AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl trans...AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.展开更多
BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,...BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.展开更多
Rhizobia, crucial for nitrogen fixation in leguminous plants, play a vital role in soybean cultivation. This study, conducted in Mexico, a major soybean importer, aimed to identify bacteria from nodules of five soybea...Rhizobia, crucial for nitrogen fixation in leguminous plants, play a vital role in soybean cultivation. This study, conducted in Mexico, a major soybean importer, aimed to identify bacteria from nodules of five soybean varieties in high-production regions. Multilocus sequence analysis (MLSA) was employed for enhanced species resolution. The study identified six Bradyrhizobium species: Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 6, Bradyrhizobium elkanii USDA 76, Bradyrhizobium neotropicale, Bradyrhizobium lablabi, and Bradyrhizobium icense. Bradyrhizobium japonicum USDA 110 predominated in the soils, displaying symbiotic preference for the Huasteca 400 variety. However, phylogenetic analysis didn't reveal a clear association between strains, soil, and soybean variety. This research sheds light on the diversity of rhizobia in Mexican soybean cultivation, contributing to the understanding of symbiotic relationships in soybean production systems.展开更多
BACKGROUND Sleep breathing,one of the basic human needs,is a physiological need that affects cardiac functions,body temperature,daily vitality,muscle tone,hormone secretion,blood pressure,and many more.In the internat...BACKGROUND Sleep breathing,one of the basic human needs,is a physiological need that affects cardiac functions,body temperature,daily vitality,muscle tone,hormone secretion,blood pressure,and many more.In the international literature,studies reported that patients have had sleep problems in the hospital since the 1990s,but no measurement tool has been developed to determine the causes of hospitalacquired insomnia in individuals.These findings suggest that sleep remains in the background compared to activities such as nutrition and breathing.Although patients generally experience hospital-acquired sleep problems,there is no measurement tool to determine hospital-acquired sleep problems.These features show the originality of the research.AIM To develop a measurement tool to determine the sleep problems experienced by patients in the hospital.METHODS A personal information form,hospital-acquired insomnia scale(HAIS),and insomnia severity index(ISI)were used to collect research data.The study population consisted of patients hospitalized in the internal and surgical clinics of a research hospital in Turkey between December 2021 and March 2022.The sample consisted of 64 patients in the pilot application stage and 223 patients in the main application stage.Exploratory factor analysis and confirmatory factor analysis(CFA)analyses were performed using the SPSS 20 package program and the analysis of moment structure(AMOS)package program.Equivalent forms method used.RESULTS The HAIS consisted of 18 items and 5 subscales.The Cronbach alpha values of the subscales ranged between 0.672 and 0.842 and the Cronbach alpha value of the overall scale was 0.783.The scale explained 58.269%of the total variance.The items that constitute the factors were examined in terms of content integrity and named as physical environmental,psychological,safety,socioeconomic,and nutritional factors.CFA analysis of the 5-factor structure was performed in the AMOS package program.The fit indices of the obtained structure were examined.It was determined that the values obtained from the fit indices were sufficient.A significant correlation was determined between the HAIS and the ISI,which was used for the equivalent form method.CONCLUSION The HAIS is a valid and reliable measurement tool for determining patients’level of hospitalacquired insomnia.It is recommended to use this measurement tool to determine the insomnia problems of patients and to adapt it in other countries.展开更多
To screen the stable expression genes related to the stress （strong light, dehydration and temperature shock） we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test ...To screen the stable expression genes related to the stress （strong light, dehydration and temperature shock） we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y（II） and Fv/Fm were significantly affected when stress was imposed on the thalli of Porphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress （at salinity 90） on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-la showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.展开更多
