Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds

Involvement of Host Cell Integrin α2 in Cryptosporidium parvum Infection

Cryptosporidium parvum is an opportunistic pathogen in AIDS patients. It is an intracellular but extracytoplasmic parasite residing in a host cell-derived parasitophorous vacuole. It is still poorly understood how this parasite interacts with host cells. We observed that expression of the integrin α2 (ITGA2) gene in host cells was significantly upregulated upon C. parvum infection, and a higher level of ITGA2 protein was present in the parasite infection sites. The infection could be reduced by the treatment of antibodies against ITGA2 and integrin β1 (ITGB1) subunits, as well as by type I collagen (an integrin α2β1 ligand). We also generated stable knockdown of ITGA2 gene expression in HCT-8 cells and observed consistent reduction of parasite infection in these knockdown cells. Collectively, our evidence indicates that host cell ITGA2 might be involved in interacting with Cryptosporidium during infection, probably acting as part of the regulatory elements upstream of the reported recruiting and reorganization of F actin at the infection sites

Transcriptome analysis reveals unique metabolic features in the Cryptosporidium parvum Oocysts associated with environmental survival and stresses

BACKGROUND: Cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. Little is known on the metabolic dynamics of the parasite, and study is hampered by the lack of molecular and genetic tools. Here we report the development of the first Agilent microarray for C. parvum (CpArray15K) that covers all predicted ORFs in the parasite genome. Global transcriptome analysis using CpArray15K coupled with real-time qRT-PCR uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite. RESULTS: Oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. The proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. Energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase (LDH) gene. We also studied parasite responses to UV-irradiation, and observed complex and dynamic regulations of gene expression. Notable changes included increased transcript levels of genes involved in DNA repair and intracellular trafficking. Among the stress-related genes, TCP-1 family members and some thioredoxin-associated genes appear to play more important roles in the recovery of UV-induced damages in the oocysts. Our observations also suggest that UV irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking. CONCLUSIONS: CpArray15K is the first microarray chip developed for C. parvum, which provides the Cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. CpArray15K has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to UV-irradiation. These observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as UV irradiation

Proteomic analysis of spinal cord tissue in a rat model of cancer-induced bone pain

BackgroundCancer-induced bone pain (CIBP) is a moderate to severe pain and seriously affects patients’ quality of life. Spinal cord plays critical roles in pain generation and maintenance. Identifying differentially expressed proteins (DEPs) in spinal cord is essential to elucidate the mechanisms of cancer pain.MethodsCIBP rat model was established by the intratibial inoculation of MRMT-1 cells. Positron emission tomography (PET) scan and transmission electron microscopy (TEM) were used to measure the stats of spinal cord in rats. Label free Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) were used to analyze the whole proteins from the lumbar spinal cord. Differentially expressed proteins (DEPs) were performed using Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, and verified using Western blot and immunofluorescence assay.ResultsIn the current study, CIBP rats exhibited bone damage, spontaneous pain, mechanical hyperalgesia, and impaired motor ability. In spinal cord, an hypermetabolism and functional abnormality were revealed on CIBP rats. An increase of synaptic vesicles density in active zone and a disruption of mitochondrial structure in spinal cord of CIBP rats were observed. Meanwhile, 422 DEPs, consisting of 167 up-regulated and 255 down-regulated proteins, were identified among total 1539 proteins. GO enrichment analysis indicated that the DEPs were mainly involved in catabolic process, synaptic function, and enzymic activity. KEGG pathway enrichment analysis indicated a series of pathways, including nervous system disease, hormonal signaling pathways and amino acid metabolism, were involved. Expression change of synaptic and mitochondrial related protein, such as complexin 1 (CPLX1), synaptosomal-associated protein 25 (SNAP25), synaptotagmin 1 (SYT1), aldehyde dehydrogenase isoform 1B1 (ALDH1B1), Glycine amidinotransferase (GATM) and NADH:ubiquinone oxidoreductase subunit A11 (NDUFA11), were further validated using immunofluorescence and Western blot analysis.ConclusionThis study provides valuable information for understanding the mechanisms of CIBP, and supplies potential therapeutic targets for cancer pain

Impacts of the Degraded Alpine Swamp Meadow on Tensile Strength of Riverbank: A Case Study of the Upper Yellow River

