metabolism tends to be more insulin resistant during first trimester pregnancy than healthy controls, no matter whether she has PCOS or not. Insulin resistance might be one of the direct causes that lead to recurrent abortion.

Rapid identification of Streptococcus iniae by specific PCR assay utilizing genetic markers in ITS rDNA.

The 16S-23S intergenic spacers (ITS) of ribosomal DNA from ten independent isolates of Streptococcus iniae and one reference strain ATCC29178 were sequenced, aligned and used to design a polymerase chain reaction (PCR) primer set for rapid and specific detection and identification of S. iniae. This primer set amplified a 377-bp DNA fragment specifically from S. iniae, but not from other common bacterial pathogens of fish or from non-fish pathogens. The PCR conditions were optimized to allow detection of the organism from agar, broth culture or infected fish tissue. The sensitivity of the PCR assay was established by the detection of DNA as low as 0.02 ng or as few as 10 CFU bacterial cells. The establishment of the specific PCR assay provides a useful tool for the identification and diagnosis of fish infection with S. iniae.

[Epidemiological characteristics of hemorrhagic stroke among residents in Tianjin from 1999 to 2018].

Objective: To explore the incidence characteristics of hemorrhagic stroke among residents in Tianjin from 1999 to 2018, to provide data for formulating accurate prevention and control strategies. Methods: The incidence rate of hemorrhagic stroke and its distribution in different ages, gender, urban and rural areas were analyzed by monitoring new hemorrhagic stroke cases in Tianjin from 1999 to 2018. The standardized incidence rate was calculated based on the sixth national population census in 2010. χ2 test was used to compare the incidence rate. The trend of age and trend of age were analyzed by trend χ2 test. Joinpoint software was used to analyze the change in the annual incidence rate. Results: During the study period, hemorrhagic stroke accounted for 13.08% of all stroke cases. The incidence rate of a hemorrhagic stroke in Tianjin residents was 75.38/100 000-46.70/100 000, showing a downward trend (Z=-28.63, P<0.01, APC=-1.45%). After adjustment of age and gender, the standardized rate was 57.93/100 000-27.55/100 000, still showing a downward trend (Z=-54.72, P<0.01, APC=-3.38%). The incidence rate of male and female hemorrhagic stroke decreased, and the incidence rate of males in all ages and age groups was higher than that in females (P<0.01). The age of onset is younger, and the standardized incidence rate of an urban hemorrhagic stroke in the city decreases (P<0.01), and the city descends faster. Conclusions: The characteristics of hemorrhagic stroke in Tianjin are similar to those in developed countries. The lower age group and rural population are the key groups of intervention. The study of incidence characteristics and trends is of great significance for accurate prevention and control of stroke, reducing disease and medical costs.

Newborn dopaminergic neurons are associated with the migration and differentiation of SVZ-derived neural progenitors in a 6-hydroxydopamin-injected mouse model.

The use of the existing endogenous neural progenitor cells (NPCs) in the brains of adult mammalian animals is challenging for cell therapy in treating Parkinson's disease (PD). Previous studies have indicated that there is a low level of neurogenesis in the substantia nigra (SN) of adult mice. To assess the regenerative/neurogenic capacity of NPCs following an intranigral injection of 6-hydroxydopamine (6-OHDA), the proliferation and differentiation of subventricular zone (SVZ)- and midbrain-derived NPCs were investigated, and the origin of SN newborn dopaminergic neurons was traced by using Nestin-CreERTM::ROSA26-LacZ mice and constructing a plasmid CD133-Promoter2-Cre. Our results showed that an intranigral injection of 6-OHDA-induced loss of dopaminergic neurons produced a significant increase in the SVZ-derived NPCs of the third ventricle (3V), cerebral aqueduct (Aq), and their surrounding regions. The SN newly generated dopaminergic neurons might contribute a little to an incomplete recovery of the nigrostriatal system. In addition, we found that SN newborn dopaminergic neurons were mainly derived from the migration and differentiation of the NPCs in the 3V- and Aq-SVZ and their adjacent regions. Thus, it will become an ideal strategy to treat PD by promoting the proliferation and differentiation of endogenous NPCs.

[Chronic fungal rhinosinusitis:clinical and pathologic characteristics of 40 case].

