Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.     

泌尿生殖道支原体感染检测及药敏试验

目的了解本地区泌尿生殖道解脲脲原体（Uu）和人型支原体（Mh）感染状况及药物敏感性,指导临床医生合理应用抗生素。方法应用法国生物梅里埃公司提供的IST试剂盒进行支原体鉴定及9种药物敏感检测,并对结果进行统计分析。结果1210例门诊患者检出支原体阳性683例,总感染率为56.4%,其中Uu单独感染占628例（占51.9%）,Mh单独感染14例（占1.2%）,Uu和Mh混合感染41例（占3.4%）。交沙霉素和原始霉素敏感率最高,对Uu分别为98.5%和97.0%,对Uu和Mh混合感染率都为100%;氧氟沙星敏感率最低,分别为1.5%和0.0%。结论泌尿生殖道系统感染主要由解脲支原体引起,交沙霉素和原始霉素敏感率最高,氧氟沙星敏感率最低。临床应选用培养敏感的抗菌药物,提高治疗效果。     

Determination of serum triglycerides using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads

A method for determination of serum triglycerides (Tgs) using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling was developed and evaluated. The minimum detection limit of the method was 0.54 mM. The analytical recovery of added triolein in the serum was 97.55 +/- 1.5% (mean +/- S.D.). The mean value of serum Tgs, determined by the present method showed a good correlation (r = 0.984) with the Bayer's kit method, employing free enzymes. The within and between batch coefficients of variation (CV) were < 2.25% and < 1.35% respectively. No significant loss of activity was observed, when co-immobilized enzymes were reused for about 200 times and stored at 4 degrees C in distilled water. The cost of Tg determination for 200 serum samples was less, as compared with Bayer's kit method.     

Characteristics of Mycoplasma meleagridis sp. n., Isolated from Turkeys.

Yamamoto, R. (University of California, Davis), C. H. Bigland, and H. B. Ortmayer. Characteristics of Mycoplasma meleagridis sp. n., isolated from turkeys. J. Bacteriol. 90:47-49. 1965.-A designation is proposed for a pathogenic Mycoplasma species isolated from turkeys. The organism originally was recovered from the air-sac lesion of a turkey poult in 1957, and was designated the "N" strain. Mycoplasma species with identical characteristics have since been recovered from the sinus, trachea, oviduct, vagina, semen, and bursa of Fabricius of turkeys. The organism has been recovered from many turkey flocks throughout the country. Many investigators have confirmed the original finding that this organism is antigenically distinct from other known serotypes of Mycoplasma found in poultry. The species proposed is Mycoplasma meleagridis sp. n.     

Evaluation of the NucliSens miniMAG RNA extraction and real-time NASBA applications for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in throat swabs

Nucleic acids were extracted from 215 throat swabs from patients with community-acquired pneumonia by the manual Boom extraction, the NucliSens miniMAG and the Qiagen DNA blood kit and amplified respectively by in-house real-time NASBA, NucliSens EasyQ reagents, and real-time PCR for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae. Five out of 215 throat swabs were found to be C. pneumoniae positive by all techniques used. A total of 11 out of 215 throat swabs were positive for M. pneumoniae; 10/215 by Qiagen extraction and PCR amplification and 9/215 by NucliSens miniMAG and NucliSens EasyQ amplification. The NucliSens miniMAG and NucliSens EasyQ applications were successfully coupled to detect both organisms in throat swabs.     

Histochemical Observations on Mycoplasma after Staining with Acridine Orange

Mycoplasma colonies and Mycoplasma cells in preparations from infected milk and lymph nodes were observed for their fluorescent qualities after treatment with acridine orange. Mycoplasma colonies fluoresced brilliant red or red-orange. When treated after exposure to ribonuclease, the colonies fluoresced lime-green. There was no fluorescence when both ribonucleic acid and deoxyribonucleic acid were destroyed by perchloric acid. Detection of Mycoplasma in smears of mastitic milk or smears of infected lymph nodes was not definitive because of the large amount of nonspecific ribonucleic acid-rich material present during inflammatory reactions.     

Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR

The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.     

Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum.

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.     

儿科呼吸道疾病中肺炎支原体感染的结果分析

目的探讨儿科肺炎支原体（MP）感染特点,辅助临床医师早期诊断,合理用药。方法测定我院一年来2013例儿科呼吸道疾病患儿的肺炎支原体抗体（IgM）。结果2013例呼吸道感染患儿,检出肺炎支原体抗体（IgM）阳性者769例,占38.2%,769例阳性分别表现为肺炎369例（48%）,支气管炎238例（31%）,咽炎92例（12%）,哮喘70例（9%）。其中,肺炎组与各组相比较,具有统计学意义。MP IgM感染的检出率明显高于其他各组（P〈0.01）。结论MP感染是患儿不可忽视的病原体,检测患儿血清MP抗体能够及早诊断,指导治疗。     

Effect of cholesterol and lanosterol on the structure and dynamics of the cell membrane of Mycoplasma capricolum. Deuterium nuclear magnetic resonance study.

