Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting

Introduction

Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent.

Methods

In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed.

Results

There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both.

Conclusions

Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used.     

Performance of three LED-based fluorescence microscopy systems for detection of tuberculosis in Uganda

Background

Direct smear microscopy using Ziehl-Neelsen (ZN) staining is the mainstay of tuberculosis (TB) diagnosis in most high burden countries, but is limited by low sensitivity in routine practice, particularly in high human immunodeficiency virus (HIV) prevalence settings.

Methods

We compared the performance of three commercial light emitting diode (LED)-based microscopy systems (Primostar™ iLED, Lumin™ and AFTER®) for fluorescent detection of Mycobacterium tuberculosis with ZN microscopy on slides prepared from sputum of TB suspects. Examination time for LED-based fluorescent microscopy (LED FM) and ZN slides was also compared, and a qualitative user appraisal of the LED FM systems was carried out.

Results

LED FM was between 5.6 and 9.4% more sensitive than ZN microscopy, although the difference was not statistically significant. There was no significant difference in the sensitivity or specificity of the three LED FM systems, although the specificity of Fraen AFTER was somewhat lower than the other LED FM methods. Examination time for LED FM was 2 and 4 times less than for ZN microscopy. LED FM was highly acceptable to Ugandan technologists, although differences in operational performance of the three systems were reported.

Conclusions

LED FM compares favourably with ZN microscopy, with equivalent specificity and a modest increase in sensitivity. Screening of slides was substantially quicker using LED FM than ZN, and LED FM was rated highly by laboratory technologists. Available commercial systems have different operational characteristics which should be considered prior to programmatic implementation.     

Comparing the physical demands of friendly matches and small-sided games in semiprofessional soccer players

This study compared the physical demands of friendly matches (FMs) and small-sided games (SGs) in semiprofessional soccer players by means of global positioning system technology. Twenty-seven semiprofessional soccer players were monitored during 7 FMs and 9 sessions involving different SGs. Their physical profile was described on the basis of 20 variables related to distances and frequencies at different running speeds, the number of accelerations, and through global indicators of workload such as the work:rest ratio, player workload, and the exertion index. Results showed significant differences (p < 0.01) between SGs and FMs for the following variables: overall workload (SG > FM); the distribution of the distance covered in the speed zones 7.0-12.9 km·h(-1) (SG > FM) and >21 km·h(-1) (FM > SG); the distribution of time spent in certain speed zones (FM > SG: 0.0-6.9 and >21 km·h(-1); FM > SG: 7.0-12.9 km·h(-1)). More sprints per hour of play were performed during FMs, with greater mean durations and distances, greater maximum durations and distances, and a greater frequency per hour of play for sprints of 10-40 and >40 m (p < 0.01). The frequency of repeated high-intensity efforts was higher during FM (p < 0.01). The results show that coaches and strength and conditioning professionals should consider FMs during their training routine to foster specific adaptations in the domain of high-intensity effort.     

Term human fetal membranes have a weak zone overlying the lower uterine pole and cervix before onset of labor

The etiology of fetal membrane (FM) rupture is unknown. A hypothesis that the FM weakens by a process of collagen remodeling and apoptosis to facilitate rupture has been proposed. Human FMs reportedly exhibit a zone of altered histology, postulated to be the FM rupture site, but concomitant FM weakness has not been demonstrated. We hypothesized that a discrete zone of FM with marked weakness, histological change, and evidence of remodeling and apoptosis, develops in late gestation in the FM overlying the cervix. FM tissue from women undergoing prelabor cesarean delivery were perioperatively marked to identify the FM overlying the cervix, cut with a procedure that facilitates remapping the rupture strength of FM pieces to their former location and orientation on a three-dimensional model, and tested for strength. A 10-cm FM zone centered at the cervical mark was compared with the remaining FM. Mean rupture strength within the cervical zone was 55% of the remaining FM. The cervical zone also exhibited increased MMP-9 protein, decreased tissue inhibitor of metalloproteinases-3 (TIMP-3) protein, and increased PARP cleavage coincident with the previously reported zone of altered histology. A discrete zone of weakness is present in term prelabor FMs overlying the cervix and has biochemical characteristics consistent with tissue remodeling and apoptosis.     

Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli

To promote the decomposition of sugarcane bagasse (SCB) for conversion into value-added products and to reduce waste, the capability of fungal mixes (FMs) to degrade SCB was examined. A total of 169 isolates from SCB and non-SCB were categorized as thermotolerant and thermoresistant. Thirty-six fungal candidates were screened for the presence of polyphenol oxidase, endoglucanase (EDN) and xylanase (XLN) activities, and EDN and XLN activities were quantitated. Five identified isolates (Aspergillus flavus AG10; Aspergillus niger AG68 & NB23; and Penicillium citrinum AG93 & AG140) were selected as the best enzyme producers, and 15 moderately to highly xylolytic, cellulolytic and ligninolytic isolates were added to construct FMs. Using a Taguchi design, the top ten reducing sugar-producing FMs (no. 12 showed the maximum amount of reducing sugar, at 2.11 mg g⁻¹, followed by no. 7, 15, 2, 16, 11, 13, 6, 4, & 8) were selected as potential agents for decomposition durations of 1, 2 and 3 months. The maximum decrease in SCB materials compared with the control was generated by FM 6 (9.08% cellulose reduction); FM 13 (21.03% hemicellulose reduction); and FM 16 (9.21% lignin reduction). These results indicate the potential use of SCB as a substrate for synergistic FMs. These FMs could be applied to the large-scale composting of SCB and other related agricultural residues, thus improving the biological pretreatment of lignocellulose.     

Paradoxes of Hymenoptera flight muscles,extreme machines

In the Carboniferous, insects evolved flight. Intense selection drove for high performance and approximately 100 million years later, Hymenoptera (bees, wasps and ants) emerged. Some species had proportionately small wings, with apparently impossible aerodynamic challenges including a need for high frequency flight muscles (FMs), powered exclusively off aerobic pathways and resulting in extreme aerobic capacities. Modern insect FMs are the most refined and form large dense blocks that occupy 90% of the thorax. These can beat wings at 200 to 230 Hz, more than double that achieved by standard neuromuscular systems. To do so, rapid repolarisation was circumvented through evolution of asynchronous stimulation, stretch activation, elastic recoil and a paradoxically slow Ca²⁺ reuptake. While the latter conserves ATP, considerable ATP is demanded at the myofibrils. FMs have diminished sarcoplasmic volumes, and ATP is produced solely by mitochondria, which pack myocytes to maximal limits and have very dense cristae. Gaseous oxygen is supplied directly to mitochondria. While FMs appear to be optimised for function, several unusual paradoxes remain. FMs lack any significant equivalent to the creatine kinase shuttle, and myofibrils are twice as wide as those of within cardiomyocytes. The mitochondrial electron transport systems also release large amounts of reactive oxygen species (ROS) and respiratory complexes do not appear to be present at any exceptional level. Given that the loss of the creatine kinase shuttle and elevated ROS impairs heart function, we question how do FM shuttle adenylates at high rates and tolerate oxidative stress conditions that occur in diseased hearts?     

Cloning, expression, purification and activation by Na ion of halophilic alkaline phosphatase from moderate halophile Halomonas sp. 593

We have succeeded in the cloning of alkaline phosphatase gene, haalp, from moderate halophile Halomonas sp. 593. A deduced amino acid sequence showed a high ratio of acidic to basic amino acids, characteristic of halophilic proteins. The gene product was efficiently expressed in Escherichia coli BL21 Star (DE3) pLysS, but in an inactive form. The purified recombinant HaALP was separated into four fractions by gel filtration. When they were dialyzed against 50 mM Tris-HCl (pH 8.0)/2 mM MgCl? buffer containing 3 M NaCl, one of these four fractions was activated to almost full activity. This fraction contained a folding intermediate that was converted to the native structure by the salt. Among the additional salts tested, i.e., KCl, KBr, LiCl, MgCl?, (NH?)?SO?, and Na?SO?, only Na?SO? was effective, suggesting the importance of Na ion.     

