Selection of reference genes for quantitative real-time PCR normalization in the coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae).

The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.

Lead contamination alters enzyme activities and microbial composition in the rhizosphere soil of the hyperaccumulator Pogonatherum crinitum.

Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator; however, the effects of Pb contamination on P. crinitum rhizosphere soil enzymatic activities and microbial composition remain largely unexplored. Thus, an indoor experiment was conducted by cultivating P. crinitum seedlings and exposing them to four Pb concentrations (0, 1,000, 2000 and 3000 mg/kg Pb). Protease, urease, acid phosphatase and invertase activities were determined using standard methods while soil bacterial composition was determined by 16 S rDNA sequencing. The results showed that rhizosphere soil acid phosphatase activity significantly increased with increasing Pb concentration, while urease activity was significantly greater in rhizosphere soil contaminated with 1000 and 2000 mg/kg than in the control. There was a clear shift in bacterial composition during phytoremediation by P. crinitum. Compared to the control, Bacteroidetes was more abundant in all Pb-contaminated soils, Actinobacteria was more abundant in 1000 mg/kg Pb-treated soil, and Firmicutes was more abundant in 3000 mg/kg Pb-treated soil. Positive correlations were observed between dominant bacterial phyla and soil enzyme activities. Metabolic pathways, such as ABC transporter, quinine reductase, and ATP-binding protein were significantly increased in rhizosphere soil bacteria with Pb contamination. In conclusion, Pb contamination differentially influenced the activities of rhizosphere soil enzymes, specifically increasing acid phosphatase and urease activities, and alters the dominance of soil bacteria through up-regulation of genes related to some metabolic pathways. The strong correlations between dominant bacterial phyla and enzymatic activities suggest synergetic effects on the growth of P. crinitum during Pb contamination.

First identification and genotyping of Enterocytozoon bieneusi in humans in Myanmar.

BACKGROUND: Intestinal pathogen infections are widespread among impoverished populations. Enterocytozoon bieneusi is the most common pathogen of intestinal microsporidian species in humans worldwide. However, no epidemiological information is available on E. bieneusi infection in humans in Myanmar. The present study comprised the first identification and genotyping of E. bieneusi in humans conducted in Myanmar. RESULTS: A total of 172 fecal specimens were collected from the Wa people (one each) in four villages of Pangsang Township of the Matman District of Shan State, Myanmar, and each participant completed a questionnaire. E. bieneusi was identified and genotyped using polymerase chain reaction (PCR) and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. The average prevalence of E. bieneusi was 8.72% (15/172), ranging from 3.85 to 13.89% by village. E. bieneusi infection was not related to any of the risk factors studied. Six genotypes were identified, comprising two known genotypes Peru6 (n = 10) and D (n = 1) and four novel genotypes (MMR23, MMR25, MMR86, and MMR87) (one each), and two people infected with genotype Peru6 were from the same family. A phylogenetic analysis based on a neighbor-joining tree of the ITS sequences of E. bieneusi indicated that all the six genotypes were clustered into group 1. CONCLUSIONS: This is the first identification and genotyping of E. bieneusi in humans in Myanmar. The observations that the two people infected with genotype Peru6 were from the same family, and that all six genotypes obtained in the present study fell into zoonotic group 1, showed the potential for anthropogenic and zoonotic transmissions. The present data argue for the importance of epidemiological control and prevention from medical sectors.

Boronic acid sensors with double recognition sites: a review.

Boronic acids reversibly and covalently bind to Lewis bases and polyols, which facilitated the development of a large number of chemical sensors to recognize carbohydrates, catecholamines, ions, hydrogen peroxide, and so on. However, as the binding mechanism of boronic acids and analytes is not very clear, it is still a challenge to discover sensors with high affinity and selectivity. In this review, boronic acid sensors with two recognition sites, including diboronic acid sensors, and monoboronic acid sensors having another group or binding moiety, are summarized. Owing to double recognition sites working synergistically, the binding affinity and selectivity of sensors can be improved significantly. This review may help researchers to sort out the binding rules and develop ideal boronic acid-based sensors.