Deuterium nuclear magnetic resonance (NMR) techniques were employed to study the effect of sterols on the composition and dynamics of the membrane lipids of Mycoplasma capricolum, a natural fatty acid auxotroph that requires sterols for growth. The membrane lipids of cells grown in modified Edwards medium supplemented with cholesterol, oleic acid (OA), and palmitic acid (PA) were composed primarily of phosphatidylglycerol (PG) (60%) and cardiolipin (CL) (35%). The incorporation of cholesterol and the cellular OA/PA ratio increased nonlinearly with increases in exogenous cholesterol level, whereas the levels of phospholipid increased only slightly. At the growth temperature, 37 degrees C, the residual deuterium quadrupole splittings were found to be 43-46 kHz for cells grown with (7,7,8,8-2H4) PA and 1.25 micrograms/ml (30 mol%) to 10 micrograms/ml (50 mol%) cholesterol, respectively, similar to that found in the cholesterol/lecithin binary dispersions of similar cholesterol contents. Deuterium T2e of these samples were found to be 170 +/- 10 microseconds and were independent of cellular cholesterol content. In comparison, T2e of the corresponding lipid extracts were longer (320-420 microseconds) and dependent on cholesterol content. Thus, lipid-protein interactions in the cell membrane is the dominant mechanism responsible for the reduced T2e. At lower temperatures, spectra indicative of the coexistence of gel and liquid-crystalline states were observed for cells having low cholesterol levels. For both cell membrane and membrane lipid extract containing 50 mol% cholesterol, T2e was found to be constant at the temperature range from 15 to 40 degrees C. On the other hand, T2e of cell membrane containing 30 mol% cholesterol decreased linearly at 3.2 microseconds/degrees C. T2e of the corresponding lipid extract showed much stronger temperature variation. Cells containing 39 mol% lanosterol were found to have a quadrupole splitting of 39 kHz, broader than that of the cholesterol-free lecithin dispersion (less than 30 kHz) but less than that of cell membrane containing 30 mol% cholesterol (43 kHz). T2e of the lanosterol sample was found to be 130 +/- 10 microseconds which decreased linearly at a slope similar to that observed for the low cholesterol sample. Therefore, although lanosterol appeared to be capable of modulating cell membrane physical properties it is less effective than cholesterol. When growth rates were correlated with NMR parameters, we found that the membranes of faster growing cells were also more ordered. In contrast, the T2e of the cells of M. capricolum seemed to be maintained at a relatively constant value around 170 microseconds.     

Molecular characterization of the pig<Emphasis Type="Italic"> C3</Emphasis> gene and its association with complement activity

The complement system catalyzes direct lysis of micro-organisms and modulates phagocytosis, inflammation, humoral and cellular immune responses. Since the complement protein C3 is the central component within all pathways of complement activation, C3 is a candidate gene for complement activity and also for improved protection against many pathogens. The pig C3 gene was sequenced, screened for polymorphisms, and analyzed for association with hemolytic complement activity of the alternative and classical pathway (AH(50), CH(50)). C3c serum levels and haptoglobin (HP) serum concentrations were measured before and after vaccination against Mycoplasma hyopneumoniae, Aujeszky virus, and porcine reproductive and respiratory syndrome virus in F2 animals of a pig resource population based on crossbreeding of Duroc and Berlin Miniature Pig. The genomic C3 sequence covers 444 bp of promoter region, 41 exons and 40 introns, as well as 881 bp of the 3'-flanking region. The cDNA codes for a 1,661-amino acid precursor C3. Five polymorphic sites were detected in the 5'-UTR, intron 13, exon 15, exon 30, and the 3'-UTR. Within the resource population two haplotypes were found to segregate. Analysis of variance applying a repeated measures model revealed a significant effect of the interaction of C3 genotype and time of measurement relative to immunization on CH(50), AH(50,)and C3c that is likely to be due to variation of C3 expression. In contrast, the time course of the HP acute-phase reaction is not associated with C3 genomic variation. The association of C3 with complement activity indicates the importance of C3 as a candidate gene for natural resistance to micro-organisms, although the causative polymorphism modulating the expression of C3 remains to be delineated.     

