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PubMed2002

Effect of vessel size on the flow efficiency of the total cavopulmonary connection: in vitro studies.

The total cavopulmonary connection (TCPC) creates a passive system of blood flow into the pulmonary circulation. We hypothesize that the efficiency differences found in models with superior vena cava-inferior vena cava (SVC-IVC) offsets is dependent on vessel size, with flow efficiency improving with larger size vessels. Two sets of in vitro TCPC models (TCPC-3 and TCPC-15) were constructed corresponding to average vessel diameters of 3- and 15-year-old patients. The model with full SVC-IVC offset was the most efficient in TCPC-3 models. There was no significant difference between geometric arrangements in TCPC-15 models; however, the average efficiencies were significantly higher. Among the models representing vessel sizes of the younger age group, the model with the full-diameter SVC-IVC offset was the most efficient. The models representing vessel sizes of the older age group showed marked improvement in efficiencies throughout without obvious differences between various geometric arrangements. This suggests that geometric considerations in TCPC surgical techniques may be of lower than expected significance over the life span of a patient. More important, after balancing the effects of improved flow efficiency with larger vessels against the effects of chronic volume overload, the trend of performing the Fontan surgery on increasingly younger patients may need to be reconsidered after further studies.

Adolescent↗

PubMed2005

Eco-Efficiency Analysis of biotechnological processes.

Eco-Efficiency has been variously defined and analytically implemented by several workers. In most cases, Eco-Efficiency is taken to mean the ecological optimization of overall systems while not disregarding economic factors. Eco-Efficiency should increase the positive ecological performance of a commercial company in relation to economic value creation--or to reduce negative effects. Several companies use Eco-Efficiency Analysis for decision-making processes; and industrial examples of best practices in developing and implementing Eco-Efficiency have been reviewed. They clearly demonstrate the environmental and business benefits of Eco-Efficiency. An instrument for the early recognition and systematic detection of economic and environmental opportunities and risks for production processes in the chemical industry began use in 1997, since when different new features have been developed, leading to many examples. This powerful Eco-Efficiency Analysis allows a feasibility evaluation of existing and future business activities and is applied by BASF. In many cases, decision-makers are able to choose among alternative processes for making a product.

Biotechnology↗

PubMed2000

Non-invasive estimation of myocardial efficiency using positron emission tomography and carbon-11 acetate--comparison between the normal and failing human heart.

The clearance kinetics of carbon-11 acetate, assessed by positron emission tomography (PET), can be combined with measurements of ventricular function for non-invasive estimation of myocardial oxygen consumption and efficiency. In the present study, this approach was applied to gain further insights into alterations in the failing heart by comparison with results obtained in normals. We studied ten patients with idiopathic dilated cardiomyopathy (DCM) and 11 healthy normals by dynamic PET with 11C-acetate and either tomographic radionuclide ventriculography or cine magnetic resonance imaging. A "stroke work index" (SWI) was calculated by: SWI = systolic blood pressure x stroke volume/body surface area. To estimate myocardial efficiency, a "work-metabolic index" (WMI) was then obtained as follows: WMI = SWI x heart rate/k(mono), where k(mono) is the washout constant for 11C-acetate derived from monoexponential fitting. In DCM patients, left ventricular ejection fraction was 19%+/-10% and end-diastolic volume was 92+/-28 ml/m2 (vs 64%+/-7% and 55+/-8 ml/m2 in normals, P<0.001). Myocardial oxidative metabolism, reflected by k(mono), was significantly lower compared with that in normals (0.040+/-0.011/min vs 0.060+/-0.015/min; P<0.003). The SWI (1674+/-761 vs 4736+/-895 mmHg x ml/m2; P<0.001) and the WMI as an estimate of efficiency (2.98+/-1.30 vs 6.20+/-2.25 x 10(6) mmHg x ml/m2; P<0.001) were lower in DCM patients, too. Overall, the WMI correlated positively with ejection parameters (r=0.73, P<0.001 for ejection fraction; r=0.93, P<0.001 for stroke volume), and inversely with systemic vascular resistance (r=-0.77; P<0.001). There was a weak positive correlation between WMI and end-diastolic volume in normals (r=0.45; P=0.17), while in DCM patients, a non-significant negative correlation coefficient (r=-0.21; P=0.57) was obtained. In conclusion non-invasive estimates of oxygen consumption and efficiency in the failing heart were reduced compared with those in normals. Estimates of efficiency increased with increasing contractile performance, and decreased with increasing ventricular afterload. In contrast to normals, the failing heart was not able to respond with an increase in efficiency to increasing ventricular volume. The present data support the usefulness of the WMI for non-invasive characterization of cardiac efficiency and may serve as a background for improved evaluation of medical therapy for heart failure.