Objective:To further improve diagnosis and treatment for chronic fungal rhinosinusitis(CFRS). Method:Forty patients with CFRS were treated with nasal endoscopic surgrey(ESS). The key-points of surgery were as follows: ①for single fungal sphenoid sinusitis, fracturing middle turbinate ourward or resecting one-third of posterior middle turbinate, enlarging the osseous ostium of sinus, removing sinus fungal ball and polypoid nucosa, and flipping mucosa of ostium inside sphenoid sinus to avoid narrow of ostium;②for fungal maxillary sinusitis, retaining uncinate process or resecting tail of uncinate process, enlarging ostium of maxillary sinus according to surgical field, and resecting lesion tissue;③for frontal-ethmoidal sinus lesion, routine treatment was performed; ④iodoform gauze packing the surgical cavity after soaking it with diluted iodine solution(1∶1) for 5 min. Result:Pathological examination indicated that 39 cases were infected with fungus(38 cases with aspergillus and 1 case with mucor, respectively), including 4 cases in which lesion mucosa was invaded by fungus. Of these 39 cases,34 cured after receiving one operation, 4 cases relapsed and finally cured after receiving re-operation or outpatient treatment, 1 case had concomitant sphenoid sinus fibroblastoma with skull base invaded and was remitted after radiation therapy. In addition,1 case was diagnosed clinically as allergic fungal sinusitis although no fungus was found by culture and pathological examination, and the disease was not still controlled completely after several times of recurrence. No complication was observed for all of the cases. Conclusion:ESS is an effective treatment for CFRS, and intraoperative multi-point biopsy may be ignored easily.Confirming whether mucosa of sinus is invaded by fungus as well as pathogenic fungi types will have an important value for postoperative further treatment and reduction of recurrence. Concomitant disease should be considered for the cases with poor curative effect.

Brachial artery flow-mediated dilatation and carotid intima-media thickness in young ED patients with insulin resistance.

The evidence of a close relationship between cardiovascular disease and erectile dysfunction (ED) is well documented. The aim of this study is to investigate whether there is an early asymptomatic impairment of the peripheral vasculature in young ED patients without obvious cardiovascular disease. We studied a total of 261 ED patients (19-40 years old) and 40 age-matched healthy controls. All participants received questionnaires of cardiovascular risk factors and erectile function assessment, were subjected to lab tests of fasting blood sample, and underwent the ultrasonographic examination of brachial artery flow-mediated dilation (FMD) and carotid intima-media thickness (c-IMT). Insulin resistance (IR) was measured by the homeostasis model assessment of insulin resistance (HOMA-IR). Compared with normal human controls, FMD was significantly lower, whereas the average c-IMT was significantly greater in ED patients. An inverse correlation was found between FMD and mean c-IMT. The ED patients had significantly higher levels of fasting glucose, fasting insulin and HOMA-IR index, but showed relatively lower total testosterone and prolactin levels than the controls. Both FMD and c-IMT showed a significant correlation with International Index of Erectile Function-5 questionnaire (IIEF-5) score, age and HOMA-IR. Multivariate stepwise regression analysis demonstrated that age, HOMA-IR and IIEF-5 score were the risk factors associated with FMD and c-IMT. In conclusion, young ED patients in association with IR display diminished FMD and increased c-IMT. Furthermore, ED, HOMA-IR and age are independent predictors of the two subclinical atherosclerotic markers.

Allopregnanolone enhances the neurogenesis of midbrain dopaminergic neurons in APPswe/PSEN1 mice.