黄曲霉毒素B_1抗独特型抗体的制备和应用研究II.抗独特型抗体应用

以抗独特型抗体(anti-idiotypeantibody,Ab2)的酶切片段Fab2替代黄曲霉毒素B1(AFB1)建立一种不需要使用AFB1的无毒酶联免疫吸附(Enzyme-LinkedImmunosorbentAssay,ELISA)试剂盒,研究该试剂盒特异性、稳定性和AFB1的加标回收,并将该试剂盒用于农产品和饲料中AFB1的检测。结果表明,该试剂盒具有和常规ELISA试剂盒一样的特异性和加标回收能力,无毒试剂盒和常规试剂盒一样都可以用于农产品和饲料中AFB1的检测,并且两种试剂盒的检测结果并无显著差异,无毒ELISA试剂盒在适当处理后,40C放置3个月和-200C放置5个月,以间接非竞争ELISA测定的吸光值分别是起始时的85%和87%。     

Production of thuringiensin by fed-batch culture of Bacillus thuringiensis subsp. darmstadiensis 032 with an improved pH-control glucose feeding strategy

A improved pH-control fed-batch strategy for Bacillus thuringiensis subsp. darmstadiensis 032 producing thuringiensin was developed based on the analysis of the batch culture, constant rate fed-batch cultures and the original pH-control fed-batch. Having considered the pH variation and the glucose consumption status, the pH was adjusted from 6.5 to 7.0 by adding base in the late cultivation period of batch culture, and then the pH was kept at 7.0 by glucose feeding. The feeding was terminated when the pH could not be controlled by glucose feeding anymore. The proposed fed-batch strategy effectively avoided underfeeding or overfeeding, and it increased the thuringiensin yield and Y_P/X by 89.51% and 103.2% compared to that of the batch culture, respectively.     

Response to “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues”

I found the research paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues” by Price ME and colleagues in the June 2012 issue of Epigenetics to be an interesting read, but it contains errors in regards to the use of the MethylFlash Methylated DNA Quantification Kit. The text contained in the “Global DNA Methylation” paragraph under the “Methods” section of the paper claims that the kit was used by “following the manufacturer’s protocol.” I find that this is quite misleading to the reader as we have identified, based on the original electronic publication ahead of print, that the steps had not been correctly carried out.

The original text was the following: “DNA was hybridized to wells treated to have a high affinity for DNA. The wells were washed with a 5-mC-specific capture antibody followed by a detection antibody. The absorbance (or optical density) of each well was measured using a microplate spectrophotometer. Correlation of technical replicates was poor (r = 0.09, p = 0.72) using this kit.” However, the user guide of this kit clearly states that DNA is bound, not hybridized, to the strip wells and that the wells should be washed with the included Wash Buffer rather than a “5-mC-specific capture antibody.”

Therefore, it is not surprising that the result or correlation of replicates was poor and that the authors’ “results were variable” due to improper use of the kit. Based on our quality control tests and feedback from the vast amount of users of this popular kit, the variation between replicates should be less than 10% with R value > 0.9 (p < 0.01), assuming proper user performance according to the product manual.

I hope this helps to correct the misinformation presented in the paper as I feel that it is important to promote accuracy on behalf of the epigenetic research community. I also kindly encourage any users of an Epigentek product to work with our very knowledgeable technical support team should they have any difficulties.     

微波等离子体溅射诱变选育花生四烯酸高产菌及补料工艺研究

采用微波等离子（N^ ,15w,3min)溅射高山被孢霉T105,筛选到高产菌R254,该菌最高生物量29．3g/L,油脂11．5g/L,花生四烯酸4．20g/L,花生四烯酸产量是原出发菌株1．35倍。通过对突变株R245的代谢情况、继代稳定性以及脂肪酸组成分析,认为微波等离子溅射诱变育种是获得花生四烯酸高产菌株的有效方法。并采用补糖工艺可进一步提高其产量,花生四烯酸产量为7．43g/L,是未补糖时花生四烯酸产量的1．76倍,且为出发菌株花生四烯酸最高产量的2．40倍。     

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvB(FH)] and M. genitalium [RuvB(Mge)], respectively) are described. Both RuvB(FH) and RuvB(Mge) were found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvB(Mge), however, was significantly lower than that of RuvB(FH). Interestingly, we found RuvB(FH) to be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvB(M129)]) that differs from RuvB(FH) in a single amino acid residue (at position 140). In contrast to RuvB(FH), RuvB(M129) displayed only marginal levels of DNA-unwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.     