Acetates↗

PubMed2004

The labeling efficiency of human telomeres is increased by double-strand PRINS.

Telomeres are composed of tandem repeated sequences, TTAGGG, that can be detected either by fluorescence in situ hybridization (FISH), more efficiently by using a peptide nucleic acid (PNA) probe, or by the primed in situ (PRINS) technique. However, the efficiency of human telomere labeling using PRINS is somewhat lower than the efficiency using PNA-FISH. To solve this problem, we developed a double-strand PRINS technique, which uses two primers, (TTAGGG)(7) and (CCCTAA)(7), to label both forward and reverse telomeric DNA strands. A total of 120 lymphocyte metaphases obtained from three normal adults were scored to evaluate the labeling efficiency based upon the telomere signal frequency present in chromatid ends and chromosome arms. As a comparison, 30 metaphases from the same three individuals were evaluated using PNA-FISH. The average labeling efficiency of PRINS was increased to a level very close to that obtained with PNA-FISH. Therefore, we demonstrated that the low labeling efficiency of human telomeres with regular PRINS was likely caused by uneven annealing of primers at the relatively short human telomere sequences, resulting in some telomere sites with very weak or absent labeling. We suggest that the present double-strand labeling protocol is critical to maximize the labeling efficiency of the human telomere sequence when using the PRINS technique.

Adult↗

PubMed1999

Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in mismatch correction efficiencies between prokaryotic and eukaryotic cells.

Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies--but much more efficiently than transversion mispairs--as revealed by mutation rate increases in MMR mutants. To assess the relative efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold, respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction.

Base Pair Mismatch↗

PubMed1976

Relative efficiencies of a soluble and immobilized two-enzyme system of glucose oxidase and catalase.

Dual catalysts of varying glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6) activities were constructed by immobilization of the enzymes to silanized nickel silica alumina with glutaraldehyde. The amount of product formed and the efficiency of the systems were determined using either of the enzymes to initiate the cyclic reaction. The former depends on the activity of both enzymes in the system while efficiency describes how well the second enzyme in the sequence utilizes the intermediate of the reaction. Efficiency was a function both of the ratio of the two activities and their absolute values. An increase in the activity of the second enzyme of the reaction sequence of a decrease in the activity of the first enzyme increased the efficiency. A higher efficiency was observed with higher absolute activities of the enzymes when the ratios of the enzyme increased the efficiency. A higher efficiency was observed with higher absolute activities of the enzymes when the ratio of the enzymes was constant. Dual catalysts were compared to mixed catalysts (each enzyme immobilized to separate particles) and to soluble, homogeneous systems at equal total activities. In general, the dual catalysts were superior to either of the others. Computer simulations of the reaction rates indicated that all experiments were performed in the lag period of the two-step reaction.

Aluminum Silicates↗

PubMed1995

The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice.

Various combinations of promoters, introns and transcription terminators were used to drive the expression of bovine growth hormone (bGH) cDNA in different cell types. In constructs containing the human cytomegalovirus (hCMV) promoter and the SV40 late genes terminator, the intron from SV40 genes (VP1) was much more efficient, than the intron from the early genes (t). The synthetic intron SIS generated by the association of an adenovirus splice donor and an immunoglobulin G splice acceptor showed the highest activity. The respective potency of these introns was similar in several mammalian (CHO, HC11 and COS) and fish (TO2 and EPC) cells. The rabbit whey acidic protein (WAP) gene promoter was highly efficient to drive the expression of bGH gene in the HC11 mammary cell lines. In contrast, the bGH cDNA under the control of the same promoter was much less efficiently expressed when the SV40 VP1 intron and transcription terminator were used. The rabbit WAP gene and the human GH gene terminators did not or only moderately enhanced the expression of the construct WAP bGH cDNA. Introduction of a promoter sequence from the mouse mammary tumor virus (MMTV) LTR in the VP1 intron increased very significantly the expression of the WAP bGH cDNA. Although several of these vectors showed high potency when expressed stably in HC11 cells, all of them were only moderately efficient in transgenic mice. These data indicate that the VP1 and the SIS introns may be used to express foreign cDNAs with good efficiency in different cell types. The addition of an enhancer within an intron may still reinforce its efficiency. However, transfection experiments, even when stable expression is carried out, are poorly predictive of the potential efficiency of a vector in transgenic animals.

