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At least 19 recordsLinked to original sources
PubMed1991

99Tcm-exametazime-labelled leucocytes: effect of volume and concentration of exametazime on labelling efficiency, and clinical protocol for high efficiency multi-dose radiolabelling.

We have investigated the effect of volume and concentration of exametazime on the labelling efficiency of 99Tcm-exametazime-labelled leucocytes. The first study examined the effect of varying the volume of exametazine solution whilst the concentration remained constant. A vial of Ceretec (Amersham Ltd plc) was reconstituted with sodium chloride injection BP. Aliquots of 0.25-2.0 ml were removed, added to sodium pertechnetate injection BP and incubated with 2 x 10(7)-5 x 10(7) leucocytes from 25 ml blood. The labelling efficiency decreased from 65 +/- 10% S.D. (0.25 ml) to 45 +/- 8% (2.0 ml) (n = 4). In a second study different concentrations of exametazime solution were used whilst the volume remained constant. A vial of Ceretec was reconstituted with sodium chloride injection BP. Aliquots of 25-200 micrograms were removed, made up to a fixed volume of 0.6 ml, added to pertechnetate and incubated with the plug of leucocytes as before. The labelling efficiency decreased as the concentration of ligand decreased. Thus for 200 micrograms/0.6 ml the labelling efficiency was 64 +/- 5% and for 25 micrograms/0.6 ml the labelling efficiency was 43 +/- 18% (n = 4). Clinical studies were performed using 50 ml blood from patients with a wide range of inflammatory disorders including inflammatory bowel disease, abdominal abscess and vasculitis. The concentration of ligand used was 83 micrograms/0.25 ml. The labelling efficiency was found to be 82 +/- 7% (n = 36).

Butanones↗
PubMed2026

Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.

Brassica napus↗
PubMed1991

The developmentally regulated shift from membrane to secreted mu mRNA production is accompanied by an increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency.

To determine whether there are any developmental changes in the efficiencies of cleavage-polyadenylation or splicing reactions that could affect the usage of weak (suboptimal) processing signals and thus provide a basis for the regulated production of mu m versus mu s mRNA during B-lymphocyte maturation, we studied the expression of transfected mu genes in which the natural competition between cleavage-polyadenylation and splicing was replaced by alternative usage of tandem weak and strong poly(A) sites or by competition between suboptimal and optimal 5' splice junctions. Our results indicate that there is a 50 to 100% increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency as maturation proceeds from the B-cell to plasma cell stage.

Adenovirus Early Proteins↗
PubMed1975

A transforming marker that produces merodiploids with high efficiency and stable transformants with low efficiency in Streptococcus.

A mutation (ery-r8) conferring a high level of resistance to erythromycin in the Challis strain of Streptoccus sanguis can be transferred to wild-type erythromycin-sensitive recipients via single molecules of donor DNA. The transformants thus produced are of two types: (1) cells slightly more resistant to erythromycin than wild-type and capable of segregating (at a frequency of 2 X 10(-4)/bacterium/generation) either wild-type or highly-resistant cells like the original donor type; (2) cells phenotypically and genotypically identical to the original donor type. The unstable diploids (ery-r8/+) occur with a frequency equivalent to that obtained with high-efficiency (HE) markers, whereas the stable donor-type (ery-r8) transformants occur with about five hundred times lower frequency. Penetration of the wild-type recipient by more than one molecule of DNA bearing the ery-r8 marker increases by as much as seven times the incidence of stable transformants. UV-irradiation of molecules bearing the ery-r8 marker diminishes their ability to cooperate in producing a stable transformant, although the UV sensitivity of stable transformant production by a single DNA molecule is not different from that of diploid production. Hence, stable transformants do not appear to be produced by a process typical of low efficiency (LE) markers, which are generally highly sensitive to ultraviolet irradiation. Moreover, stable ery-r8 transformants are produced with equally low frequencies in strains of S. pneumoniae that discriminate (hex+) and fail to discriminate (hex--) between HE and LE markers. We postulate that all transformations by the ery-r8 marker result in ery-r8/+ diploids, and that segregation results in the infrequent stable transformants of the original donor type. This hypothesis is supported by the observations that rifampin treatment of ery-r8/+ populations increases the frequency of segregation and similar treatment of wild-type recipients under-going transformation by the ery-r8 marker increases the frequency of stable transformants.--In producing the ery-r8/+ transformant the r8 allele is integrated close to the site of its wild-type homolog, since single molecules of DNA from this transformant can be shown to carry both alleles. Segregation of either the ery-r8 or + allele is not detectably enhanced by acridine orange or thymidine deprivation.--The ery-r8 marker occurs close to a site of mutation (ery-r2) which confers erythromycin resistance upon ribosomes. When the r2 and r8 markers are jointly transferred, ery-r2-r8/+ genomes are produced in which the r2 marker is stably integrated but the r8 marker is unstably adjoined to its wild-type homolog. Thus, the duplicated region can be quite short. When the ery-r8 marker is stably integrated, the region of the marker is refractory to subsequent transformation. Markers with properties like ery-r8 are not particularly rare, being found with a frequency of about 4% among spontaneous mutations to erythromycin resistance.