覆膜对土壤-莴苣体系氮素分布和植物吸收的影响

覆膜种植作为全球广泛采用的农作物栽培方式,能改变土壤的水热生态效应,并影响元素的赋存状态。本文采用野外栽培实验,研究了覆膜对土壤—莴苣体系中氮素分布和植物吸收的规律。相比于不覆膜方式,覆膜对土壤含水率、pH值及脲酶活性的影响不明显,但减少了6.0%的土壤有机质、10.4%的硝态氮和1.3%的全氮含量,增加了6.5%的土壤铵态氮。单因素方差分析表明,土壤有机质及全氮含量的组间差异达到了显著性水平。覆膜方式下,氮素生理群落微生物中反硝化细菌占生理群落总数的77.8%—96.2%,氨化细菌、亚硝化细菌及硝化细菌各占1.8%—16.5%、0.6%—5.1%和0.4%—2.8%。与不覆膜相比,覆膜使氨化细菌平均值减少了28.2%,亚硝化、硝化及反硝化细菌平均值分别增加了119.8%、26.7%和48.7%。莴苣植株中的全氮含量变化规律为叶片>茎部>根部。覆膜使根部全氮含量降低了2.8% ,茎部与叶部则分别增加了10.5%和6.8%。覆膜使莴苣根部全氮的富集系数平均值降低了1.5%,迁移系数TF1（莴苣茎部和根部全氮的比值）和TF2（莴苣叶部和根部全氮的比值）平均值分别提高了12.5%和9.5%,影响较小。相关性分析表明,有机质与土壤全氮呈显著正相关（P < 0.05）,而脲酶、铵态氮及无机氮三者与亚硝化细菌均呈显著负相关（P < 0.05）。     

Nested Association Mapping for Identification of Functional Markers

Identification of functional markers (FMs) provides information about the genetic architecture underlying complex traits. An approach that combines the strengths of linkage and association mapping, referred to as nested association mapping (NAM), has been proposed to identify FMs in many plant species. The ability to identify and resolve FMs for complex traits depends upon a number of factors including frequency of FM alleles, magnitudes of their genetic effects, disequilibrium among functional and nonfunctional markers, statistical analysis methods, and mating design. The statistical characteristics of power, accuracy, and precision to identify FMs with a NAM population were investigated using three simulation studies. The simulated data sets utilized publicly available genetic sequences and simulated FMs were identified using least-squares variable selection methods. Results indicate that FMs with simple additive genetic effects that contribute at least 5% to the phenotypic variability in at least five segregating families of a NAM population consisting of recombinant inbred progeny derived from 28 matings with a single reference inbred will have adequate power to accurately and precisely identify FMs. This resolution and power are possible even for genetic architectures consisting of disequilibrium among multiple functional and nonfunctional markers in the same genomic region, although the resolution of FMs will deteriorate rapidly if more than two FMs are tightly linked within the same amplicon. Finally, nested mating designs involving several reference parents will have a greater likelihood of resolving FMs than single reference designs.THE primary purpose for identifying functional markers (FMs) associated with complex traits in plant species is to provide molecular genetic information underlying variability upon which both artificial and natural selection are based. FMs are defined as polymorphic sites within genomes that causally affect phenotypic trait variability (Andersen and Lubberstedt 2003). This definition is a pragmatic recognition that phenotypic variability can be due to genomic variability located outside of open reading frames. Forward genetics approaches to associate naturally occurring structural genomic variants with phenotypic variability can be broadly categorized as (1) linkage mapping, also referred to as quantitative trait locus (QTL) mapping, (2) association genetic mapping, also known as linkage disequilibrium (LD) mapping, and (3) designs that combine linkage and LD mapping.The third approach based on the concept of combining LD with QTL mapping is a natural extension of the multifamily QTL approach and has been referred as joint linkage and linkage disequilibrium mapping (JLLDM) (Xiong and Jin 2000; Farnir et al. 2002; Wu et al. 2002; Perez-Enciso 2003; Jung et al. 2005) in samples from natural populations. The combined approach also has been applied to designed mapping families sampled from plant breeding populations (Xu 1998a; Jannink and Jansen 2000; Jannink and Wu 2003; Jansen et al. 2003). A special case of designed mapping families that are interconnected, known as nested association mapping (NAM), was proposed by Yu et al. (2008). As originally proposed, a NAM population consists of multiple families of recombinant inbred lines (RILs) derived from multiple inbred lines crossed to a single reference inbred line. Implicitly, genomic information is composed of high-density genotypes of parental inbred lines and low-density genotypes from segregating progeny. If the segregating progeny are RILs or doubled haploid lines (DHLs), then the genomic information can be “immortalized” for associations with phenotypes obtained through long-term longitudinal studies (Nordborg and Weigel 2008).A NAM population consisting of 25 families with 200 RILs for each family has been developed and released as a genetic resource for identification of FMs in maize (Yu et al. 2008). Other publicly available NAM populations are being developed for several species including Arabidopsis thaliana (Buckler and Gore 2007), barley (R. Wise, personal communication), sorghum (J. Yu, personal communication), and soybean (B. Diers, personal communication).The power, accuracy, and precision of identifying FMs in experimental NAM populations have not been investigated for complex genetic architectures. These statistical properties depend upon a number of factors including the following:

1. Data analysis method: Some methods are more powerful than others; however, experimental biologists prefer methods implemented in existing software packages. Are least-squares methods sufficiently powerful to identify FMs in established and developing NAM populations?
2. Frequency of functional markers and magnitudes of genetic effects: Development of a NAM population will change the allele frequencies of the FM relative to the reference population from which the lines are sampled. How will allele frequency and magnitude of genetic effects in a typical NAM population affect the ability to identify FMs?
3. Disequilibrium among functional and nonfunctional markers: Disequilibrium may exist among alleles within subpopulations even when there is no physical basis for genetic linkage. To what extent can the NAM design address consequences of gametic disequilibrium (population structure) in the reference population?
4. Multiple FMs in the same genomic region: If multiple FMs are physically located in the same genomic region, will equilibrium among the parental lines enable resolution of multiple FMs?
5. Mating design: An appropriate mating design can maximize the number of families that are informative for FMs. Will multiple-reference mating designs improve the probability of identifying FMs?

These five questions were addressed.     

Validity and Reliability of A-Mode Ultrasound for Body Composition Assessment of NCAA Division I Athletes

This study evaluated the validity and reliability of the BodyMetrix™ BX2000 A-mode ultrasound for estimating percent body fat (%BF) in athletes by comparing it to skinfolds and the BOD POD. Forty-five (22 males, 23 females) National Collegiate Athletic Association (NCAA) Division-I athletes volunteered for this study. Subjects were measured once in the BOD POD then twice by two technicians for skinfolds and ultrasound. A one-way repeated-measures ANOVA revealed significant differences between body composition methods (F = 13.24, p < 0.01, η² = 0.24). This difference was further explained by a sex-specific effect such that the mean difference between ultrasound and BOD POD was large for females (~ 5% BF) but small for males (~ 1.5% BF). Linear regression using the %BF estimate from ultrasound to predict %BF from BOD POD resulted in an R² = 0.849, SEE = 2.6% BF and a TE = 4.4% BF. The inter-rater intraclass correlation (ICC) for skinfold was 0.966 with a large 95% confidence interval (CI) of 0.328 to 0.991. The inter-rater ICC for ultrasound was 0.987 with a much smaller 95% CI of 0.976 to 0.993. Both skinfolds and ultrasound had test-retest ICCs ≥ 0.996. The BX2000 ultrasound device had excellent test-retest reliability, and its inter-rater reliability was superior to the skinfold method. The validity of this method is questionable, particularly for female athletes. However, due to its excellent reliability, coaches and trainers should consider this portable and easy to use A-mode ultrasound to assess body composition changes in athletes.     