In the meandering riverbank of the Upper Yellow River (UYR), the native alpine swamp meadow (AS) has continuously degenerated into an alpine meadow (AM) due to climate change and intensified grazing. Its implication on river morphology is still not well known. This study examined this effect by in situ measurings of (1) physical properties of roots and their distribution in the soil-root mixture of the upper bank layer, and (2) the tensile strength in terms of excavating tests for triggering cantilever collapses of AS and AM riverbanks. The results showed that the root number in AS was significantly greater than that in AM, though the root distribution in both was similar. Also, the average tensile strength of individual roots in AS was 31,310 kPa, while that in AM was only 16,155 kPa. For the soil-root mixture, it decreased from 67.39 to 21.96 kPa. The weakened mechanical property was mainly ascribed to the lessened root number and the simpler root structure in the soil-root mixture of AM that reduces its ability to resist the external force. These findings confirmed that healthy AS can enhance bank stability and delay the development of tensile cracks in the riverbank of the meandering rivers in the UYR

Gene discovery, evolutionary affinity and molecular detection of Oxyspirura petrowi, an eye worm parasite of game birds

BACKGROUND: Oxyspirura petrowi appears to be emerging as a nematode parasite that could negatively impact Northern Bobwhite quail individuals and populations within Texas and other regions of the United States. Despite this eye worm's potential importance in the conservation of wild quail, little is known about the general biology and genome composition of O. petrowi. To fill the knowledge gap, we performed a small scale random genome sequence survey, sequenced its 18S rRNA and the intergenic region between the 18S and 28S rRNA genes, studied its phylogenetic affinity, and developed a PCR protocol for the detection of this eye worm. RESULTS: We have generated ~240 kb of genome sequence data derived from 348 clones by a random genome survey of an O. petrowi genomic library. The eye worm genome is AT-rich (i.e., 62.2% AT-content), and contains a high number of microsatellite sequences. The discovered genes encode a wide-range of proteins including hypothetical proteins, enzymes, nematode-specific proteins. Phylogenetic analysis based on 18S rRNA sequences indicate that the Spiruroidea is paraphyletic, in which Oxyspirura and its closely related species are sisters to the filarial nematodes. We have also developed a PCR protocol based on the ITS2 sequence that allows sensitive and specific detection of eye worm DNA in feces. Using this newly developed protocol, we have determined that ~28% to 33% of the fecal samples collected from Northern Bobwhites and Scaled Quail in Texas in the spring of 2013 are O. petrowi positive. CONCLUSIONS: The O. petrowi genome is rich in microsatellite sequences that may be used in future genotyping and molecular fingerprinting analysis. This eye worm is evolutionarily close to the filarial nematodes, implying that therapeutic strategies for filariasis such as Loa loa would be referential in developing treatments for the Thelazoidea parasites. Our qPCR-based survey has confirmed that O. petrowi infection is of potential concern to quail managers in Texas

National incidence of traumatic fractures in China: a retrospective survey of 512 187 individuals

Background Traumatic fractures place a substantial burden on health-care systems worldwide. Although detailed information about incidence, distribution, and risk factors for traumatic fractures is vital for planning and prevention, in China, national data are unavailable. We aimed to do an up-to-date national survey on the population-weighted incidence of traumatic fractures in China. Methods The China National Fracture Study (CNFS) was a retrospective epidemiological study that recruited a nationally representative sample from eight provinces, 24 urban cities, and 24 rural counties in China using stratified random sampling and the probability proportional to size method. All eligible household members who had lived in their current residence for 6 months or longer were personally interviewed by trained research teams about traumatic fractures of the trunk, arms, or legs (not including the skull, sternum, and ribs) that had occurred in 2014. Telephone surveys were used for participants who were non-contactable after repeated visits. Fracture cases were verified by clinical records, medical history, and radiographs by orthopaedic surgeons and radiologists. We estimated incidence rates for traumatic fractures for the overall population and for subgroups by age and sex, as well as by demographic factors such as ethnic origin, occupation, geographical region, and residency category. We also studied potential associations between fractures and various factors of interest, such as age, ethnic origin, education, smoking, alcohol drinking, sleep time per day, and history of previous fracture. Data were weighted during statistical analysis to ascertain the national incidence rate. This study is registered with the Chinese Clinical Trial Registry, number ChiCTR-EPR-15005878. Findings Between Jan 19, 2015, and May 16, 2015, 535 836 individuals were selected and invited to participate in the study. Questionnaires from 23 649 (4%) individuals were excluded due to missing items, insufficient responses, or logical errors. Following exclusions, 512 187 (96%) individuals participated in the CNFS, consisting of 259 649 (51%) boys and men and 252 538 (49%) girls and women. Of these individuals, 1763 individuals had experienced traumatic fractures during 2014 (n=1833). The population-weighted incidence rate of traumatic fractures of the trunk, arms, or legs was 3·21 (95% CI 2·83–3·59) per 1000 population in 2014 (3·65, 3·12–4·18 in men and 2·75, 2·46–3·04 in women). For all ages, sleeping less than 7 h per day was identified as a risk factor for traumatic fractures. We identified previous fracture history as a risk factor for adults aged 15 years and older. Alcohol consumption incurred a risk effect for men aged 15 years and older and women aged 15–64 years. Interpretation Our results provide detailed information about fracture incidence, distribution, and risk factors, which can now be used as an up-to-date clinical evidence base for national health-care planning and preventive efforts in China and elsewhere. Specific public health policies that focus on decreasing alcohol consumption, prohibiting drunk driving, promoting smoking cessation, and encouraging individuals to obtain sufficient sleep and maintain a healthy bodyweight should be urgently implemented to help reduce the risk of traumatic fractures