Diploidy↗
PubMed1992

Energy conversion efficiency in human left ventricle.

BACKGROUND: Left ventricular mechanical efficiency is one of the most important measures of left ventricular pump performance. Several clinical studies, however, have shown that mechanical efficiency does not fall substantially as the heart fails. To clarify the insensitivity of mechanical efficiency to the change in pump performance, we analyzed human left ventricular mechanical efficiency, applying the concept of left ventricular systolic pressure-volume area (PVA). METHODS AND RESULTS: PVA correlates linearly with myocardial oxygen consumption per beat (MVO2): MVO2 = a.PVA+b, and represents the total mechanical energy of contraction. We determined MVO2-PVA relation and external work in 11 patients with different contractile states. We also calculated the energy transfer from MVO2 to PVA (PVA/MVO2 efficiency), that from PVA to external work (work efficiency), and mechanical efficiency (external work/MVO2). Left ventricular pressure-volume loops were constructed by plotting the instantaneous left ventricular pressure against the left ventricular volume at baseline and during pressure loading. The contractile properties of the ventricle were defined by the slope of the end-systolic pressure-volume relation (Ees). Pressure elevation raised external work by 41.4%, PVA by 71.2%, and MVO2 by 54.5%. These changes were associated with a decrease in work efficiency and an increase in PVA/MVO2 efficiency. The opposite directional changes in these two efficiencies rendered the mechanical efficiency constant. The slope, a, of the relation between MVO2 and PVA was relatively constant (2.46 +/- 0.33) over the range of 0.8-8.8 mm Hg/ml of Ees, but the oxygen axis intercept, b, tended to decrease with the reduction in Ees. PVA/MVO2 efficiency correlated inversely (r = -0.66, p less than 0.05) with Ees, whereas work efficiency correlated linearly with Ees (r = 0.91, p less than 0.01). CONCLUSIONS: Mechanical efficiency is not appreciably affected by changes in loading and inotropic conditions as long as the left ventricular contractility is not severely depressed.

Cardiac Catheterization↗
PubMed1985

The relative translation efficiencies of reovirus messenger RNAs.

The relative translation efficiencies of reovirus messenger RNAs were measured by infecting BHK cells with serotype 1, 2, or 3 virus and measuring the rate of formation of each viral protein species and the amount of each viral single-stranded (ss) RNA species present in the infected cells throughout the multiplication cycle. The translation efficiency of each ssRNA species was then calculated as a percentage of the species that was translated most efficiently. The relative translation efficiencies of the various ssRNA species did not change significantly during the multiplication cycle and cognate mRNA species of serotype 1, 2, and 3 virus were generally translated with similar efficiencies. However, the relative translation efficiencies of the individual ssRNA species differed greatly. The most efficiently translated ssRNA species was usually species s4, followed by species m2 which, on average, was translated about two-thirds as efficiently as species s4. Species s2 and s3 were translated slightly less than one-half as efficiently as species s4; species m3, l2, and l3 one-quarter to one-third as efficiently; and species s1 one-twentieth to one-tenth as efficiently. Finally, species l1 and m1 were translated very inefficiently under all conditions; their translation efficiencies were usually no more than 1% of that of species s4.

Animals↗
PubMed1990

The defibrillation success rate versus energy relationship: Part I--Curve fitting and the most efficient defibrillation energy.