An earlier study has demonstrated that exogenous allopregnanolone (APα) can reverse the reduction of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) of 3-month-old male triple transgenic Alzheimer's disease mouse (3xTgAD). This paper is focused on further clarifying the origin of these new-born TH-positive neurons induced by exogenous APα treatment. We performed a deeper research in another AD mouse model, 4-month-old male APPswe/PSEN1 double transgenic AD mouse (2xTgAD) by measuring APα concentration and counting immunopositive neurons using enzyme-linked immunosorbent assay (ELISA) and unbiased stereology. It was found that endogenous APα level and the number of TH-positive neurons were reduced in the 2xTgAD mice, and these reductions were present prior to the appearance of ß-amyloid (Aß)-positive plaques. Furthermore, a single 20mg/kg of exogenous APα treatment prevented the decline of total neurons, TH-positive neurons and TH/bromodeoxyuridine (BrdU) double-positive neurons in the SNpc of 2xTgAD mice although the decreased intensity of TH-positive fibers was not rescued in the striatum. It was also noted that exogenous APα administration had an apparent increase in the doublecortin (DCX)-positive neurons and DCX/BrdU double-positive neurons of subventricular zone (SVZ), as well as in the percentage of neuronal nuclear antigen (NeuN)/BrdU double-positive neurons of the SNpc in the 2xTgAD mice. These findings indicate that a lower level of endogenous APα is implicated in the loss of midbrain dopaminergic neurons in the 2xTgAD mice, and exogenous APα-induced a significant increase in the new-born dopaminergic neurons might be derived from the proliferating and differentiation of neural stem niche of SVZ.

The radiation of Thaparocleidus (Monogenoidea: Dactylogyridae: Ancylodiscoidinae): phylogenetic analyses and taxonomic implications inferred from ribosomal DNA sequences.

The monophyly of Thaparocleidus Jain 1952 and its phylogenetic relationship with Pseudancylodiscoides were assessed using the D1-D2 domains of the large-subunit ribosomal deoxyribonucleic acid sequences, and taxonomic implications were discussed concerning the relative taxonomic importance of the characters of the reproductive complex and those of the haptoral scleties for future genus erection and combination within the Ancylodiscoidinae. Thaparocleidus contained divergent genetic lineages and was not resolved as a monophyletic group. The first lineage was represented by two species found on Pangasium sutchi (Pangasidae), and the second one contained 12 species all from Silurus astus (Siluridae). Three clades were observed for species from S. astus, consistent with results of morphological analyses, indicating that 12 Thaparocleidus species could be divided into three groups by morphological examinations of the male copulatory organ (MCO). Pseudancylodiscoides spp. was more closely related to Thaparocleidus spp. from S. astus, and morphologically, it was found that shapes of MCOs of these species were the same type but different from that of Thaparocleidus spp. from P. sutchi. Therefore, based on results of molecular phylogenetic analyses and morphological examinations, we propose to abolish Pseudancylodiscoides and to erect a new genus to accommodate Thaparocleidus species from S. astus and Pseudancylodiscoides spp. It is also suggested to erect another new genus to accommodate two Thaparocleidus species from the P. sutchi, whose MCO shapes are different from other Thaparocleidus species.

Some characteristics of host-parasite relationship for Cryptocaryon irritans isolated from South China.

A survey on the host range for the parasitic ciliate Cryptocaryon irritans was carried out among the major maricultured fish species in the Huizhou region of Guangdong Province in South China, and some characteristics of its host-parasite relationship were described. The survey showed that all ten investigated species of fish (representing six different families) were infected with C. irritans with similar susceptibility. In chemoattraction assays, sera and mucus collected from investigated fish strongly attracts C. irritans theronts. Sera collected from infected orange-spotted groupers and yellow spotted grunts (Plectorhynchus cinctus) could immobilize C. irritans theronts, and their immobilization titers were 1:40 and 1:6.7, respectively. The surface antigens of C. irritans were demonstrated by indirect immunofluorescence and immunostaining assays using immune orange-spotted grouper serum and a monoclonal antibody against grouper IgM.

Development of specific PCR assays for the detection of Cryptocaryon irritans.

Cryptocaryon irritans is one of the most important protozoan pathogens of marine fish, causing the "white spot" disease and posing a significant problem to marine aquaculture. In the present study, a C. irritans-specific reverse primer (S15) was designed based on the published sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of C. irritans and used together with the conserved forward primer P1 to develop a specific polymerase chain reaction (PCR) assay for direct, rapid, and specific detection of C. irritans. The specificity of these primers was tested with both closely and distantly related ciliates (Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, Ichthyophthirius multifiliis, Pseudourostyla cristata, and Paramecium caudaium), and only C. irritans was detected and no product was amplified from any other ciliates examined in this study using the specific primer set P1-S15. The specific PCR assay was able to detect as low as 45 pg of C. irritans DNA and a nested PCR assay using two primer sets (P1/NC2, P1/S15) increased the sensitivity, allowing the detection of a single C. irritans. The species-specific PCR assays should provide useful tools for the diagnosis, prevention, and molecular epidemiological investigations of C. irritans infection in marine fish.