Validation of Asia-Pacific Colorectal Screening Score for Asymptomatic Participants and Outpatients in Northwest District of China.

INTRODUCTION: Asia-Pacific Colorectal Screening (APCS) score has been implemented for colorectal cancer screening in asymptomatic cohort in many regions. However, no study has validated its efficiency in Asian outpatients with mild-self-limited gastrointestinal symptoms yet. The purpose of this study was to validate its efficiency in asymptomatic subjects and outpatients in Ningxia. METHODS: The records of 329 asymptomatic participants and 300 outpatients were collected and analyzed from database in the General Hospital of Ningxia Medical University from September 2017 to April 2018. These 2 main groups were divided into 3 tiers based on the scores calculated by the category of APCS score. The detection rates of advanced colorectal neoplasia (ACRN) were further compared according to histopathological classifications of tissues acquired during colonoscopy. RESULTS: Among the 329 participants screened in the asymptomatic cohort, 78 subjects (23.7%) were in the low-risk (LR) tier, 187 subjects (56.8%) in the moderate-risk (MR) tier, and 64 subjects (19.5%) in the high-risk (HR) tier. The percentage of ACRN in the LR, MR, and HR groups was 1.3, 8.6, and 20.3%, respectively. In the 300 outpatient cohorts, 78 patients (26%) were in the LR tier, 140 patients (46.7%) in the MR tier, and 82 patients (27.3%) in the HR tier. The detection rates of ACRN in the LR, MR, and HR groups were 0, 10, and 39%, respectively. CONCLUSION: APCS score is an effective method for ACRN screening in asymptomatic and also the outpatient subjects. Individuals with HR scores should be given priority for colonoscopy.

Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae).

Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, ß-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, ß-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

Molecular detection and genetic characterizations of Cryptosporidium spp. in farmed foxes, minks, and raccoon dogs in northeastern China.

Cryptosporidium spp. are common intestinal protozoa causing diarrhea in humans and a variety of animal species. With the recent development of fur industry, a large number of fur animals are farmed worldwide, especially in China. The existence of identical Cryptosporidium species/genotypes in humans and fur animals suggests zoonotic potential. In order to assess the presence of zoonotic Cryptosporidium species and/or genotypes in farmed fur animals, 367 fecal specimens were collected from 213 foxes, 114 minks and 40 raccoon dogs farmed in Heilongjiang, Jilin, and Liaoning provinces, northeastern China, during the period from June 2014 to October 2016. By PCR and sequencing of the partial small subunit (SSU) rRNA gene of Cryptosporidium, 20 of 367 (5.4%) animal samples were found to be infected, corresponding to 12 of 213 fox samples (5.6%) and 8 of 114 mink samples (7.0%) screened. Three Cryptosporidium species/genotypes were identified: C. canis (n = 17), C. meleagridis (n = 1) and Cryptosporidium mink genotype (n = 2). Two host-adapted C. canis types (C. canis dog genotype and C. canis fox genotype) were found. By PCR and sequencing of the partial 60 kDa glycoprotein (gp60) encoding gene, one mink genotype isolate was successfully subtyped as XcA5G1R1. The three Cryptosporidium species/genotypes identified in this study have been previously reported in humans suggesting that fur animals infected with Cryptosporidium spp. may pose a risk of zoonotic transmission of cryptosporidiosis, especially for the people working in fur animal farming and processing industry.

Prevalence of Hymenolepis nana and H. diminuta from Brown Rats (Rattus norvegicus) in Heilongjiang Province, China.