Transcriptome Analysis in Chicken Cecal Epithelia upon Infection by Eimeria tenella In Vivo

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chickens. However, our understanding on how chickens respond to coccidian infection is highly limited at both molecular and cellular levels. The present study employed the Affymetrix chicken genome array and performed transcriptome analysis on chicken cecal epithelia in response to infection for 4.5 days in vivo by the cecal-specific species E. tenella. By Significance Analysis of Microarrays (SAM), we have identified 7,099 probe sets with q-values at <0.05, in which 4,033 and 3,066 genes were found to be up- or down-regulated in response to parasite infection. The reliability of the microarray data were validated by real-time qRT-PCR of 20 genes with varied fold changes in expression (i.e., correlation coefficient between microarray and qRT-PCR datasets: R (2) = 0.8773, p<0.0001). Gene ontology analysis, KEGG pathway mapping and manual annotations of regulated genes indicated that up-regulated genes were mainly involved in immunity/defense, responses to various stimuli, apoptosis/cell death and differentiation, signal transduction and extracellular matrix (ECM), whereas down-regulated genes were mainly encoding general metabolic enzymes, membrane components, and some transporters. Chickens mustered complex cecal eipthelia molecular and immunological responses in response to E. tenella infection, which included pathways involved in cytokine production and interactions, natural killer cell mediated cytotoxicity, and intestinal IgA production. In response to the pathogenesis and damage caused by infection, chicken cecal epithelia reduced general metabolism, DNA replication and repair, protein degradation, and mitochondrial functions

Streptococcal Toxic Shock Syndrome Caused by Streptococcus suis Serotype 2

BACKGROUND: Streptococcus suis serotype 2 ( S. suis 2, SS2) is a major zoonotic pathogen that causes only sporadic cases of meningitis and sepsis in humans. Most if not all cases of Streptococcal toxic shock syndrome (STSS) that have been well-documented to date were associated with the non-SS2 group A streptococcus (GAS). However, a recent large-scale outbreak of SS2 in Sichuan Province, China, appeared to be caused by more invasive deep-tissue infection with STSS, characterized by acute high fever, vascular collapse, hypotension, shock, and multiple organ failure. METHODS AND FINDINGS: We investigated this outbreak of SS2 infections in both human and pigs, which took place from July to August, 2005, through clinical observation and laboratory experiments. Clinical and pathological characterization of the human patients revealed the hallmarks of typical STSS, which to date had only been associated with GAS infection. Retrospectively, we found that this outbreak was very similar to an earlier outbreak in Jiangsu Province, China, in 1998. We isolated and analyzed 37 bacterial strains from human specimens and eight from pig specimens of the recent outbreak, as well as three human isolates and two pig isolates from the 1998 outbreak we had kept in our laboratory. The bacterial isolates were examined using light microscopy observation, pig infection experiments, multiplex-PCR assay, as well as restriction fragment length polymorphisms (RFLP) and multiple sequence alignment analyses. Multiple lines of evidence confirmed that highly virulent strains of SS2 were the causative agents of both outbreaks. CONCLUSIONS: We report, to our knowledge for the first time, two outbreaks of STSS caused by SS2, a non-GAS streptococcus. The 2005 outbreak was associated with 38 deaths out of 204 documented human cases; the 1998 outbreak with 14 deaths out of 25 reported human cases. Most of the fatal cases were characterized by STSS; some of them by meningitis or severe septicemia. The molecular mechanisms underlying these human STSS outbreaks in human beings remain unclear and an objective for further study