Animals↗

PubMed1990

Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system.

We have characterized the promoter region of the human elongation factor 1 alpha-encoding gene (EF-1 alpha) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1 alpha gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1 alpha gene: (i) ligated to the end of the TATA box (pEF220-CAT); (ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and (iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.

Animals↗

PubMed1994

Improved hemodynamic function and mechanical efficiency in congestive heart failure with sodium dichloroacetate.

OBJECTIVES: The purpose of this study was to determine whether sodium dichloroacetate improves hemodynamic performance and mechanical efficiency in congestive heart failure. BACKGROUND: Congestive heart failure is associated with impaired hemodynamic performance and reduced mechanical efficiency. Dichloroacetate stimulates pyruvate dehydrogenase activity by inhibition of pyruvate dehydrogenase kinase, which results in inhibition of free fatty acid metabolism and stimulation of high respiratory quotient glucose and lactate consumption by the heart. Facilitation of glucose and lactate consumption with dichloroacetate should improve mechanical efficiency of the failing ventricle. METHODS: Ten patients with New York Heart Association functional class III to IV congestive heart failure were studied. Dichloroacetate (50 mg/kg body weight) was administered intravenously for 30 min, with measurements of hemodynamic variables, coronary sinus blood flow and blood gas, glucose and lactate levels for 2 h. The same patients were also given dobutamine (5 to 12.5 micrograms/kg per min) for comparison. RESULTS: Therapeutic levels of dichloroacetate were achieved (100 to 160 micrograms/liter of plasma). Myocardial consumption of lactate was stimulated from 29% to 37.4%. Forward stroke volumes increased (+5.3 ml/beat, p < 0.02), as did left ventricular stroke work (+1.8 g-m/m2 per beat, p < 0.02) and left ventricular minute work (from 1.38 to 1.55 kg-m/m2 per min, p < 0.01). Myocardial oxygen consumption decreased (from 19.3 to 16.5 ml/min, p = 0.06) as left ventricular minute work increased. Left ventricular mechanical efficiency thus improved from 15.2% to 20.6% (p = 0.03). Dobutamine administration resulted in the opposite trend with respect to myocardial lactate extraction (from 34% to 15.3%, p < 0.02). Stroke volume increased (+7.4 ml/beat, p = NS vs. dichloroacetate), as did left ventricular minute work (from 1.29 to 1.59 g-m/m2 per min, p < 0.01 vs. dichloroacetate) and myocardial oxygen consumption (from 18.6 to 21.0 ml/min, p = 0.06 vs. dichloroacetate). Left ventricular mechanical efficiency did not change with dobutamine administration (from 16.4% to 15.8%, p = NS). CONCLUSIONS: Dichloroacetate administration stimulates myocardial lactate consumption and improves left ventricular mechanical efficiency. Forward stroke volume and left ventricular minute work increase significantly, with a simultaneous reduction in myocardial oxygen consumption. Dobutamine administration results in similar hemodynamic improvements but with no change in left ventricular mechanical efficiency and with opposite effects on lactate metabolism. The opposing metabolic actions, yet similar hemodynamic responses, of dichloroacetate and dobutamine suggest that these agents may be complementary in the treatment of congestive heart failure.

Dichloroacetic Acid↗

PubMed2005

Pyridinium cationic lipids in gene delivery: an in vitro and in vivo comparison of transfection efficiency versus a tetraalkylammonium congener.

Cationic lipids provide a promising alternative to the use of viruses for delivering genes therapeutically. Among the several classes of lipidic vectors, those bearing a heterocyclic cationic head have shown important advantages, such as low cytotoxicity and improved efficiency across different cell lines. We recently reported a simple and efficient strategy for obtaining pyridinium cationic lipids, starting from pyrylium salts and primary amines. The present study is aimed to compare the cellular toxicity and transfection efficiency generated by the pyridinium polar head versus the tetramethylammonium one on several tumor cell lines and also in experimental animals, delivered via intratumor injections. Thus, the lead compound 1-(2,3-dioleoyloxypropyl)-2,4,6-trimethylpyridinium lipid (2Oc), coformulated with different helper lipids in various molar ratios, was tested against its ammonium congener DOTAP-a standard transfection reagent. The results revealed that when formulated with cholesterol at 1:1 molar ratio, the pyridinium lipid 2Oc was able to transfect several cancer cell lines with similar or better efficiency than its tetraalkylammonium congener DOTAP, while producing lower cytotoxicity. The NCI-H23 lung cancer cell line was found to be the most susceptible to be transfected. Therefore, we designed an in vivo assay based on this type of carcinoma in nude mice, which were injected intratumoral with 2Oc- and DOTAP-based lipoplexes. The red fluorescent protein reporter revealed that the pyridinium cationic lipid was superior to its tetraalkylammonium congener, transfecting the tissue on a higher area and with higher efficiency. These encouraging findings, together with the simple and efficient synthetic strategy, lay the foundation for further development of pyridinium lipids for gene therapy with improved transfection efficiency in vivo and even further reduced cytotoxicity.