Hymenolepis nana and Hymenolepis diminuta are globally widespread zoonotic cestodes. Rodents are the main reservoir host of these cestodes. Brown rats (Rattus norvegicus) are the best known and most common rats, and usually live wherever humans live, especially in less than desirable hygiene conditions. Due to the little information of the 2 hymenolepidid species in brown rats in China, the aim of this study was to understand the prevalence and genetic characterization of H. nana and H. diminuta in brown rats in Heilongjiang Province, China. Total 114 fecal samples were collected from brown rats in Heilongjiang Province. All the samples were subjected to morphological examinations by microscopy and genetic analysis by PCR amplification of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene. In total, 6.1% (7/114) and 14.9% (17/114) of samples were positive for H. nana and H. diminuta, respectively. Among them, 7 and 3 H. nana isolates were successfully amplified and sequenced at the COX1 and ITS2 loci, respectively. No nucleotide variations were found among H. nana isolates at either of the 2 loci. Seventeen H. diminuta isolates produced 2 different COX1 sequences while 7 ITS2 sequences obtained were identical to each other. The present results of H. nana and H. diminuta infections in brown rats implied the risk of zoonotic transmission of hymenolepiasis in China. These molecular data will be helpful to deeply study intra-specific variations within Hymenolepis cestodes in the future.

Increasing rate of inflammatory bowel disease: a 12-year retrospective study in NingXia, China.

BACKGROUND: In China, the incidence of Inflammatory bowel disease (IBD) has shown a significant growth trend. Analysis of the epidemiology, clinical manifestations, diagnostic means, and treatment of IBD will further improve the clinician's understanding of IBD, improve knowledge and further enable early diagnosis and standardized therapeutic management. The purpose of this study was to analyze the clinical characteristics of IBD inpatients in General Hospital of NingXia Medical University over a 12-year period to identify trends in clinical and epidemiological features, clinical manifestations, and treatment programs. METHODS: By excluding188 patients with incomplete information or incompatible with the 2012 Guidlines cases, we retrospectively analyzed the case records of 567 inpatients with a diagnosis of IBD admitted to the General Hospital of NingXia Medical University between January 2002 and December 2014. The clinical epidemiological features, clinical manifestations, diagnostic methods, and therapeutic status were analyzed. RESULTS: Over the study period, IBD hospitalization rates in 2002 and 2014 groups was 1.96 % and 4.05 %, increased 2.07 times. Of 567 cases of IBD, 483 (85.19 %) cases were categorized as ulcerative colitis (UC) and 84 as Crohn's disease (CD) (14.81 %). Total male cases were 321 (56.61 %). Mean age of cases was 49.06 ± 14.92 years for UC and 44.84 ± 14.67 years for CD. The majority of UC was located in the colon, with a moderate level of disease activity. A combination of clinical manifestations and colonoscopy was mostly used to make a diagnosis; relatively the rate of pathological diagnosis was low, with a small proportion of patient's diagnosed based on radiology. Treatment with SASP/5ASA and steroids was applied to the majority of inpatients and 47.83 % were treated with antibiotics; in contrast, only 1.86 % cases were treated with immunosuppressive therapy. CONCLUSIONS: An increasing trend of admissions for IBD can be observed in our study; there are some differences in clinical features and treatment compared with Western countries, and further research into this is required.

Evaluation of NIN/RPN12 binding protein inhibits proliferation and growth in human renal cancer cells.

The targeted delivery of small interfering RNA (siRNA) to specific tumor tissues and tumor cells remains as one of the key challenges in the development of RNA interference as a therapeutic application. The ribosome assembly factor NIN/RPN12 binding protein (NOB1) has been suggested to be essential for processing of the 20S pre-rRNA to the mature 18S rRNA, and is also reported to participate in proteasome biogenesis. However, it is unclear whether NOB1 is involved in tumor cells growth. The aim of this study was to determine whether the suppression of lentivirus mediated NOB1 siRNA inhibits the growth of human clean cell carcinoma (ccRCC) cells, further focused on NOB1 as a possible therapeutic target for renal cell carcinoma treatment. NOB1 deletion that caused significant decline in cell proliferation was observed in both 786-O and ACHN cell lines as investigated by MTT assay. Further, the number and size of the colonies formed were also significantly reduced in the absence of NOB1. Moreover, NOB1 gene knockdown arrested the cell cycle and inhibited cell cycle-related protein expression. The Kaplan-Meier survival curves revealed that low NOB1 expression was associated with poor prognosis in ccRCC patients. Collectively, these results indicate that NOB1 plays an essential role in renal cell cancer cell proliferation, and its gene expression could be a therapeutic target.