In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells

Background aimsIn this study, the authors performed an in-house abbreviated qualification of a commercially available real-time polymerase chain reaction (PCR) kit for limit of detection (LOD), matrix interference and ruggedness of mycoplasma detection in a human bone marrow-derived mesenchymal stromal cell (MSC(M)) investigational cell product (NCT02351011). The approach used was similar to an abbreviated qualification the authors previously conducted for endpoint PCR, which was accepted by Canadian regulators for final product release of the same MSC(M) investigational cell product for treatment of osteoarthritis patients (NCT02351011). With patient consent, biobanked MSCs(M) were re-analyzed by real-time PCR for mycoplasma detection to conduct in-house qualification of the kit.MethodsLOD was determined by spiking MSCs(M) with a series of 10-fold dilutions of two commercially available genomic DNA (gDNA) reference standards for Mycoplasma arginini (M. arginini) and Mycoplasma hominis (M. hominis). Matrix interference was tested by using 10-fold dilutions of MSC(M)s down to 4500 cells/mL. Polyadenylic acid (poly[A]) was used to improve DNA recovery in samples with 4500–45 000 MSCs(M)/mL. Real-time PCR tests performed on different days were compared to evaluate ruggedness.ResultsReal-time PCR analysis showed a conservative LOD of 40 genome copies (GCs)/mL and 240 GCs/mL, which are equivalent to 10 colony-forming units (CFUs)/mL, for M. arginini and M. hominis, respectively. According to a less conservative manufacturer-based criterion for positivity, the kit detected 0.4 GC/mL (0.1 CFU/mL) and 24 GCs/mL (1 CFU/mL) M. arginini and M. hominis, respectively. Real-time PCR with different MSC(M) dilutions did not show matrix interference. However, DNA recovery was compromised at MSC(M) concentrations at or below 45 000 cells/mL. The addition of poly(A) as a DNA carrier improved DNA recovery and allowed an LOD, considered here to be equivalent to 10 CFUs/mL, to be achieved, which was not possible in diluted MSC(M) samples (≤45 000 cells/mL) in the absence of poly(A). Ruggedness was demonstrated with tests (n = 18) performed on different days, with an average overall inter-assay percent coefficient of variation of less than 4 for M. arginini (3.62 [400 GCs/mL], 3.61 [40 GCs/mL]) and less than 3 for M. hominis (2.83 [2400 GCs/mL], 1.95 [240 GCs/mL]).ConclusionsA commercially available real-time PCR mycoplasma detection kit was qualified for evaluating mycoplasma contamination in investigational MSC(M) products and met the criteria used previously (and accepted by Canadian regulators) for in-house qualification of an endpoint PCR mycoplasma detection kit, and the addition of poly(A) addressed the poor recovery of mycoplasma gDNA in samples with low cell numbers.     

Effects of electromagnetic field on the batch cultivation and nutritional composition of Spirulina platensis in an air-lift photobioreactor

Spirulina platensis was batch cultivated in a novel 3.5l magnetic air-lift photobioreactor with external-loop on which 0-0.55 T electromagnetic field (EMF) was imposed to investigate the EMF effects on the algal growth and nutrition composition. At the same time, the correlative mechanism was discussed mainly based on C, N, P uptake. It was found that, EMF has double-side effects on S. platensis cultivation depend on EMF intensity. 0.25 T EMF stress was found to be suitable for the algal cultivation enhancement increasing the maximum cell dry weight by 22% in a time period two days less than that of the control at 35 degrees C and 252 micromol photonsm(-2)S(-1). In addition, the nutritional composition of S. platensis was improved in both essential amino acids such as histidine and trace elements Ni, Sr, Cu, Mg, Fe, Mn, Ca, Co and V. It was suggested it was possible to optimize the cultivation process of S. platensis by imposing suitable weak EMF which may enhance the nutrition assimilation e.g. C, N, P and minerals by S. platensis.     

Novel evolved immunoglobulin (Ig)-binding molecules enhance the detection of IgM against hepatitis C virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases.     

pH-controlled continuous cultivation of mycoplasmas

The continuous cultivation of mycoplasmas in a pH-controlled metabolistat was investigated with the fermentative strain Mycoplasma mobile 163K and the nonfermentative strain Mycoplasma arthritidis ISR1. The addition of medium and the removal of culture suspension were regulated by acid production from glucose by M. mobile 163K and by ammonium production from arginine by M. arthritidis ISR1, respectively. For both strains the optimal pH for continuous growth was 7.0. The steady state could be maintained for at least 21 days. With CFU of 8.4 X 10(9) ml-1 (M. mobile 163K) and 3.2 X 10(9) ml-1 (M. arthritidis ISR1), the cell concentrations were slightly higher than those obtained in batch cultures. The dependence on the adjusted pH values was measured for several parameters, such as flow rate, CFU, glucose fermentation or production of ammonia, and gliding velocity. Since the long lag phases of batch cultures can be avoided, pH-controlled continuous cultures provide an appropriate system for the production of mycoplasma cells.