Background： Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this stud...Background： Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results： Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were： beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A （cyclophilin A）, ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein--zeta polypeptide The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were （most to least stable） succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, sucdnate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions： BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.展开更多
The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can ...The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.展开更多
Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without val...Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.展开更多
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the...Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.展开更多
Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(...Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill)stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.展开更多
Bacillus coagulans can help ameliorate or prevent gastrointestinal diseases, but the genetic relationships among B. coagulans isolates are not well studied. Multilocus sequence typing analysis was conducted on 57 isol...Bacillus coagulans can help ameliorate or prevent gastrointestinal diseases, but the genetic relationships among B. coagulans isolates are not well studied. Multilocus sequence typing analysis was conducted on 57 isolates of B. coagulans from 22 provinces or autonomous regions in China. B. coagulans isolates were highly diverse and a total of 33(sequence typings) STs were found. These isolates had a weak clonal population structure and strong indications of intraspecies recombination. The evolution direction of B. coagulans was not correlated with geography or isolation source. Fifteen strains were selected for further analysis based on proximity relationships from the phylogenetic tree. Five isolates(B. coagulans-1, B. coagulans-10, B. coagulans-39, B. coagulans-70 and B. coagulans-71) with good spore-forming ability relative to the rest of the isolates were evaluated for constipation relief. B. coagulans-39 significantly relieved constipation symptoms in mice by regulating intestinal flora, increasing the production of short-chain fatty acids and restoring the level of gastrointestinal regulatory peptides. Comparative genomic analysis showed the beneficial effects of B. coagulans-39 might be associated with specific functional genes that are involved in the utilization of various carbohydrates as primary substrates and short-chain fatty acid production.展开更多
Purpose: The aim of this study was to isolate and identify potentially pathogenic airborne fungi from Hospital das Clínicas Samuel Libanio in the city of Pouso Alegre-MG and evaluate their susceptibility to natur...Purpose: The aim of this study was to isolate and identify potentially pathogenic airborne fungi from Hospital das Clínicas Samuel Libanio in the city of Pouso Alegre-MG and evaluate their susceptibility to natural and industrial products. Methods: The air collection was performed by passive sedimentation in the morning during the autumn and winter seasons. Petri dishes were open as the location of air conditioning. The isolates were subjected to pathogenicity test. The identification of the fungi was performed according to the macroscopic evaluation and micromorphology. Potentially pathogenic isolates were susceptibility tested by disc diffusion method. The agents used were insecticides and industrial cleaning products and essential oils of citronella plants, lemon grass, eucalyptus and Melaleuca extracted by steam distillation method. Results: We obtained a total of 356 fungal isolates. The inside door environments were 126 (35.39%) and the outside environments were 230 (64.6%) isolates. The 22 (6.18%) isolates from the inside and 25 (6.18%) outside the hospital showed pathogenic potential. This isolates were identified as Acremonium spp., A. niger, A. terreus, A. versicolor, Curvularia sp., Penicillium sp. and Scopulariopsis sp. Susceptibility testing it was observed that most of the isolates were susceptible to the principle product containing sodium hypochlorite. Citronella oil showed enormous potential inhibition against all isolates. Already lemon grass oil was effective only against isolates of Penicillium spp. Conclusions: All genres identified are significant allergens, which can cause respiratory disease in both immunocompromised individuals such as asthmatics and people with any immune deficit. The monitoring of environmental sources should be performed, especially in special areas with immunocompromised patients. Despite efforts to try to reduce fungal infections hospital there are still flaws in the strategies employed.展开更多
Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an en- dogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in ...Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an en- dogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in inflam- matory conditions;however information about the stability and expression of HKGs in chronic inflammatory joint dis- ease such as rheumatoid arthritis (RA) is scarce. The expressional stability of 10 commonly used HKGs was analyzed in the neuronal (spinal cord, dorsal root ganglia) and in the musculoskeletal tissues (tendon, muscle, epiphysis, capsule, periosteum and ankle joint) using RT-qPCR in the rat model of RA. In individual tissues, suitable HKGs were selected by | △Ct| (│Ct control-Ct arthritis│) and further analyzed by using software programs;geNorm and normfinder. We found hypoxanthine-guanine phosphoribosyl tranferase (HPRT) as the most stable gene except ankle joint while glyce-raldehyde-3-phosphate dehydrogenase (GAPDH) was found as the least stable gene in musculoskeletal tissues. In in-flamed ankle joint where no reference gene was found to be stably expressed, an inflammatory cell marker CD3 was used to normalize peptidylprolyl isomerase B (PPIB), the most homogenous HKG identified among the 10 HKGs. The normalized PPIB was then used to analyze the gene expression of neurokinin 1 (NK1), receptor of substance P, a potent pro-inflammatory mediator. We observed a 3.5 fold increase (p = 0.009) in NK1 expression in inflamed ankle joint compared to control. Our results indicate that reference genes stability should be evaluated before using them as refer- ence during inflammatory conditions. In tissues with intense inflammatory cell infiltration, an inflammatory cell marker should be used to normalize the selected reference gene to avoid erroneous results.展开更多
Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosor...Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits;however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.展开更多
The effectiveness of celebrity endorsements has been a topic that has interested many advertising researchers. It has been long debated on which advertising method is most effective. The purpose of this study was to c...The effectiveness of celebrity endorsements has been a topic that has interested many advertising researchers. It has been long debated on which advertising method is most effective. The purpose of this study was to conclude whether or not advertising agencies and social cause organizations are obtaining the results of these studies and using them in magazine advertisement campaigns and which one of these advertisements is more prevalent in magazines. Advertisements were examined from two American magazines, W Magazine and Good Housekeeping. The results of the study showed that celebrtiy endoresments have been featured in magazine advertisements more in recent years that products are endorsed by celebrities more than social causes and a majority of these endoresments are for products that affect a consumer＇s self-image.展开更多
When he left his uncle in Nor-bu Linka, the Dalai Lama’s summer palace in Lhasa, on the evening of March 10,1959, Qiampa Getsang never expected one day he would do the same job as his late uncle did to manage the pal...When he left his uncle in Nor-bu Linka, the Dalai Lama’s summer palace in Lhasa, on the evening of March 10,1959, Qiampa Getsang never expected one day he would do the same job as his late uncle did to manage the palace of the Dalai Lama.展开更多
The ZDPS-1A pico-satellite designed in Zhejiang University with a mass of 3.5 kg and a power consumption of less than 3.5 W is the smallest satellite in China up to now. The housekeeping system (HKS) is the core part ...The ZDPS-1A pico-satellite designed in Zhejiang University with a mass of 3.5 kg and a power consumption of less than 3.5 W is the smallest satellite in China up to now. The housekeeping system (HKS) is the core part of ZDPS-1A. The reliability of HKS has an important influence on the safety of the satellite. Traditional fault-tolerant methods do not apply to ZDPS-1A due to such pico-satellite characteristics as light weight, compactness in size, energy saving, and high integration. This paper deals with a highly-reliable, low-cost design for HKS using industrial devices. The reliable strategies of HKS include a dual modular redundancy scheme, CPU warm backup, a static triple modular redundancy scheme, and two-level watchdogs. Recursive experiments, special tests, and environmental tests show that this system meets the design target. This design has already been applied to ZDPS-1A, which was launched to execute in-orbit tasks on Sept. 22, 2010. To date, the satellite has been in a proper state for more than 15 months.展开更多