Enterocytozoon bieneusi in Dairy Cattle in the Northeast of China: Genetic Diversity of ITS Gene and Evaluation of Zoonotic Transmission Potential.

Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans. It has been found in a wide range of animals and is considered an important zoonotic pathogen. To date, little information is available on the role that cattle play in the epidemiology of human microsporidiosis caused by E. bieneusi in China. In this study, 133 fecal specimens from dairy cattle were collected in Heilongjiang Province, China. Enterocytozoon bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA gene, with 30.1% positive. Nine ITS genotypes were identified: six known genotypes-O (n = 26), EbpA (n = 2), I (n = 2), J (n = 2), D (n = 1) and BEB4 (n = 1)-and three novel genotypes named as CC-I to CC-III (two each). Genotype O was identified in cattle for the first time. The observation of all the six known genotypes here reported previously in humans, and also the fact of all the three novel genotypes (CHN-DC1 to CHN-DC3) falling into zoonotic group 1, indicate the possibility of cattle in the transmission of E. bieneusi to humans.

Human cystic echinococcosis in Heilongjiang Province, China: a retrospective study.

BACKGROUND: Cystic echinococcosis (CE) is one of emerging zoonotic parasitic diseases throughout the world, having significant medical and economic importance in developing countries. The western and northwestern China is considered as CE endemic areas. In northeastern China's Heilongjiang Province, the increasing number of sporadic human CE cases has attracted more and more attention. The aims of the present study were to understand the clinical characteristics of human CE in the investigated area and to compare the coincidence rates of CT, ultrasound and serological test against the histopathology results among CE patients. METHODS: Hospital data of 183 human CE cases in the period from January 2004 to July 2013 were collected from the two largest hospitals in Heilongjiang Province. Clinical data were analyzed, including age, gender, occupation and living residence of CE patients and localization, size and number of CE cysts as well as the diagnosis methods of CE before operation. RESULTS: The results revealed that the incidence of CE reached a peak in the age group of 41-50 years. Among the 183 CE patients, the females were observed to have a higher percentage of CE patients (60.66%, 111/183) than males (39.34%, 72/183). The majority of CE patients were farmers, followed by workers, employees, public servants, students and so on. CE cysts were most commonly found in the livers, with a 30 cm cyst in diameter being detected. CT showed the highest coincidence rate (96.64%) for hepatic CE among the three common diagnosis methods (CT, ultrasound imagine and serological test) compared against the histopathology results. CONCLUSIONS: This is the first retrospective analysis of human CE cases in Heilongjiang Province in recent ten years. Clinical characteristics of human CE were described here. CT appeared to be the most effective diagnosis method for hepatic CE.

High prevalence of Enterocytozoon bieneusi in asymptomatic pigs and assessment of zoonotic risk at the genotype level.