The effect of applying an energy pulse to the heart during ventricular fibrillation is described by the probability of successful defibrillation or success rate. Seven to ten (8.60 +/- 0.84: mean +/- standard deviation) defibrillation trials per energy were randomly attempted at energies which span the defibrillation success rate versus energy curve. We obtained 70.0 +/- 8.4 episodes per dog. We fit the defibrillation success rate versus energy relationship from ten dogs (20.5 +/- 1.5 kg) to four types of curves: linear, exponential, probit transformed linear, and logit transformed linear. The correlation coefficients for each fit are 0.917 +/- 0.057, 0.944 +/- 0.014, 0.926 +/- 0.51, and 0.889 +/- 0.098, respectively. We therefore conclude that the exponential curve best describes the DSRE relationship. This suggests the existence of an energy below which defibrillation does not occur. At higher energies, the exponential curve asymptotically approaches a 100% success rate, which indicates that increasing the energy produces a diminishing benefit to defibrillation success rate. The estimated energies with a 0% defibrillation success rate are surprisingly consistent among dogs, with 2.072 +/- 0.553 J. The estimated energy with an 80% defibrillation success rate is 5.217 +/- 1.091 J. The estimated defibrillation success rate corresponding to the defibrillation threshold of 3.59 +/- 1.06 J is consistent with 0.516 +/- 0.144. The estimated energies with a 0% success rate correlate well with the defibrillation thresholds with R = 0.772; P = 0.0088. Since implantable defibrillators have a limited energy supply, we determined energy efficiency by dividing defibrillation success rate by the applied energy and energy consumption by dividing the applied energy by the defibrillation success rate. The most efficient defibrillation energy occurs at the maximum energy efficiency and the minimum energy consumption. The most efficient defibrillation energy of 4.34 +/- 0.97 J determined from the exponential fit has a success rate of 0.70 +/- 0.06. The most efficient defibrillation energy can be predicted from the defibrillation threshold. Clinically, a 70% success rate may not be adequate. We, therefore, compared the energy efficiency and consumption of energies with 90% and 95% success rates to the most efficient defibrillation energy. About a 50% increase in energy from the most efficient defibrillation energy is necessary for a 90% success rate which results in about a 13% loss in energy efficiency and about a 16% increase in energy consumption. About an 84% energy increase is necessary for a 95% success rate which results in about a 24% loss in energy efficiency and about a 33% increase in energy consumption.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1975

Muscular efficiency during steady-rate exercise: effects of speed and work rate.

In a comparison of traditional and theoretical exercise efficiency calculations male subjects were studied during steady-rate cycle ergometer exercises of "0," 200, 400, 600, and 800 kgm/min while pedaling at 40, 60, 80, and 100 rpm. Gross (no base-line correction), net (resting metabolism as base-line correction), work (unloading cycling as base-line correction), and delta (measurable work rate as base-line correction) efficiencies were computed. The result that gross (range 7.5-20.4%) and net (9.8-24.1%) efficiencies increased with increments in work rate was considered to be an artifact of calculation. A LINEAR OR SLIGHTLY EXPONENTIAL RELATIONSHIP BETWEEN CALORIC OUTPUT AND WORK RATE DICTATES EITHER CONSTANT OR DECREASING EFFICIENCY WITH INCREMENTS IN WORK. The delta efficiency (24.4-34.0%) definition produced this result. Due to the difficulty in obtaining 0 work equivalents, the work efficiency definition proved difficult to apply. All definitions yielded the result of decreasing efficiency with increments in speed. Since the theoretical-thermodynamic computation (assuming mitochondrial P/O = 3.0 and delta G = -11.0 kcal/mol for ATP) holds only for CHO, the traditional mode of computation (based upon VO2 and R) was judged to be superior since R less than 1.0. Assuming a constant phosphorylative-coupling efficiency of 60%, the mechanical contraction-coupling efficiency appears to vary between 41 and 57%.

Adult↗
PubMed1989

Predictions of thermodynamic efficiency in a pumped biochemical reaction.

We propose and analyze a possible experimental system for the investigation of the thermodynamic efficiency of generating biochemical gradients. We investigate the efficiency of a model pump that uses 6-phosphofructokinase (EC 2.7.1.11, an enzyme that exhibits highly nonlinear kinetics), chromatophores from Rhodobacter sphaeroides, and light to generate a biochemical gradient. We analyze the experimental system and an equivalent alternative configuration and show that the establishment and maintenance of a concentration gradient across a membrane is thermodynamically equivalent to the establishment and maintenance of a stationary state in a single-phase, isothermal, open, homogeneous reaction system. With a constant input of light, the system can exist in a stable node (a stable steady state), a stable focus (upon perturbation from its steady state, the system returns to its steady state with an oscillatory component), and a stable limit cycle (at steady state the system exhibits stable oscillations). We investigate the efficiency of the system with both steady and oscillatory light input and observe efficiency changes that depend upon the autonomous state of the system and the frequency and amplitude of the periodic light input. When the system is in a stable focus, an efficiency maximum is seen when the system is perturbed at its resonant frequency. When the system is in a stable limit cycle, efficiency increases are seen near the 1:3, 1:2, and 2:1 entrainment regions and an efficiency decrease is seen near the 1:1 entrainment region. We further calculate various contributions to the efficiency: the phase shift of the force and flux, the magnitude of the response to the perturbation, and changes in the average values of the force and flux during a perturbation. We show that all three changes contribute to the overall changes in efficiency, but increases and decreases in the average force make the largest contributions.