Internal standards are critical for quantitative RNA analyses.Housekeeping genes are often used as internal standards with the assumption that their express-ion levels remain relatively constant in different experi-me...Internal standards are critical for quantitative RNA analyses.Housekeeping genes are often used as internal standards with the assumption that their express-ion levels remain relatively constant in different experi-mental conditions.In this study,four commonly used housekeeping genes,Glyceraldehyde-3-phosphate dehy-drogenase(GAPDH),β-actin,28S rRNA and 18S rRNA were selected to test whether this assumption is tenable under hypoxic conditions.We tested the RNA expression level of these four genes in different hypoxic conditions.Rats subjected to acute hypoxia for 2 hours were used for tissue detection.Primary cultured neural stem cells from E13 fetal rats were treated with 3%O_(2) or 10%O_(2) for 24 hours for in vitro experiments.In both experiments,expression levels of 28S rRNA and 18S rRNA were constant,independent of hypoxia types.However,expression levels of GAPDH and b-actin were all changed in all kinds of hypoxic conditions.In particu-lar,the mRNA expression level of GAPDH was increased by 43.4%under 3%O_(2) hypoxic conditions.These results suggest that 18S rRNA and 28S rRNA are reliable internal controls for comparative analyses of transcrip-tion under hypoxia.GAPDH appears particularly unfa-vorable for this purpose in hypoxic conditions.展开更多
Blood-based mieroRNA （miRNA） signatures as biomarkers have been reported for various pathologies, including cancer, neurological disorders, cardiovascular diseases, and also infections. The regulatory mechanism behi...Blood-based mieroRNA （miRNA） signatures as biomarkers have been reported for various pathologies, including cancer, neurological disorders, cardiovascular diseases, and also infections. The regulatory mechanism behind respective miRNA patterns is only partially understood. Moreover, ＂preserved＂ miRNAs, i.e., miRNAs that are not dysregulated in any disease, and their biological impact have been explored to a very limited extent. We set out to systematically determine their role in regulatory networks by defining groups of highly-dysregulated miRNAs that contribute to a disease signature as opposed to preserved housekeeping miRNAs. We further determined preferential targets and pathways of both dysregulated and preserved miRNAs by computing multi-layer networks, which were compared between housekeeping and dysregulated miRNAs. Of 848 miRNAs examined across 1049 blood samples, 8 potential housekeepers showed very limited expression variations, while 20 miRNAs showed highly-dysregulated expression throughout the investigated blood samples. Our approach provides important insights into miRNAs and their role in regulatory networks. The methodology can be applied to systematically investigate the differences in target genes and pathways of arbitrary miRNA sets.展开更多
INTRODUCTION The new Glen Allen High School was built in 2010 by Henrico County Public Schools(HCPS)to serve the growing student population in the Glen Allen area of Henrico County,Virginia.As a suburban area northwes...INTRODUCTION The new Glen Allen High School was built in 2010 by Henrico County Public Schools(HCPS)to serve the growing student population in the Glen Allen area of Henrico County,Virginia.As a suburban area northwest of the City of Richmond,Glen Allen’s population doubled between 1980 and 2000,and grew an additional 18%between 2000 and 2010.The new high school is one of nine in the county and can accommodate approximately 1,800 students and 150 staff.The school facility is located on a 95-acre parcel of land that was previously wooded and undeveloped.展开更多
基金Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education(MoHE)600-IRMI/FRGS 5/3(101/2019).
文摘AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.
基金Supported by São Paulo Research Foundation(FAPESP),No.2010/08918-9 and 2020/11564-6the KBSP Young Investigator Fellowship,No.2011/00204-0+2 种基金the DBF Fellowship,No.2019/27492-7the LMG Fellowship,No.2014/01395-1the CFB Fellowship,No.2014/14278-3.
文摘BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.
文摘Rhizobia, crucial for nitrogen fixation in leguminous plants, play a vital role in soybean cultivation. This study, conducted in Mexico, a major soybean importer, aimed to identify bacteria from nodules of five soybean varieties in high-production regions. Multilocus sequence analysis (MLSA) was employed for enhanced species resolution. The study identified six Bradyrhizobium species: Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 6, Bradyrhizobium elkanii USDA 76, Bradyrhizobium neotropicale, Bradyrhizobium lablabi, and Bradyrhizobium icense. Bradyrhizobium japonicum USDA 110 predominated in the soils, displaying symbiotic preference for the Huasteca 400 variety. However, phylogenetic analysis didn't reveal a clear association between strains, soil, and soybean variety. This research sheds light on the diversity of rhizobia in Mexican soybean cultivation, contributing to the understanding of symbiotic relationships in soybean production systems.