Animals↗

PubMed2006

An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency.

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.

Amino Acid Motifs↗

PubMed2006

Optimization of 25 kDa linear polyethylenimine for efficient gene delivery.

A 25-kDa linear polyethylenimine (25 kDa L-PEI) has proven to be efficient and versatile agent for gene delivery. Therefore, we determined the optimal transfection conditions of 25 kDa L-PEI and examined whether it has comparable transfection efficiency with other commercially available reagents, ExGen 500, LipofectAMINE 2000, and Effectene by using EGFP expression vector in different cell lines. Transfection efficiency and cytotoxicity were measured by flow cytometry. First of all, we determined the optimal ratio of nitrogen to phosphorous (N/P) and DNA concentration. With the increase of N/P ratio and DNA amounts, transfection efficiency increased with a slight variation in cell types. The optimal amounts of 25 kDa L-PEI were determined at N/P ratio 40 and DNA concentration varied among the cell types. In addition, 25 kDa L-PEI worked efficiently and was less toxic than other reagents. However, the efficiency and toxicity of all these reagents varied according to cell types as well as the ratio of DNA to reagents and the amounts of DNA. Our finding illustrates the importance of optimal transfection conditions of 25 kDa L-PEI to obtain maximal transgene expression with less cytotoxicity. Importantly, the optimization of those conditions may make possible to perform transfection cost-effectively and efficiently.

Animals↗

PubMed2005

The initiating radical yields and the efficiency of polymerization for various dental photoinitiators excited by different light curing units.

OBJECTIVES: To evaluate the efficiency of the photopolymerization of dental resins it is necessary to know to what extent the light emitted by the light curing units is absorbed by the photoinitiators. On the other hand, the efficiency of the absorbed photons to produce species that launch the polymerization process is also of paramount importance. Therefore, the previously determined PAE (photon absorption efficiency) is used in conjunction with the polymerization quantum yields for the photoinitiators, in order to be able to compare the total process on an equivalent basis. This parameter can be used to identify the best performance for the photochemical process with specific photoinitiators. METHODS: The efficiency of LED (Ultrablue IS) and QTH (Optilux 401) lamps were tested comparing their performances with the photoinitiators camphorquinone (CQ); phenylpropanedione (PPD); monoacylphosphine oxide (Lucirin TPO); and bisacylphosphine oxide (Irgacure 819). The extent of photopolymerization per absorbed photon was determined from the polymerization quantum yields obtained by using the photoinitiators to polymerize methyl methacrylate, and afterwards combined with the previously determined PAEs. RESULTS: Although CQ presents a rather low polymerization quantum yield, its photopolymerization efficiency is practically the highest when irradiated with the Ultrablue LED. On the other hand, Lucirin is much more efficient than the other photoinitiators when irradiated with a QTH lamp, due to its high quantum yield and the overlap between its absorption spectrum and the output of the visible lamp light. SIGNIFICANCE: Difference in photopolymerization efficiencies arise when combinations of photoinitiators are used, and when LED sources are used in preference to QTH. Mechanistic understanding is essential to optimal initiator formulation.

4-Aminobenzoic Acid↗

PubMed2006

Determining the best ventilatory efficiency measure to predict mortality in patients with heart failure.