The radiation of Haliotrema (Monogenea: Dactylogyridae: Ancyrocephalinae): molecular evidence and explanation inferred from LSU rDNA sequences.

The D1-D2 domains of LSU rDNA were used to reconstruct the phylogenetic relationships within the Ancyrocephalinae (Monogenea: Dactylogyridae) utilizing maximum-parsimony (MP), maximum-likelihood (ML), minimum evolution (ME) and neighbour-joining (NJ) methods. A total of 32 monogenean taxa were examined in the present study, including 9 Haliotrema species and 13 other species representing the Ancyrocephalinae, 4 Thaparocleidus species representing the Ancylodiscoididae, and 6 species representing the Diplectanidae which were used as multiple outgroups. All 4 analyses (i.e. MP, ML, ME and NJ) inferred the same interrelationship pattern: (Diplectanidae, (Ancylodiscoididae, Dactylogyridae)) with high bootstrap support. However, 9 Haliotrema species were dispersed to form 4 clades together with species from other genera, indicating the apparent non-monophyly of Haliotrema. Three major groups were defined based on reconstructed phylogenetic trees to explain the radiation of Haliotrema species. The morphology of the reproductive organ, particularly the male copulatory organ (MCO), was discussed to further understand the formation of each group. (1) Results of the present study indicated an intimate relationship among Metahaliotrema (2 species), Protogyrodactylus (4 species) and Haliotrema (2 of 9 species), and notably, all these species share vagina-absence. (2) Based on the present molecular analyses and the morphological characters of the MCO, we propose to transfer H. spirotubiforum and the undetermined Haliotrema sp. ZHDDb to Euryhaliotrema as new combinations. (3) We propose to erect a new genus to accommodate the Haliotrema species with horn-like shaped MCO. Taxonomic implications of the present molecular phylogenetic analyses are discussed. A wider range of taxa and more DNA markers displaying various evolutionary rates should be used to estimate phylogenetic relationships among species within the Ancyrocephalinae and Ancylodiscoididae in further studies.

Characterization of Cryptocaryon irritans isolates from marine fishes in Mainland China by ITS ribosomal DNA sequences.

Seven isolates of Cryptocaryon irritans from different host species and geographical locations in Mainland China were characterized by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) using two isolates of Ichthyophthirius multifiliis for comparative purposes. The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S sequences were amplified by polymerase chain reaction and the amplicons were sequenced directly. The ITS-1, 5.8S, and ITS-2 sequences were 129, 160, and 190 bp in length, respectively, for all seven C. irritans isolates, whereas the corresponding sequences for the two I. multifiliis isolates were 142, 153, and 194 bp, respectively. While sequence variation among the seven C. irritans isolates ranged from 0 to 1.6% in both the ITS-1 and ITS-2, and the two I. multifiliis isolates differed by 1.4% in the ITS-1 and 1.0% in the ITS-2; C. irritans differed from I. multifiliis by 57.1-60.9% in the ITS-1 and 79.4-83.0% in the ITS-2, indicating that ITS sequences provide reliable genetic markers for the identification and differentiation of the two species. Phylogenetic analysis using the sequence pairwise-distance data using the neighbor-joining method inferred that the seven C. irritans isolates from Mainland China and two other isolates (T.A and Aus.C) from other countries clustered together to show monophyly, which could be readily distinguished from the other monophyletic group all from other regions. Therefore, ITS sequence data and phylogenetic analysis provided strong support that C. irritans isolates from Mainland China represent a single species. The definition of genetic markers in the ITS rDNA provide opportunities for studying the ecology and population genetic structures of the C. irritans from Mainland China and elsewhere and is also relevant to the diagnosis and control of fish diseases they cause.

Molecular and morphological evidence indicates that Pseudorhabdosynochus lantauensis (Monogenea: Diplectanidae) represents two species.