Enterocytozoon bieneusi is an emerging and clinically significant enteric parasite infecting humans and animals and can cause life-threatening diarrhea in immunocompromised people. Pigs are considered to be one of the main reservoir hosts of E. bieneusi based on their high prevalence rates and zoonotic genotypes in pigs. As an opportunistic pathogen, E. bieneusi infection of pigs can be inapparent, which leads to neglect in detecting this parasite in pigs and assessing the epidemiological role of pigs in the transmission of human microsporidiosis. In the present study, 95 healthy pigs aged 2 or 3 months were randomly selected from three areas in Heilongjiang Province, China. E. bieneusi isolates were identified and genotyped based on the small-subunit (SSU) rRNA and internal transcribed spacer (ITS) regions of the rRNA gene by PCR and sequencing. A high prevalence of E. bieneusi was observed, 83.2% (79/95) at the SSU rRNA locus versus 89.5% (85/95) at the ITS locus. Ten ITS genotypes were obtained, comprising six known genotypes­EbpA (n = 30), D (n = 19), H (n = 18), O (n = 11), CS-1 (n = 1), and LW1 (n = 1)­and four novel genotypes named HLJ-I to HLJ-IV; 70.6% (60/85) of E. bieneusi genotypes were zoonotic (genotypes EbpA, D, and O). The findings of a high prevalence of E. bieneusi in pigs and a large percentage of zoonotic genotypes indicate that pigs may play a role in the transmission of E. bieneusi to humans and may become an important source of water contamination in our investigated areas.

Cryptosporidium andersoni as a novel predominant Cryptosporidium species in outpatients with diarrhea in Jiangsu Province, China.

BACKGROUND: Cryptosporidium hominis and C. parvum are usually considered to be the major pathogens responsible for human cryptosporidiosis. However, there have been few studies regarding the molecular epidemiology of Cryptosporidium in human infections in China. Here we investigated Cryptosporidium infection in patients with diarrhea, in Danyang Hospital of Jiangsu Province, China, at the genotype level. METHODS: A total of 232 stool specimens were collected from outpatients with diarrhea in Danyang Hospital of Jiangsu Province, China, from February 2012 to January 2013. Each specimen was stained from direct fecal smears and examined for Cryptosporidium using modified acid fast staining and microscopy. Moreover, genomic DNA of each fecal sample was screened for the presence of Cryptosporidium with nested PCR, which was genotyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA). RESULTS: The average infection rate of Cryptosporidium was 1.3% (3/232) by microscopy and subjected to PCR amplification of the SSU rRNA gene of Cryptosporidium, with 9.91% (23/232) being positive for Cryptosporidium with a significant peak in autumn. Based on the SSU rRNA gene, two Cryptosporidium spp. were identified, including C. andersoni (n =21) and C. hominis (n =2). Two types of C. andersoni, designated as A370 + and A370 - , were found in the SSU rRNA gene in our present study, which was 100% homologous to C. andersoni infections derived from dairy calves and goats, respectively. The clinical questionnaires showed no significant difference in age, gender and frequency of diarrhea, but duration of diarrhea was shorter for C. andersoni than that of C. hominis (mean, 2 vs. 4 days; p <0.01). CONCLUSIONS: C. andersoni is the dominant species in Danyang City of Jiangsu Province. The fact that SSU rRNA sequences of C. andersoni obtained from human stools exhibited 100% homologous to those derived from dairy calves and goats supported that C. andersoni infection might be attributable to animal origin. The difference in the duration of diarrhea of C. andersoni and C. hominis indicated that different Cryptosporidium species might cause different clinical manifestations.

Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervus elaphus): deer specificity and zoonotic potential of ITS genotypes.

As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in a variety of biomes, and the distribution of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmission of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 31.9% (29/91) was observed in deer, with 32.6% (28/86) for sika deer, and 20% (1/5) for red deer. Six ITS genotypes were identified: one known genotype BEB6 (n = 20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n = 5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD-II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans.

Molecular characterization and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in humans and domestic animals in Heilongjiang Province, China.