BACKGROUND: Ventilatory efficiency, the relationship between ventilation (VE) and carbon dioxide production (VCO2), predicts mortality in heart failure patients, but its determination has not been standardized. Additionally, it is unclear if the prognostic power of ventilatory efficiency is independent of exercise intensity. Therefore, we investigated the relative prognostic power of different measures of ventilatory efficiency calculated from maximal and sub-maximal exercise in patients with heart failure. METHODS AND RESULTS: Heart failure patients (n = 355, 72% males, age 51 +/- 10 years) had follow-up for at least 5 years from an exercise test. There were 145 events (133 deaths and 12 emergent cardiac transplants). Ventilatory efficiency calculations were not equivalent. Of the different measures of ventilatory efficiency, the VE/VCO2 slope to peak exercise was the most significant predictor of mortality in a multivariable Cox model, including ejection fraction, systolic blood pressure, peak oxygen consumption (VO2), gender, etiology, and heart rate. A 5 U increment of the VE/VCO2 slope to peak exercise corresponded to a 9% increase in mortality risk. When tests were grouped by peak exercise intensity, by quartiles of peak respiratory exchange ratio, the VE/VCO2 slope to peak exercise was always a better predictor than peak VO2. CONCLUSION: Peak and sub-maximal measures of ventilatory efficiency were not equivalent, and the VE/VCO2 slope to peak exercise was the best predictor of mortality in patients with heart failure. Thus, the prognostic power of ventilatory efficiency is enhanced when exercise extends beyond the ventilatory threshold and includes all of the available exercise data.

Adult↗

PubMed2007

Ionic separation efficiency of a novel electric-field-assisted membrane module comprising an array of microchannel units.

The ionic separation efficiency of a novel membrane module comprising an array of microchannel units is analyzed. Under the Debye-Hückel approximation, we derive a semianalytical expression for the ionic separation efficiency. Analyses reveal that the effects of the size of the microchannel, the fixed charge density in the membrane layer, and the permittivity of the membrane layer on ionic separation efficiency depend strongly on the valence type of electrolyte in treated water. Under the condition of a symmetric electrolyte, the ionic separation efficiency is found to be about unity and unresponsive to variation of system parameters. If the valence of the cation is higher than that of the anion, the ionic separation efficiency is larger than unity, and decreases to unity as the size of the microchannel increases. In contrast, if the valence of the cation is lower than that of the anion, the ionic separation efficiency is smaller than unity and increases to unity as the size of the microchannel increases. Under the latter two conditions, the effects of both fixed charge density in the membrane layer and permittivity of the membrane layer on the ionic separation efficiency are found to be reversed.

Journal Article↗

PubMed2005

Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation.

Chitosans of defined molecular weight (Mw 10-213 kDa) and degree of deacetylation (DD 46-88%) were synthesized, complexed with pEGFP-C2 plasmid into nanoparticles (NP) and evaluated for cellular uptake and transfection efficiency in the A549 cell model. DNA condensation of >90% was achieved at the N/P ratio of 6, independent of the chitosan Mw and DD. However, chitosan vectors of lower Mw or DD were less efficient at retaining the DNA upon dilution, and consequentially, less capable of protecting the condensed DNA from degradation by DNase and serum components. A549 cellular uptake of the NP was also significantly reduced by decreasing the Mw or DD of the chitosan vector. These factors contributed to the low transfection efficiencies for chitosan vectors of low Mw or DD. There was good correlation between transfection efficiency, cellular uptake and zeta potential of the NP, suggesting that cellular uptake mediated by electrostatic interactions with the cell membrane preceded efficient transfection. NP produced with chitosan of Mw 213 kDa and DD of 88% showed the highest zeta potential (+23 mV), cellular uptake (4.1 microg/mg protein) and transfection efficiency (12.1%), while chitosan vector with Mw of 213 kDa and DD of 46% showed the lowest cellular uptake (0.4 microg/mg protein) and transfection efficiency (0.05%). Confocal microscopy images suggested that the chitosan-complexed DNA successfully escaped from the endo-lysosomal compartment for nuclear translocation and expression. Intracellular DNA disassembly appeared to occur at different locations depending on the retentive capacity of the chitosan vector.

Acetylation↗

PubMed2006

Making V(D)J rearrangement visible: quantification of recombination efficiency in real time at the single cell level.

V(D)J recombination is of fundamental importance for the diversity of immunoglobulin and T cell receptor genes. An enhanced green fluorescent protein (EGFP) based assay was successfully developed to monitor V(D)J recombination efficiency. This assay makes V(D)J recombination visible at the single cell level in real time. Surprisingly, despite a high (60% to 90%) transfection efficiency, the EGFP based V(D)J recombination efficiency was found to be low ( approximately 1%) in 293 cells. The EGFP based V(D)J recombination efficiency correlated well with that achieved by the classical V(D)J recombination assay. The EGFP based V(D)J recombination efficiency depended on the relative RAG (recombination activating gene)-1 and RAG-2 but not Artemis expression vector concentrations used for co-transfection. A rise of RAG-1 dosage increased recombination efficiency. In contrast, a surplus of RAG-2 inhibited V(D)J recombination efficiency. The test differentiates RAG null mutants as seen in human severe combined immunodeficiency (SCID).

Base Sequence↗

PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