Quality assurance of canine vaginal cytology: A preliminary study

Regulatory controls of quality assurance in veterinary laboratories are less common than in human reproduction laboratories and the intra- and inter-technician variation in the assessment of canine vaginal cytology has not been reported. This study was designed to determine whether variation in classification of vaginal epithelial cells and interpretation of vaginal cytology smears existed within and between technicians in a canine reproductive laboratory.Sixteen vaginal cytology smears representing different known stages of the oestrous cycle were examined twice by one experienced technician and three inexperienced technicians in a blinded random order study design. Seven assessments were made; counting and classifying one hundred vaginal epithelial cells into four morphological classifications and assessment of three cellular categories. Technicians also interpreted their results and reported the stage of the cycle they thought each slide represented. In addition, selected samples were sent to four external commercial laboratories for interpretation.For the experienced technician, intra-technician variation was low for the morphological classifications and cellular assessments (r = 0.69-0.95). There was more intra-technician variation between results from Examination One and Examination Two for the inexperienced technicians (r = 0.53-0.92 where correlations were found). When inexperienced technicians' results were compared to results from the experienced technician, the inter-technician variation was low; results were correlated for 17 of the 21 observations (four morphological classifications and three cellular assessments across the three technicians) (r = 0.38-0.87). When technician interpretations of stage of the oestrous cycle were compared to the known stage of the cycle for each smear, the experienced technician correctly interpreted 19 of the 32 smears, whilst the three inexperienced technicians correctly interpreted 14, 16, and 18 of the 32 smears. The interpretation of vaginal smears by external laboratories was varied and sometimes inconclusive; 50% of laboratories incorrectly identified metoestrus smears as proestrus and 25% of the laboratories incorrectly identified an oestrus smear as proestrus.The results of this study are highly important for clinicians undertaking canine reproductive assessments since they demonstrate the potential for variability of results. While the greatest precision was found when vaginal smears were examined by an experienced technician (who, on a daily basis, examines many smears), more variability in both the reporting of different cell types and interpretation of the smears was observed by inexperienced technicians and when samples were sent to external commercial laboratories. These findings suggest that suitable quality control programmes should be implemented for laboratories that are undertaking routine assessments of canine reproductive function.     

Attractive Effects of American Serpentine Leafminer, Liriomyza trifolii (Burgess), to Light-Emitting Diodes

Phototactic responses of Liriomyza trifolii adults to six different light-emitting diodes (LEDs) were investigated, and their responses were compared to that using a luring lamp (BLB) under laboratory conditions. Based on the attraction rate under optimal light conditions, the green LED (520?±?5 nm) showed the highest attraction rate (99.7 %), followed by the yellow LED (590?±?5 nm, 96.1 %), the red LED (625?±?10 nm, 91.4 %), the blue LED (470?±?10 nm, 91.2 %), the UV LED (365 nm, 71.0 %), and the IR LED (730 nm, 5.6 %). Moreover, the green LED was approximately 1.4 times more attractive than BLB (71.1 %) to L. trifolii adults. These results suggest that the green LED was the most useful for monitoring of L. trifolii adults under optimal conditions.     

In Vivo Transfer of cellular immunity to primates with transfer factor prepared from human or primate leucocytes

Dialyzable transfer factor (TF_d) prepared from a human donor and transfer factor (TF) from baboon whole cell lysates was administered to 3 species of nonhuman primates: baboons, cebus monkeys and marmosets. In vivo transfer was evaluated with in vivo skin test and in vitro blastogenic responses to multiple antigens. Transfer of cellular reactivity in all three nonhuman primate species was demonstrated with both human TF_d and baboon TF. A cumulative conversion rate of 45% for skin test responses and 65% for lymphocyte blastogenesis was demonstrated following human TF_d injection while conversion was 17% and 33% respectively following baboon TF. Specificity was supported by the absence of conversion to TF donor negative antigens. There were no signficant differences observed between the 3 recipient primate species.     