BACKGROUND: Cryptosporidiosis and giardiasis are significant parasitic diseases shared between humans and domestic animals. Due to the close contact between humans and domestic animals in rural areas, it is important to consider the potential transmission of zoonotic parasites from infected domestic animals to humans. This investigation aimed to determine the prevalence and molecular characteristics of Cryptosporidium spp. and Giardia duodenalis in domestic animals and villagers. METHODS: A total of 116 fecal samples from villagers and 686 fecal samples from domestic animals in Heilongjiang Province, China, were analyzed for two parasites using nested polymerase chain reaction (PCR) targeting various genetic loci and DNA sequence analysis of the PCR products. RESULTS: By sequence analysis of the SSU rRNA gene, the prevalence of Cryptosporidium in humans was 0.9% (1/116), with one species of C. parvum (n = 1) detected; among domestic animals, the prevalence was 2.6% (18/686), with five species identified: C. suis (n = 7) and C. scrofarum (n = 7) in pigs, C. meleagridis (n = 1) in chickens, C. andersoni (n = 1) in cattle, and C. canis (n = 2) in foxes. C. parvum and C. canis were further subtyped as IIdA19G1 and XXa4 on the basis of gp60 gene. Regarding G. duodenalis, based on the SSU rRNA, bg, gdh, and tpi genes, the prevalence in domestic animals was 5.1% (31/608), with three assemblages identified: A (n = 1) in pigs, D (n = 1) in foxes, and E (n = 27) in geese, cattle, pigs, ducks, and sheep, along with mixed infection of A + E (n = 1) in one pig and B + E (n = 1) in one sheep. No G. duodenalis was detected in humans (0/116). CONCLUSIONS: The present results show that no overlap of subtypes between animals and villagers was found in Cryptosporidium spp. and G. duodenalis, indicating a minor role of domestic animals in infecting humans in this population. However, the presence of zoonotic protozoa in domestic animals highlights the need for special attention to high-risk individuals during close contact with domestic animals.

Occurrence and genetic characterization of Enterocytozoon bieneusi in pet dogs in Yunnan Province, China.

Enterocytozoon bieneusi is the most common microsporidian species in humans and can affect over 200 animal species. Considering possible increasing risk of human E. bieneusi infection due to close contact with pet dogs and identification of zoonotic E. bieneusi genotypes, 589 fresh fecal specimens of pet dogs were collected from Yunnan Province, China to determine the occurrence of E. bieneusi, characterize dog-derived E. bieneusi isolates, and assess their zoonotic potential at the genotype level. Enterocytozoon bieneusi was identified and genotyped by PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. Twenty-nine specimens (4.9%) were positive. A statistical difference was observed in occurrence rates of E. bieneusi in pet dogs among 11 sampling sites by Fisher's exact test. Fifteen genotypes were identified and all of them phylogenetically belonged to zoonotic group 1, including four known genotypes (EbpC, D, Peru 8, and Henan-III) and 11 novel genotypes. Genotype Henan-III was reported in dogs for the first time. The finding of known genotypes found previously in humans and novel genotypes falling into zoonotic group 1 indicates that dogs may play a role in the transmission of E. bieneusi to humans in the investigated areas.

Title: Occurrence et caractérisation génétique d'Enterocytozoon bieneusi chez les chiens de compagnie dans la province du Yunnan, Chine. Abstract: Enterocytozoon bieneusi est l'espèce de microsporidies la plus répandue chez l'homme et peut affecter plus de 200 espèces animales. Compte tenu du risque accru possible d'infection humaine à E. bieneusi en raison d'un contact étroit avec des chiens de compagnie et de l'identification de génotypes zoonotiques d'E. bieneusi, 589 échantillons fécaux frais de chiens de compagnie ont été collectés dans la province du Yunnan, en Chine, pour déterminer la présence d'E. bieneusi, caractériser les isolats obtenus de chiens, et évaluer leur potentiel zoonotique au niveau du génotype. Enterocytozoon bieneusi a été identifié et génotypé par PCR et séquençage de la région d'espacement transcrit interne (ITS) du gène de l'ARN ribosomal (ARNr). Vingt-neuf échantillons (4,9%) étaient positifs. Une différence statistique a été observée dans les taux de présence d'E. bieneusi chez les chiens de compagnie parmi 11 sites d'échantillonnage par le test exact de Fisher. Quinze génotypes ont été identifiés et tous appartenaient phylogénétiquement au groupe zoonotique 1, dont quatre génotypes connus (EbpC, D, Peru 8 et Henan-III) et 11 nouveaux génotypes. Le génotype Henan-III est signalé pour la première fois chez le chien. La découverte de génotypes connus précédemment trouvés chez l'homme et de nouveaux génotypes appartenant au groupe zoonotique 1 indique que les chiens peuvent jouer un rôle dans la transmission d'E. bieneusi aux humains dans les zones étudiées.