文摘BACKGROUND Sleep breathing,one of the basic human needs,is a physiological need that affects cardiac functions,body temperature,daily vitality,muscle tone,hormone secretion,blood pressure,and many more.In the international literature,studies reported that patients have had sleep problems in the hospital since the 1990s,but no measurement tool has been developed to determine the causes of hospitalacquired insomnia in individuals.These findings suggest that sleep remains in the background compared to activities such as nutrition and breathing.Although patients generally experience hospital-acquired sleep problems,there is no measurement tool to determine hospital-acquired sleep problems.These features show the originality of the research.AIM To develop a measurement tool to determine the sleep problems experienced by patients in the hospital.METHODS A personal information form,hospital-acquired insomnia scale(HAIS),and insomnia severity index(ISI)were used to collect research data.The study population consisted of patients hospitalized in the internal and surgical clinics of a research hospital in Turkey between December 2021 and March 2022.The sample consisted of 64 patients in the pilot application stage and 223 patients in the main application stage.Exploratory factor analysis and confirmatory factor analysis(CFA)analyses were performed using the SPSS 20 package program and the analysis of moment structure(AMOS)package program.Equivalent forms method used.RESULTS The HAIS consisted of 18 items and 5 subscales.The Cronbach alpha values of the subscales ranged between 0.672 and 0.842 and the Cronbach alpha value of the overall scale was 0.783.The scale explained 58.269%of the total variance.The items that constitute the factors were examined in terms of content integrity and named as physical environmental,psychological,safety,socioeconomic,and nutritional factors.CFA analysis of the 5-factor structure was performed in the AMOS package program.The fit indices of the obtained structure were examined.It was determined that the values obtained from the fit indices were sufficient.A significant correlation was determined between the HAIS and the ISI,which was used for the equivalent form method.CONCLUSION The HAIS is a valid and reliable measurement tool for determining patients’level of hospitalacquired insomnia.It is recommended to use this measurement tool to determine the insomnia problems of patients and to adapt it in other countries.
基金Supported by the National Natural Science Foundation of China(Nos.41176134,30830015)the Prospective Joint Research Project of Jiangsu Province(No.BY2011188)+1 种基金the National Marine Public Welfare Research Project(Nos.201105008-2,201105023-7)the National Basic Research Program of China(973 Program)(No.2011CB411908)
文摘To screen the stable expression genes related to the stress （strong light, dehydration and temperature shock） we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y（II） and Fv/Fm were significantly affected when stress was imposed on the thalli of Porphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress （at salinity 90） on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-la showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.
文摘Background： Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results： Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were： beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A （cyclophilin A）, ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein--zeta polypeptide The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were （most to least stable） succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, sucdnate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions： BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.
基金the National 973 Program of China(2010CB126200)the Genetically Modified Organism Breeding Project,Ministry of Agriculture,China(2009ZX08001-002B)
文摘The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.
基金Supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-EW-G-12B)the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KZCX2-EW-Q213)the National High Technology Research and Development Program of China (863 Program)(No.2012AA10A412)
文摘Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.
基金Supported by the National Natural Science Foundation of China(Nos.41476140,41306151,41676157,41506172)the Strategic Leading Science and Technology Projects of Chinese Academy of Sciences(No.XDA11020404)+1 种基金the China Postdoctoral Science Foundation(No.2015M582153)the Science and Technology Plan of Jiangsu Province(No.BE2016330)
文摘Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.
基金Supported by the National Natural Science Foundation of China(No.41666006,31702379)the Nanhai Famous Youth Project
文摘Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill)stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.
基金supported by the National Natural Science Foundation of China Program[No.31871773 and No.31820103010]Projects of Innovation and Development Pillar Program for Key Industries in Southern Xinjiang of Xinjiang Production and Construction Corps[2018DB002]+2 种基金National First-Class Discipline Program of Food Science and Technology[JUFSTR20180102]the BBSRC Newton Fund Joint Centre AwardCollaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province。
文摘Bacillus coagulans can help ameliorate or prevent gastrointestinal diseases, but the genetic relationships among B. coagulans isolates are not well studied. Multilocus sequence typing analysis was conducted on 57 isolates of B. coagulans from 22 provinces or autonomous regions in China. B. coagulans isolates were highly diverse and a total of 33(sequence typings) STs were found. These isolates had a weak clonal population structure and strong indications of intraspecies recombination. The evolution direction of B. coagulans was not correlated with geography or isolation source. Fifteen strains were selected for further analysis based on proximity relationships from the phylogenetic tree. Five isolates(B. coagulans-1, B. coagulans-10, B. coagulans-39, B. coagulans-70 and B. coagulans-71) with good spore-forming ability relative to the rest of the isolates were evaluated for constipation relief. B. coagulans-39 significantly relieved constipation symptoms in mice by regulating intestinal flora, increasing the production of short-chain fatty acids and restoring the level of gastrointestinal regulatory peptides. Comparative genomic analysis showed the beneficial effects of B. coagulans-39 might be associated with specific functional genes that are involved in the utilization of various carbohydrates as primary substrates and short-chain fatty acid production.