Kinetics↗
PubMed1989

"Worst case" aerosol testing parameters: I. Sodium chloride and dioctyl phthalate aerosol filter efficiency as a function of particle size and flow rate.

The efficiency of filter media is dependent on the characteristics of the challenge aerosol and the filter's construction. Challenge aerosol parameters, such as particle size, density, shape, electrical charge, and flow rate, are influential in determining the filter's efficiency. In this regard, a so-called "worst case" set of conditions has been proposed for testing respirator filter efficiency in order to ensure wearer protection. Data collected on various types of filters (dust and mist; dust, fume, and mist; paint, lacquer, and enamel mist; and high efficiency) challenged with a worst case-type sodium chloride (NaCl) and dioctyl phthalate (DOP) aerosol are presented. The particle size of maximum penetration varies as a function of filter type and was less than 0.25-micron count mean diameter (CMD) in all cases. The count efficiency for high efficiency filters was greater than 99.97% at worst case testing conditions, but the worst case count efficiencies for dust and mist; dust, fume and mist; and paint, lacquer and enamel mist filters were not nearly as efficient as existing test methods indicate. Also, as the test flow rate is increased, the count efficiency decreases. Thus, respirator filters were found to conform to the prediction of single-fiber filtration theory.

Aerosols↗
PubMed1990

The effect of hyperinflation on respiratory muscle strength and efficiency in healthy subjects and patients with asthma.

We evaluated respiratory muscle strength and efficiency in 15 patients with asthma. There was a significant reduction in mean Plmax (89.3 +/- 4.7 versus 110.5 +/- 9.4 cm H2O, p less than 0.001) and efficiency (2.41 +/- 0.2 versus 4.2 +/- 0.6%, p less than 0.01). This reduction in strength and efficiency was seen only in the male patients. Following bronchodilator, there was a significant increase in Plmax (from 89.3 +/- 4.7 to 96.2 +/- 5.4 cm H2O, p less than 0.005) and efficiency (from 2.41 +/- 0.2 to 3.22 +/- 0.2%, p less than 0.001). There was no correlation between the change in strength and efficiency and the degree of improvement in FEV1 following bronchodilator. However, there was a significant correlation with the fall in lung volume. To determine whether hyperinflation would result in a reduction in respiratory muscle strength and efficiency, we induced a mean increase in end-expiratory lung volume of 0.66 L by applying continuous negative pressure around the chest in 10 healthy individuals. This was associated with a significant fall in Plmax (from 110.5 +/- 9.4 to 100.5 +/- 8.93 cm H2O, p less than 0.001) and efficiency (from 4.2 +/- 0.6 to 2.6 +/- 0.5%, p less than 0.005). The data suggest that the strength and efficiency of the respiratory muscles are reduced in asthmatic males but not in the females. The strength and efficiency of the respiratory muscles improve significantly following bronchodilator, and this improvement is related to reduction in lung volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1992

Evaluation of various measures of and factors influencing feed efficiency of dairy cattle.

Early part records for milk yield and feed consumption of 2230 first lactation purebred and crossbred dairy cows were analyzed to evaluate various measures of feed efficiency. Corrected milk yield was estimated by adjusting the second 8 wk of milk yield for differences in weight of TDN consumed during wk 9 to 16, percentage of TDN derived from concentrate, and BW.75. Corrected milk yield is an estimate assuming that cows are the same size and consume the same amount of feed. Hence, it represents an expression of feed efficiency. Net feed efficiency, gross efficiency, corrected milk yield, and wk 9 to 16 milk were analyzed simultaneously. Coefficient of determination for net efficiency (.51) and gross efficiency (.72) were lower than that of milk (.82), whereas corrected milk yield had a coefficient of determination similar to that of milk. Hence, the use of ratios to define feed efficiency was less accurate than using corrected milk yield. Effects of genetic groups, stations, season of freshening, year of freshening, and heterosis were similar for gross efficiency and corrected milk yield, but different from those for milk. Therefore, corrected milk yield performed the same function as feed efficiency with higher accuracy.

