Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

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Effects of microwave and ultrasound on the extraction of pectin and its chemical characterisation of banana (Musa sapientum L.) peels

Food Research ◽

10.26656/fr.2017.4(6).248 ◽

2020 ◽

Vol 4 (6) ◽

pp. 2030-2036

Author(s):

A. Phaiphan ◽

S. Churat ◽

T. Dougta ◽

P. Wichalin ◽

W. Khanchai ◽

...

Keyword(s):

Microwave Power ◽

Extraction Time ◽

High Yield ◽

Chemical Characteristics ◽

Ultrasound Assisted Extraction ◽

Degree Of Esterification ◽

Yield And Quality ◽

Assisted Extraction ◽

High Level

Pectin is a polysaccharide that is widely used as a food additive in food industries. Banana peels that are discarded by vendors after processing bananas for food products contain a high level of pectin. The pectin can be extracted from the plant cell walls whereas the conventional method for extraction would normally take some time to get a high yield. This research was conducted to explore the effects of microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) on the extraction of pectin and its chemical characteristics found in banana peels. For MAE, the effects of microwave power and extraction time on the yield and quality of extracted pectin was studied. It was found that the pectin yield and chemical characteristics increased with the increase of microwave power and extraction time. The percentage yield ranged from 20.93% to 22.91% for microwave power of 300 W for 5-15 mins. The moisture, ash, degree of esterification, methoxyl, and galacturonic content of extracted pectin by MAE had 8.98%, 5.40%, 75.50%, 12.00%, and 57.80%, respectively. For UAE, the effects of temperature and sonication time on the pectin yield and quality of the extracted pectin were also studied. The pectin yields ranged from 1.81% to 3.49% and the moisture, ash, degree of esterification, methoxyl, and galacturonic content of extracted pectin by UAE had 8.00%, 3.39%, 88.83%, 14.50%, and 90.07%, respectively. Although the use of MAE promoted a high pectin yield it had a lower purity level when compared to UAE. Besides, the chemical characteristics of the extracted pectin by UAE is more comparable with commercial citrus pectin than the pectin extracted by MAE.

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Resistance of Cucumber Cultivars to a New Strain of Cucurbit Downy Mildew

HortScience ◽

10.21273/hortsci.47.2.171 ◽

2012 ◽

Vol 47 (2) ◽

pp. 171-178 ◽

Cited By ~ 17

Author(s):

Adam D. Call ◽

Adam D. Criswell ◽

Todd C. Wehner ◽

Kaori Ando ◽

Rebecca Grumet

Keyword(s):

North Carolina ◽

Downy Mildew ◽

The United States ◽

Leaf Damage ◽

High Yield ◽

Breeding Lines ◽

Yield And Quality ◽

Resistant Lines ◽

High Level ◽

Moderately Resistant

Downy mildew, a foliar disease caused by the oomycete Pseudoperonospora cubensis (Berk. and Curt.) Rostow, is one of the most destructive pathogens of cucurbits. From 1961 to 2003, resistant cucumber cultivars in the United States had sufficient resistance to grow a successful crop without the use of fungicides. The pathogen resurged as a major problem in 2004. Since then, the dm-1 gene has not been effective against the new strain of downy mildew, and yield losses are high without the use of fungicides. The objective of this experiment was to identify cultivars having high yield and resistance to the new downy mildew. The experiment had 86 cultivars and breeding lines (hereafter collectively referred to as cultigens) and was conducted in Clinton, NC, in 2007 and 2009, in Castle Hayne, NC, in 2008 and 2009, and in Bath, MI, in 2007 to 2009. Plots were rated weekly on a 0 to 9 scale (0 = none, 1–2 = trace, 3–4 = slight, 5–6 = moderate, 7–8 = severe, and 9 = dead). Mean ratings for downy mildew leaf damage ranged from 2.9 to 5.7 in Michigan in 2008 and 2009 and from 3.8 to 6.8 in North Carolina in 2007 to 2009. None of the cultigens tested in this study showed a high level of resistance, although differences in resistance were detected. Lines WI 2757 and M 21 and cultivar Picklet were consistently among the top resistant lines in North Carolina and Michigan. The cultivars Coolgreen, Wis. SMR 18, and Straight 8 were identified as moderately to highly susceptible. An unreleased hybrid, ‘Nun 5053 F1’, and the cultivar Cates were the top yielding lines overall. The highest yield in a single year and location was from the cultivar Cates in Clinton, NC, in 2009, with 25.6 Mg·ha−1. The best cultivars in this study were only moderately resistant and would likely require fungicide applications to achieve high yield and quality in the presence of downy mildew. Until high resistance becomes available, growers would benefit by using fungicides in combination with tolerant and moderately resistant cultigens.