文摘Purpose: The aim of this study was to isolate and identify potentially pathogenic airborne fungi from Hospital das Clínicas Samuel Libanio in the city of Pouso Alegre-MG and evaluate their susceptibility to natural and industrial products. Methods: The air collection was performed by passive sedimentation in the morning during the autumn and winter seasons. Petri dishes were open as the location of air conditioning. The isolates were subjected to pathogenicity test. The identification of the fungi was performed according to the macroscopic evaluation and micromorphology. Potentially pathogenic isolates were susceptibility tested by disc diffusion method. The agents used were insecticides and industrial cleaning products and essential oils of citronella plants, lemon grass, eucalyptus and Melaleuca extracted by steam distillation method. Results: We obtained a total of 356 fungal isolates. The inside door environments were 126 (35.39%) and the outside environments were 230 (64.6%) isolates. The 22 (6.18%) isolates from the inside and 25 (6.18%) outside the hospital showed pathogenic potential. This isolates were identified as Acremonium spp., A. niger, A. terreus, A. versicolor, Curvularia sp., Penicillium sp. and Scopulariopsis sp. Susceptibility testing it was observed that most of the isolates were susceptible to the principle product containing sodium hypochlorite. Citronella oil showed enormous potential inhibition against all isolates. Already lemon grass oil was effective only against isolates of Penicillium spp. Conclusions: All genres identified are significant allergens, which can cause respiratory disease in both immunocompromised individuals such as asthmatics and people with any immune deficit. The monitoring of environmental sources should be performed, especially in special areas with immunocompromised patients. Despite efforts to try to reduce fungal infections hospital there are still flaws in the strategies employed.
文摘Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an en- dogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in inflam- matory conditions;however information about the stability and expression of HKGs in chronic inflammatory joint dis- ease such as rheumatoid arthritis (RA) is scarce. The expressional stability of 10 commonly used HKGs was analyzed in the neuronal (spinal cord, dorsal root ganglia) and in the musculoskeletal tissues (tendon, muscle, epiphysis, capsule, periosteum and ankle joint) using RT-qPCR in the rat model of RA. In individual tissues, suitable HKGs were selected by | △Ct| (│Ct control-Ct arthritis│) and further analyzed by using software programs;geNorm and normfinder. We found hypoxanthine-guanine phosphoribosyl tranferase (HPRT) as the most stable gene except ankle joint while glyce-raldehyde-3-phosphate dehydrogenase (GAPDH) was found as the least stable gene in musculoskeletal tissues. In in-flamed ankle joint where no reference gene was found to be stably expressed, an inflammatory cell marker CD3 was used to normalize peptidylprolyl isomerase B (PPIB), the most homogenous HKG identified among the 10 HKGs. The normalized PPIB was then used to analyze the gene expression of neurokinin 1 (NK1), receptor of substance P, a potent pro-inflammatory mediator. We observed a 3.5 fold increase (p = 0.009) in NK1 expression in inflamed ankle joint compared to control. Our results indicate that reference genes stability should be evaluated before using them as refer- ence during inflammatory conditions. In tissues with intense inflammatory cell infiltration, an inflammatory cell marker should be used to normalize the selected reference gene to avoid erroneous results.
文摘Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits;however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.
文摘The effectiveness of celebrity endorsements has been a topic that has interested many advertising researchers. It has been long debated on which advertising method is most effective. The purpose of this study was to conclude whether or not advertising agencies and social cause organizations are obtaining the results of these studies and using them in magazine advertisement campaigns and which one of these advertisements is more prevalent in magazines. Advertisements were examined from two American magazines, W Magazine and Good Housekeeping. The results of the study showed that celebrtiy endoresments have been featured in magazine advertisements more in recent years that products are endorsed by celebrities more than social causes and a majority of these endoresments are for products that affect a consumer＇s self-image.
文摘When he left his uncle in Nor-bu Linka, the Dalai Lama’s summer palace in Lhasa, on the evening of March 10,1959, Qiampa Getsang never expected one day he would do the same job as his late uncle did to manage the palace of the Dalai Lama.