Animals↗
PubMed2026

Investigating mechanisms of divergent feed efficiency in dairy cows.

Objectives were to investigate the associations between residual DMI (RFI), calculated as the difference between observed minus predicted DMI, with rumen microbiome, digestion, behavior, and metabolism that might explain the differences in RFI in lactating cows. One hundred 50 genotyped Holstein cows in 3 cohorts were used in this cohort study in which exposure was RFI. Rumen microbiota from 114 cows were sequenced, and a subset of 30 cows was used for hepatic mitochondrial respiration analysis. Cows were ranked by RFI and grouped into quartiles (Q1, most efficient, to Q4, least efficient) according to phenotypic (pQ) or genomic (gQ) quartiles of RFI for data presentation. Statistical models fitted the linear and quadratic RFI as continuous explanatory variables. Increasing efficiency, i.e., from larger to smaller RFI values, whether phenotypic or genomic, were associated with reduced DMI, a 3.0 kg/d difference between Q4 and Q1 according to phenotypic RFI (pRFI) and 1.9 kg/d according to genomic RFI (gRFI) without compromising ECM or body tissue reserves. These differences between Q4 and Q1 of pRFI and gRFI resulted in increased feed conversion ratio by an additional 200 and 100 g of ECM/kg DMI, respectively. Both pRFI and gRFI were associated with FA profiles in milk fat, with decreasing proportions of de novo and mixed FA and increasing proportions of pre-formed FA, particularly monounsaturated FA, as efficiency improved. Additionally, pRFI and gRFI were moderately correlated (r = 0.48) and ranking of cows was consistent across the 2 grouping methods (ρ = 0.44). Reducing RFI was associated with less total rumination time, but greater rumination time per kg of DMI by 2.0 and 1.7 min/kg between the extreme quartiles of pRFI and gRFI, respectively. Phenotypically and genomically more efficient cows were associated with less microbial α diversity based on inverse Simpson index. A total of 57 amplicon sequence variant groups were differentially abundant between Q1 and Q4 classified based on pRFI and gRFI, with Prevotella and Succinivibrionaceae shared between phenotypic and genomic RFI classifications. Increasing phenotypic and genomic efficiency was associated with an increased concentration of ruminal NH3-N. Genomically more efficient cows tended to have reduced ruminal pH (gQ1 to gQ4; 6.42 vs. 6.47 vs. 6.43 vs. 6.53) despite eating less. Decreasing pRFI was associated with reduced microbial N yield whereas, it tended to increase microbial N yield relative to the amount of N intake. Collectively, phenotypic and genomic RFI have a moderate degree of agreement matching the estimated heritability of the trait, and mechanisms underlying improved feed efficiency were linked with differences in ruminal microbiota and fermentation, and with increased rumination per kg of DM rather than total-tract digestibility or hepatic mitochondrial respiration.

dairy cow↗
PubMed1992

Myocardial oxygen consumption and mechanical efficiency of a perfused dogfish heart preparation.

Oxygen consumption of an in-pericardium heart preparation from the spiny dogfish (Squalus acanthias) was linearly related to cardiac power output. Basal oxygen consumption, predicted from the regression, was 0.127 microliters.s-1.g ventricle mass-1 and increased by 0.189 microliters.s-1.g ventricle mass-1 per milliwatt of power generated. From the relationship between cardiac power output and mechanical efficiency, mechanical efficiency was predicted to increase with cardiac power output to a maximum of 21%. Mechanical efficiency was measured during volume loading and pressure loading at two power outputs (50% and 72% of maximum power output). At 50% of maximum power output, mechanical efficiency increased significantly by 2.87%, from 11.9 +/- 0.3% to 14.8 +/- 0.5% (n = 7), when flow was halved and output pressure doubled to achieve the same power output. Similarly, at 72% of maximum power output, mechanical efficiency increased from 14.74 +/- 0.92% to 17.61 +/- 0.84% (n = 6) when flow was halved and output pressure doubled to generate the same higher level of power output. The increased mechanical efficiency at higher output pressures is believed to result from cardiac myocytes working within a length range where they are able to generate the most tension during contraction and are most efficient. We speculate that the loss of mechanical efficiency associated with large changes in sarcomere length, when stroke volume is large, is a driving force behind the use of frequency as the principal means of increasing cardiac output as observed in more active fishes, birds and mammals.