The single biopsy approach is reliable for the measurement of muscle protein synthesis rates in vivo in older men

We aimed to assess the reliability of the single biopsy approach for calculating muscle protein synthesis rates compared with the well described sequential muscle biopsy approach following a primed continuous infusion of L-[ring-(2)H(5)]phenylalanine and GC-MS analysis in older men. Two separate experimental infusion protocols, with differing stable isotope amino acid incorporation times, were employed consisting of n = 27 (experiment 1) or n = 9 (experiment 2). Specifically, mixed muscle protein FSR were calculated from baseline plasma protein enrichments and muscle protein enrichments obtained at 90 min or 50 min (1BX SHORT), 210 min or 170 min (1BX LONG), and between the muscle protein enrichments obtained at 90 and 210 min or 50 min and 170 min (2BX) of the infusion for experiments 1 and 2, respectively. In experiment 2, we also assessed the error that is introduced to the single muscle biopsy approach when nontracer naive subjects are recruited for participation in a primed continuous infusion of isotope-labeled amino acids. In experiment 1, applying the individual plasma protein enrichment values to the single muscle biopsy approach resulted in no differences in muscle protein FSR between the 1BX SHORT (0.031 ± 0.003%·h(-1)), 1BX LONG (0.032 ± 0.002%·h(-1)), or the 2BX approach (0.034 ± 0.002%·h(-1)). A significant correlation in muscle protein FSR was observed only between the 1BX LONG and 2BX approach (r = 0.8; P < 0.001). Similar results were observed in experiment 2. In addition, using the single biopsy approach in nontracer na?ve state results in a muscle protein FSR that is negative for both the 1BX SHORT (-0.67 ± 0.051%·h(-1)) and 1BX LONG (-0.19 ± 0.051%·h(-1)) approaches. This is the first study to demonstrate that the single biopsy approach, coupled with the background enrichment of L-[ring-(2)H(5)]-phenylalanine of mixed plasma proteins, generates data that are similar to using the sequential muscle biopsy approach in the elderly population.     

Comparison of two host cell range variants of feline immunodeficiency virus.

Two molecular clones of feline immunodeficiency virus were compared. The first clone, 34TF10, was from a Petaluma, Calif., isolate; the second, PPR, was isolated from a cat in the San Diego, Calif., area. The cats from which the isolates were obtained suffered from chronic debilitating illnesses. The two molecular clones differed in their in vitro host cell range. The 34TF10 clone infected the Crandall feline kidney and G355-5 cell lines, but replicated less efficiently on feline peripheral blood leukocytes. In contrast, the PPR clone productively infected the primary feline peripheral blood leukocytes but not Crandall feline kidney or G355-5 cells. The 34TF10 and PPR clones had an overall sequence identity of 91%. The env gene was the least conserved (85% at the amino acid level). Additionally, the potential open reading frame for a Tat-like protein, ORF 2, contained a stop codon in the 34TF10 isolate which was not found in the PPR clone. This truncation did not prevent in vitro or in vivo replication of 34TF10. Two splice acceptor sites were identified in the 34TF10 clone. One was 5' to the beginning of the putative tat open reading frame, and the other was 5' to the putative vif product. Both of these acceptor sites were conserved in the PPR clone. The long terminal repeats of the viruses were 7% divergent between the two clones, with a lack of conservation in putative NF-kappa B, LBP-1, and CCAAT enhancer-promoter sites.     

Stability of structures of the epsilon subunit and terminator of thermophilic ATPase