基金supported by the Foundation for the Author of National Excellent Doctoral Dissertation of China (No. 200035)the Ministry of Education Science & Technology Research Project, China (No.104069)
文摘The ZDPS-1A pico-satellite designed in Zhejiang University with a mass of 3.5 kg and a power consumption of less than 3.5 W is the smallest satellite in China up to now. The housekeeping system (HKS) is the core part of ZDPS-1A. The reliability of HKS has an important influence on the safety of the satellite. Traditional fault-tolerant methods do not apply to ZDPS-1A due to such pico-satellite characteristics as light weight, compactness in size, energy saving, and high integration. This paper deals with a highly-reliable, low-cost design for HKS using industrial devices. The reliable strategies of HKS include a dual modular redundancy scheme, CPU warm backup, a static triple modular redundancy scheme, and two-level watchdogs. Recursive experiments, special tests, and environmental tests show that this system meets the design target. This design has already been applied to ZDPS-1A, which was launched to execute in-orbit tasks on Sept. 22, 2010. To date, the satellite has been in a proper state for more than 15 months.
基金supported by National Basic Research Program of China(973 program)(No.2006CB504100)the National Natural Science Foundation of China(Grant Nos.30393133 and 30260034).
文摘Internal standards are critical for quantitative RNA analyses.Housekeeping genes are often used as internal standards with the assumption that their express-ion levels remain relatively constant in different experi-mental conditions.In this study,four commonly used housekeeping genes,Glyceraldehyde-3-phosphate dehy-drogenase(GAPDH),β-actin,28S rRNA and 18S rRNA were selected to test whether this assumption is tenable under hypoxic conditions.We tested the RNA expression level of these four genes in different hypoxic conditions.Rats subjected to acute hypoxia for 2 hours were used for tissue detection.Primary cultured neural stem cells from E13 fetal rats were treated with 3%O_(2) or 10%O_(2) for 24 hours for in vitro experiments.In both experiments,expression levels of 28S rRNA and 18S rRNA were constant,independent of hypoxia types.However,expression levels of GAPDH and b-actin were all changed in all kinds of hypoxic conditions.In particu-lar,the mRNA expression level of GAPDH was increased by 43.4%under 3%O_(2) hypoxic conditions.These results suggest that 18S rRNA and 28S rRNA are reliable internal controls for comparative analyses of transcrip-tion under hypoxia.GAPDH appears particularly unfa-vorable for this purpose in hypoxic conditions.
基金funded by the European Union (FP7 Best Ageing,6031)Saarland University,Medical Faculty.Authors acknowledge the contribution of Comprehensive Biomarker Center (CBC),Heidelberg,in funding the study
文摘Blood-based mieroRNA （miRNA） signatures as biomarkers have been reported for various pathologies, including cancer, neurological disorders, cardiovascular diseases, and also infections. The regulatory mechanism behind respective miRNA patterns is only partially understood. Moreover, ＂preserved＂ miRNAs, i.e., miRNAs that are not dysregulated in any disease, and their biological impact have been explored to a very limited extent. We set out to systematically determine their role in regulatory networks by defining groups of highly-dysregulated miRNAs that contribute to a disease signature as opposed to preserved housekeeping miRNAs. We further determined preferential targets and pathways of both dysregulated and preserved miRNAs by computing multi-layer networks, which were compared between housekeeping and dysregulated miRNAs. Of 848 miRNAs examined across 1049 blood samples, 8 potential housekeepers showed very limited expression variations, while 20 miRNAs showed highly-dysregulated expression throughout the investigated blood samples. Our approach provides important insights into miRNAs and their role in regulatory networks. The methodology can be applied to systematically investigate the differences in target genes and pathways of arbitrary miRNA sets.
文摘INTRODUCTION The new Glen Allen High School was built in 2010 by Henrico County Public Schools(HCPS)to serve the growing student population in the Glen Allen area of Henrico County,Virginia.As a suburban area northwest of the City of Richmond,Glen Allen’s population doubled between 1980 and 2000,and grew an additional 18%between 2000 and 2010.The new high school is one of nine in the county and can accommodate approximately 1,800 students and 150 staff.The school facility is located on a 95-acre parcel of land that was previously wooded and undeveloped.