Animals↗
PubMed1989

Behavioural differences between laying hen populations selected for high and low efficiency of food utilisation.

1. The effects on behaviour of selecting light-hybrid laying hens for high and low efficiency of food utilisation were investigated. Efficiency of food utilisation was measured as the proportional deviation of observed food consumption from expected food consumption. 2. Videograms of the day-time behaviour of 48 to 53-week-old individually caged hens from the F3 generation were analysed by instantaneous sampling. 3. Low-efficiency hens spent more time food-pecking, walking, pacing, and showing escape and aggressive behaviour than did high-efficiency hens. High-efficiency hens spent more than twice as much time resting and sleeping, and were never observed pacing prior to laying. 4. These differences were larger during the 2 h before laying than at other times of day. 5. Low-efficiency hens had poorer plumage, especially on the neck and the breast, and the poorer the plumage the more agitation they showed. 6. Time spent food-pecking was negatively correlated with laying frequency. 7. Differences in the occurrence of specific behaviour patterns, particularly those symptomatic of pre-laying frustration, partly explained differences in efficiency of food utilisation. Selection for higher efficiency of food utilisation might eliminate hens which are most frustrated prior to laying in cages.

Aggression↗
PubMed2025

Robust and highly efficient transformation method for a minimal mycoplasma cell.

UNLABELLED: Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here, we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining the genome of Mycoplasma mycoides. When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, colony forming unit, and transformation efficiency, it was found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early exponential growth phase. Notably, the transformation efficiency of up to 4.4 × 10-2 transformants per cell per microgram of plasmid DNA was obtained. A method to obtain several hundred to several thousand transformants with less than 0.2 mL of culture with approximately 1 × 107-108 cells and 10 ng of plasmid DNA was developed. Moreover, a transformation method using a frozen stock of transformation-ready cells was established. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCE: Mycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand the cellular process of life and for bioengineering because of their simple metabolism, small genomes, and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here, we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtained with only 10 ng of plasmid DNA, which is around one-thousandth of the amount required for traditional mycoplasma transformations. Moreover, a convenient method using frozen stocks of transformation-ready cells was established. These improved methods play a crucial role in further studies using minimal cells.

Transformation, Bacterial↗
PubMed1991

A cellular function can enhance gene expression and plating efficiency of a mutant defective in the gene for ICP0, a transactivating protein of herpes simplex virus type 1.

ICP0 transactivates herpes simplex virus type 1 genes of all classes as well as a number of heterologous viral and cellular genes, yet it is not essential for virus replication in vitro or in vivo. Stocks of ICP0 deletion mutants, however, exhibit significantly lower plating efficiencies on standard 24-h-old Vero cell monolayers than do stocks of wild-type virus. In an attempt to determine whether the growth status of cells in the monolayer affects the ability of ICP0 mutants to initiate plaque formation, the plating efficiencies and abilities of an ICP0 null mutant (7134) and of wild-type virus (KOS) to express selected viral proteins were determined on Vero cell monolayers whose growth had been arrested either by contact inhibition-trypsinization or by isoleucine deprivation and had then been released from growth arrest. The proportion of cells cycling synchronously after release from growth arrest was assessed by flow cytometry. The results of these studies indicate that the plating efficiency of 7134 was greatest on Vero cell monolayers 8 h after release from growth arrest induced by either treatment. Monolayers of both types released from growth arrest at other times supported 7134 plaque formation less efficiently. In contrast, the plating efficiency of KOS was nearly equal on monolayers at all times after release from growth arrest. Notably, both KOS and 7134 were equally efficient in entering cells and inducing expression of the immediate-early protein ICP4 in either 8- or 24-h monolayers. Relative to KOS, however, 7134 was significantly impaired in the expression of selected early and late genes in cells at 24 h postrelease. When the plating efficiencies of 7134 and KOS were examined in 0-28 cells (Vero cells that are stably transformed with the ICP0 gene) whose growth had been arrested and then released, no differences in the plating efficiencies of the two viruses as a function of growth status were noted. These findings suggest that a cellular function expressed maximally in cells 8 h after release from growth arrest can substitute operationally for ICP0 to enhance plaque formation and viral gene expression by 7134. They further suggest that one role of ICP0 in viral infection is to facilitate virus replication in cells that do not express this function.

Animals↗