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Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657308 ◽

1983 ◽

Vol 49 (01) ◽

pp. 024-027 ◽

Cited By ~ 20

Author(s):

David Vetterlein ◽

Gary J Calton

Keyword(s):

Monoclonal Antibody ◽

Light Chain ◽

Culture Media ◽

Biological Fluids ◽

Single Step ◽

High Yield ◽

Step Method ◽

Commercial Preparation ◽

E Coli ◽

Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

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Territorial Distribution of Alcohol and Drug Addictions Mortality Concerning Regional Disparities in the Slovak Republic from Year 1996 to Year 2015

10.35198/01-2019-003-0005 ◽

2020 ◽

Keyword(s):

Mortality Rate ◽

Slovak Republic ◽

Regional Disparities ◽

Point Of View ◽

Extreme Position ◽

Data Set ◽

Female Sex ◽

Male Sex ◽

The Individual ◽

High Level

BACKGROUND: This paper deals with territorial distribution of the alcohol and drug addictions mortality at a level of the districts of the Slovak Republic. AIM: The aim of the paper is to explore the relations within the administrative territorial division of the Slovak Republic, that is, between the individual districts and hence, to reveal possibly hidden relation in alcohol and drug mortality. METHODS: The analysis is divided and executed into the two fragments – one belongs to the female sex, the other one belongs to the male sex. The standardised mortality rate is computed according to a sequence of the mathematical relations. The Euclidean distance is employed to compute the similarity within each pair of a whole data set. The cluster analysis examines is performed. The clusters are created by means of the mutual distances of the districts. The data is collected from the database of the Statistical Office of the Slovak Republic for all the districts of the Slovak Republic. The covered time span begins in the year 1996 and ends in the year 2015. RESULTS: The most substantial point is that the Slovak Republic possesses the regional disparities in a field of mortality expressed by the standardised mortality rate computed particularly for the diagnoses assigned to the alcohol and drug addictions at a considerably high level. However, the female sex and the male sex have the different outcome. The Bratislava III District keeps absolutely the most extreme position. It forms an own cluster for the both sexes too. The Topoľčany District bears a similar extreme position from a point of view of the male sex. All the Bratislava districts keep their mutual notable dissimilarity. Contrariwise, evaluation of a development of the regional disparities among the districts looks like notably heterogeneously. CONCLUSIONS: There are considerable regional discrepancies throughout the districts of the Slovak Republic. Hence, it is necessary to create a common platform how to proceed with the solution of this issue.

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Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-67-72 ◽

2019 ◽

Vol 35 (6) ◽

pp. 67-72 ◽

Cited By ~ 1

Author(s):

I.V. Manukhov ◽

L.S. Yaguzhinsky ◽

M.V. Bermeshev ◽

M.A. Zisman ◽

V.G. Pevgov ◽

...

Keyword(s):

Toxic Effect ◽

Atmospheric Oxygen ◽

Inducible Promoter ◽

Oxygen Species ◽

Bacterial Cells ◽

Unique Identifier ◽

E Coli ◽

Lux Biosensors ◽

High Level ◽

Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

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Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

International Journal of Bioassays ◽

10.21746/ijbio.2017.04.004 ◽

2017 ◽

Vol 6 (04) ◽

pp. 5347 ◽

Cited By ~ 3

Author(s):

Omar B. Ahmed* ◽

Anas S. Dablool

Keyword(s):

Dna Extraction ◽

Control Method ◽

Extraction Procedure ◽

Cost Effective ◽

High Yield ◽

Gram Negative Bacteria ◽

Bacterial Dna ◽

Gram Negative ◽

E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

BIO-PROTOCOL ◽

10.21769/bioprotoc/bio101.30 ◽

2011 ◽

Vol 1 (3) ◽

Cited By ~ 1

Author(s):

Fanglian He

Keyword(s):

Dna Extraction ◽

Plasmid Dna ◽

Alkaline Lysis ◽

E Coli

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Identification of the Optimum Environments for the High Yield and Quality Traits of Lentil Genotypes Evaluated in Multi-Location Trials

Sustainability ◽

10.3390/su13158247 ◽

2021 ◽

Vol 13 (15) ◽

pp. 8247

Author(s):

Dimitrios N. Vlachostergios ◽

Christos Noulas ◽

Anastasia Kargiotidou ◽

Dimitrios Baxevanos ◽

Evangelia Tigka ◽

...