Sequences of the first internal transcribed spacer (ITS-1) and the D1-D3 domains of the large subunit (LSU) of the ribosomal DNA (rDNA) were determined for multiple specimens of 4 operational taxonomic units (OTUs) of the monogenean, Pseudorhabdosynochus lantauensis. OTUs were defined based on their collecting localities, host and/or morphological characteristics. All P. lantauensis specimens of one group (OTUs 1 and 3) differed in their sequences of the ITS-1 and partial LSU rDNA when compared with specimens of a second group (OTUs 2 and 4) by 12% and 2%, respectively. Results of the phylogenetic analyses of the LSU rDNA sequence data showed total (100%) bootstrap support for the separation of P. lantauensis into 2 distinct clades. At least 11 of the 18 nucleotide differences in the LSU sequence between the two P. lantauensis clades were derived (i.e. autapomorphic) characters when the morphologically distinct species, P. epinepheli and P. coioidesis, were used as outgroups. Furthermore, there were several autapomorphic character states for each P. lantauensis clade. This provides sufficient evidence to reject the null hypothesis that P. lantauensis represents a single species. Morphological and morphometric differences between these two clades provided additional strong support for the separation of P. lantauensis into two species. These two parasite species were found to co-exist on one of the two species of serranid fish (i.e. Epinephelus coioides) examined in the South China Sea (Guangdong Province, China).

A new method for purification of Eimeria tenella merozoites.

A rapid and simple method for purifying second generation merozoites of Eimeria tenella was developed using a host tissue digestion fluid, containing 0.25% trypsin and 0.5% taurodeoxycholic acid, to liberate merozoites grown in chick embryos or from parasitized ceca. After filtration, the digestion procedure yielded 1.4 x 10(7) or 8.33 x 10(7) merozoites per embryo or cecum, respectively. These yields were nine-fold for embryos and three-fold for ceca in comparison to previous reports. Viability of the merozoites was normal as assessed by their ability to reinfect embryos and cell cultures. The new method has advantages in that large numbers of pure, viable merozoites can be obtained quickly and easily, and the procedures require minimal effort and supplies.

Evaluation of anticoccidial drugs in chicken embryos.

Infections of Eimeria tenella in chicken embryos were used to compare the anticoccidial activity of ten drugs. The minimal inhibitory concentration (MIC) and minimal toxic concentration (MTC) were affected by the time of inoculation into the embryos and by the chemical nature of the compounds. Some compounds (nicarbazin, amprolium) had no effect on the development of coccidia when they were injected into embryos after the day of infection. Drugs that act early in the life cycle of coccidia (robenidine, clopidol, decoquinate, diclazuril, halofuginone, monensin, salinomycin, and lasalocid) were active at 5-125 micrograms/embryo when they were injected on the day of infection. The ionophores and halofuginone were highly toxic to embryos; most synthetic compounds were nontoxic. The incubation of merozoites in drug suspensions prior to the infection of embryos did not result in embryo toxicity, but the resultant MICs were much higher than those obtained when drugs were injected directly into the embryos. Several products were essentially inactive. Neither nicarbazin nor amprolium prevented oocyst formation. The widely divergent endpoints for the MIC and MTC of anticoccidials in embryos seriously limits the application of this technique as a screen for anticoccidial drugs.

Resonance Raman spectroscopy of bilirubins: band assignments and application to bilirubin/lipid complexation.

Resonance Raman spectra of bilirubins IX alpha, III alpha, and XIII alpha and mesobilirubin XIII alpha in alkaline aqueous and chloroform solutions are reported. Partial band assignments of bilirubin IX alpha are proposed. The model compounds confirm assignments of bands of the Raman spectrum of bilirubin IX alpha to each of the two different pyrromethenones. Resonance Raman spectra of mesobilirubin IV alpha, vinylneoxanthobilirubinic acid, and vinylisoneoxanthobilirubinic acid in alkaline aqueous solution and of the tetra-n-butylammonium salt of bilirubin IX alpha are used to define markers for the presence or absence of internal hydrogen bonds. Interaction of bilirubin dianion and sphingomyelin liposomes is studied. The Raman evidence suggests that in the bilirubin dianion/liposome complex the intramolecular hydrogen bonds between the propionate groups and the lactam NH/CO are ruptured. It is proposed that in the complex the bilirubin propionates form ion pairs with the quaternary ammonium ion of the choline moiety of sphingomyelin.