Itemized determination of As, Sb, Bi, Se, and Ge in graphite sample by HG-AFS with high-temperature microwave digestion.

In this paper, we optimized a method for fast and accurate determination of five impurity elements (As, Sb, Bi, Se, and Ge) in graphite samples to overcome the shortcomings of existing methods, such as complicated equipment, cumbersome process, multiple-time preparation, separate determination, and large error in results. Graphite samples were digested with HNO3-H2SO4-HClO4-HF in a high-temperature and high-pressure microwave digestion apparatus, and the elements were extracted and determined separately by AFS (atomic fluorescence spectrometry). There is no element loss during the processing and analysis of this method. The spike recoveries (As: 90.30%-102.3%, Sb: 90.73%-110.0%, Bi: 90.00%-99.67%, Se: 93.33%-110.0%, Ge: 92.26%-104.2%) and precision (RSD%; As: 1.34%-8.96%, Sb: 2.67%-7.10%, Bi: 1.83%-4.58%, Se: 0.36%-3.25%, Ge: 4.41%-8.65%) meet the requirements of the corresponding quality specifications. The method has some advantages (such as no elemental loss, fast testing, strong element targeting, and accurate results), and thus can achieve batch determination of graphite samples. The optimized method for graphite sample and final solution preparations can be used for diverse spectrometric technologies, and that for spectrometer conditions have reference value for HG-AFS instruments.

Changes of inflammatory cytokines and neurotrophins emphasized their roles in hypoxic-ischemic brain damage.

Inflammatory cytokines and neurotrophins play crucial roles in hypoxic-ischemic brain damage (HIBD), but the expression changes of these proteins had not been systematically studied. In this article, we compared the levels of tumor necrosis factor alpha (TNF-α), intercellular adhesion molecule-1 (ICAM-1), interleukin 1beta (IL-1ß), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) in the progression of HIBD and analyzed their correlations with apoptosis. Seven-day-old pups of Sprague Dawley rats (n = 120) were randomly divided into two groups: the sham-operated (control) group and the hypoxia-ischemia (HI) group. To establish the hypoxic-ischemic encephalopathy model, the pups from the HI group were subjected to left common carotid artery ligation followed by exposure to 8% O2 and 92% N2 for 2.5 hr. Pups from both the groups were sacrificed at 6, 24, 48, 72 hr and 7 days after hypoxia. The levels of TNF-α, ICAM-1, IL-1ß, NGF, and BDNF in the brain tissues were measured by enzyme-linked immunosorbent assay. The neuronal apoptosis was examined by flow cytometry. We found that the levels of TNF-α, ICAM-1, IL-1ß, NGF, BDNF, and neuronal apoptosis rate in neonatal rats with HIBD significantly increased at 6, 24, 48, and 72 hr after hypoxia compared to the control group (p < .05) and returned back to normal by 7 days. Furthermore, neuronal apoptosis rate was positively correlated with the levels of TNF-α, ICAM-1, and IL-1ß and negatively correlated with the levels of NGF and BDNF. In neonatal rats with HIBD, the brain reaches its peak levels of damage by 24-72 hr after the injury. Inflammatory cytokines such as TNF-α, ICAM-1, and IL-1ß contribute to neuronal apoptosis induced by HIBD, whereas neurotrophins NGF and BDNF antagonize it.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.