Keyword(s):

Crude Protein ◽

Agronomic Traits ◽

High Yield ◽

Breeding Programs ◽

Growing Seasons ◽

Yield And Quality ◽

Food And Feed ◽

Superior Genotypes ◽

Selection Of ◽

The Ideal

Lentil is a versatile and profitable pulse crop with high nutritional food and feed values. The objectives of the study were to determine suitable locations for high yield and quality in terms of production and/or breeding, and to identify promising genotypes. For this reason, five lentil genotypes were evaluated in a multi-location network consisting of ten diverse sites for two consecutive growing seasons, for seed yield (SY), other agronomic traits, crude protein (CP), cooking time (CT) and crude protein yield (CPY). A significant diversification and specialization of the locations was identified with regards to SY, CP, CT and CPY. Different locations showed optimal values for each trait. Locations E4 and E3, followed by E10, were “ideal” for SY; locations E1, E3 and E7 were ideal for high CP; and the “ideal” locations for CT were E3 and E5, followed by E2. Therefore, the scope of the cultivation determined the optimum locations for lentil cultivation. The GGE-biplot analysis revealed different discriminating abilities and representativeness among the locations for the identification of the most productive and stable genotypes. Location E3 (Orestiada, Region of Thrace) was recognized as being optimal for lentil breeding, as it was the “ideal” or close to “ideal” for the selection of superior genotypes for SY, CP, CT and CPY. Adaptable genotypes (cv. Dimitra, Samos) showed a high SY along with excellent values for CP, CT and CPY, and are suggested either for cultivation in many regions or to be exploited in breeding programs.

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1671. Diagnosis and management of Escherichia coli bacteriuria at a Veterans Affairs hospital

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1849 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S821-S821

Author(s):

Niyati H Shah ◽

Brooke K Decker ◽

Gaetan Sgro ◽

Monique Y Boudreaux-Kelly ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Treatment ◽

High Yield ◽

Comorbid Conditions ◽

Provider Education ◽

Transmitted Infection ◽

Physician Practices ◽

E Coli ◽

Diagnosis And Management ◽

Tract Infections

Abstract Background The IDSA recommends against screening for and treating ASB in all patients except for those pregnant or undergoing urologic procedures. Nevertheless, antibiotic treatment of ASB is widespread. We conducted a retrospective analysis of physician practices in diagnosis and management of Escherichia coli (E. coli) ASB in a male Veteran population, and compared outcomes in ASB patients treated or not treated with antibiotics. Methods Patients with an E. coli positive urine culture during an ED visit or inpatient admission from 01/2017 to 12/2017 were screened. Patients admitted to the intensive care unit or diagnosed with a sexually transmitted infection, pyelonephritis, prostatitis, or epididymitis/orchitis were excluded. A total of 163 patients were included. Demographics, clinical comorbidities and severity of illness, and outcomes were compared in ASB patients managed with or without antibiotics. ANOVA and Chi-square or Fisher’s exact tests were utilized for comparing measurements. Results ASB was present in 92/163 patients. The majority (74%) of these patients were given antibiotics. Regardless of qSOFA score or alternate infection, there were no significant differences in outcomes between ASB patients treated or not treated with antibiotics: 3-month mortality (15% vs 21%; p = 0.53), emergence of newly resistant bacterial pathogens (7% vs 13%; p = 0.43), recurrent urinary tract infections (61% vs 50%; p = 0.72), clearance of urinary pathogens (75% vs 58%; p = 0.45), length of hospital stay (7 vs 6 days, p = 0.67). Factors that were predictive of physician treatment of ASB included patient comorbid conditions such as benign prostatic hyperplasia, pyuria, and the absence of hematuria. The incidence of adverse events with antibiotic treatment of ASB was low. Conclusion The rate of antibiotic treatment of E. coli ASB in male veterans is high. Outcomes do not differ among ASB patients managed with or without antibiotics. Future studies examining outcomes in patients prescribed antibiotics for multiple episodes of ASB may yield differences, particularly in emergence of resistant pathogens. Focusing on patients with comorbid conditions who are not critically ill would be a high yield target for provider education to reduce ASB treatment. Disclosures All Authors: No reported disclosures

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