F1-type ATPase is the central enzyme for ATP synthesis in most organisms. Because of the extreme reconstitutability of thermophilic ATPase (TF1) and diversity of the minor subunits of F1 type ATPase, an operon coding for TF1 was isolated from DNA of thermophilic bacterium PS3, and its terminal region containing the epsilon subunit (TF1 epsilon) and terminator was sequenced. The primary structure of the epsilon subunit (Mr = 14 333) was deduced from the nucleotide sequence (396 base-pairs) and amino-acid sequence of its amino terminus. The conclusions drawn from the results are as follows. Homologies: TF1 epsilon shows only 6% homology with the epsilon subunits of eight species reported, but 50% homology with Escherichia coli epsilon and 41% with chloroplast. The residues having a tendency to form reverse turns (Gly, Pro and Tyr) and His are relatively well conserved. Unlike some F1 epsilon types TF1 epsilon has no ATPase inhibitor activity and is not homologous with ATPase inhibitor. TF1 epsilon is essential to connect F1 to F0, like the b subunit, and is weakly homologous with the b subunit of F0F1. The cause of 3 beta: 1 epsilon subunit stoichiometry: The ribosome binding sequence of TF1 epsilon is TAGGN7, which is incomplete compared with that of TF1 beta. The codon usage for TF1 epsilon is similar to that for TF1 epsilon. The cause of stability of TF1 epsilon and its gene: There are 18 ionic groups at the putative reverse turns and the N- and C-termini of TF1 epsilon, but only 10 ionic groups in the corresponding sites of E. coli epsilon subunit. These ionic groups enhance the external polarity of TF1 epsilon and may intensify subunit-subunit interaction. There is a terminator at the 3' end of the TF1 epsilon gene, which is stabilized by a long (13 base-pairs) stem.     

Genetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica for removal of methyl parathion in water

In this study, the mph gene encoding methyl parathion hydrolase from Pseudomonas sp. WBC-3 was expressed in Yarrowia lipolytica and the expressed methyl parathion hydrolase was displayed on cell surface of Y. lipolytica. The activity of methyl parathion hydrolase displayed on the yeast cells of the transformant Z51 was 59.5 U mg?1 of cell dry cells (450.6 U per mL of the culture) in the presence of 5.0 mM of Co2?. The displayed methyl parathion hydrolase had the optimal pH of 9.5 and the optimal temperature of 40 °C, respectively and was stable in the pH range of 4.5-11 and up to 40 °C. The displayed methyl parathion hydrolase was also stimulated by Co2?, Cu2?, Ni2? and Mn2?, and was not affected by Fe2?, Fe3?, Na?, K?, Ca2? and Zn2?, but was inhibited by other cations tested. Under the optimal conditions (OD(600 nm) = 2.6, the substrate concentration = 100 mg L?1 and 40 °C), 90.8 % of methyl parathion was hydrolyzed within 30 min. Under the similar conditions, 98.7, 97.0, 96.5 and 94.4 % of methyl parathion in tap water (pH 9.5), tap water (pH 6.8), seawater (pH 9.5) and natural seawater (pH 8.2) were hydrolyzed, respectively, suggesting that the methyl parathion hydrolase displayed on the yeast cells can effectively remove methyl parathion in water.     

Transfer factor in chronic mucocutaneous candidiasis

Fifteen patients suffering from chronic mucocutaneous candidiasis were treated with an in vitro produced TF specific for Candida albicans antigens and/or with TF extracted from pooled buffy coats of blood donors. CMI of the patients was assessed using the LMT and the LST in presence of candidine. The aim of the study was the clinical evaluation of TF treatment and the incidence of positive tests before, during, and after therapy. Immunological data were matched using the Chi square test. 87 LMT were performed for each antigen dose and at the dilution of 1/50, 58.9% (33/56) tests were positive during non-treatment or non-specific TF treatment. On the contrary 83.9% (26/31) were positive during specific TF treatment (P<0.05). In the LST, a significant decrease of thymidine uptake in the control cultures in presence of autologous or AB serum was observed when patients were matched according to non-treatment, and both non specific (P<0.05) and specific TF treatment (P<0.01). Only during specific TF treatment was a significant increase of reactivity against the Candida antigen at the highest concentration noticed, when compared with the period of non specific treatment (P<0.01). Clinical observations were encouraging: all but one patient experienced significant improvement during treatment with specific TF. These data confirm that orally administered specific TF, extracted from induced lymphoblastoid cell-lines, increases the incidence of reactivity against Candida antigens in the LMT. LST reactivity appeared not significantly increased with respect to the periods of non treatment, but was significantly increased when it was compared to the non-specific TF treatment periods. At the same time, a clinical improvement was noticed.