Viability and reconstitution of delta-endotoxins from Bacillus thuringiensis var. israelensis extracts after forty years of storage against Aedes aegypti (Diptera: Culicidae)

Abstract Background Bacillus thuringiensis subsp. israelensis (Bti) produces insecticidal endotoxins known as Cry and Cyt. Its efficiency and specificity make it the most widely used substance as a biopesticide for controlling disease from vector insects, such as mosquitoes, responsible for important human diseases such as malaria, filariasis, dengue, and yellow fevers. To date, it is proven difficult to develop a commercial product that has more than 2 years of shelf life, and there is little information on the viability of these commercial proteins under prolonged storage conditions. Results This study aimed to evaluate biological activity of reconstituted Bti endotoxins after 40 years of storage against the mosquito Aedes aegypti larvae. Five concentrations of Bti extracts were used for bioassays against 3rd and 4th instars of A. aegypti larvae. All reconstituted endotoxins from stored extracts showed a potency increase. The strain HD-500 from extract 3260 was the most effective insecticide (LC50 = 0.0014 mg/l), followed by 3756 (LC50 = 0.0037 mg/l). These strains were particularly notable, increasing their larvicidal potency one hundredfold and one thousandfold, respectively. Protein profiles in polyacrylamide gels revealed a greater presence of Cyt toxins compared to the stored Bti extracts, which maintained their activity at high concentrations. Conclusion The reconstituted Bti strains presented a great biological activity against A. aegypti larvae, specially extract 3260 (median lethal concentration (LC50) value = 0.0014 mg/l). This considerable larvicidal activity after 40 years under storage was an encouraging signal for the development of future formulation strategies regarding their useful life. The stability of extracts of stored endotoxins produced by Bti decreased significantly, particularly Cyt1A protein, which is responsible for their synergistic activity.

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Conjugal Transfer of a Toxin-Coding Megaplasmid from Bacillus thuringiensis subsp. israelensis to Mosquitocidal Strains of Bacillus sphaericus

Applied and Environmental Microbiology ◽

10.1128/aem.72.3.1766-1770.2006 ◽

2006 ◽

Vol 72 (3) ◽

pp. 1766-1770 ◽

Cited By ~ 18

Author(s):

Katherine Gammon ◽

Gareth W. Jones ◽

Steven J. Hope ◽

Cláudia M. F. de Oliveira ◽

Lêda Regis ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Aedes Aegypti ◽

Culex Quinquefasciatus ◽

Vegetative Growth ◽

Bacillus Sphaericus ◽

Conjugal Transfer ◽

Large Plasmid ◽

Erythromycin Resistance ◽

Plasmid Loss ◽

The Stability

ABSTRACT Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.

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Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae

Toxins ◽

10.3390/toxins12060355 ◽

2020 ◽

Vol 12 (6) ◽

pp. 355 ◽

Cited By ~ 1

Author(s):

Daniel Valtierra-de-Luis ◽

Maite Villanueva ◽

Liliana Lai ◽

Trevor Williams ◽

Primitivo Caballero

Keyword(s):

Bacillus Thuringiensis ◽

Aedes Aegypti ◽

Recombinant Proteins ◽

Synergistic Activity ◽

Minor Components ◽

Parasporal Crystal ◽

Synergistic Interactions ◽

Bt Toxins ◽

Mosquitocidal Activity ◽

The Individual

Bacillus thuringiensis ser. israelensis (Bti) has been widely used as microbial larvicide for the control of many species of mosquitoes and blackflies. The larvicidal activity of Bti resides in Cry and Cyt δ-endotoxins present in the parasporal crystal of this pathogen. The insecticidal activity of the crystal is higher than the activities of the individual toxins, which is likely due to synergistic interactions among the crystal component proteins, particularly those involving Cyt1Aa. In the present study, Cry10Aa and Cyt2Ba were cloned from the commercial larvicide VectoBac-12AS® and expressed in the acrystalliferous Bt strain BMB171 under the cyt1Aa strong promoter of the pSTAB vector. The LC50 values for Aedes aegypti second instar larvae estimated at 24 hpi for these two recombinant proteins (Cry10Aa and Cyt2Ba) were 299.62 and 279.37 ng/mL, respectively. Remarkable synergistic mosquitocidal activity was observed between Cry10Aa and Cyt2Ba (synergistic potentiation of 68.6-fold) when spore + crystal preparations, comprising a mixture of both recombinant strains in equal relative concentrations, were ingested by A. aegypti larvae. This synergistic activity is among the most powerful described so far with Bt toxins and is comparable to that reported for Cyt1A when interacting with Cry4Aa, Cry4Ba or Cry11Aa. Synergistic mosquitocidal activity was also observed between the recombinant proteins Cyt2Ba and Cry4Aa, but in this case, the synergistic potentiation was 4.6-fold. In conclusion, although Cry10Aa and Cyt2Ba are rarely detectable or appear as minor components in the crystals of Bti strains, they represent toxicity factors with a high potential for the control of mosquito populations.

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Replicates Number for Drug Stability Testing during Bioanalytical Method Validation—An Experimental and Retrospective Approach

Molecules ◽

10.3390/molecules27020457 ◽

2022 ◽

Vol 27 (2) ◽

pp. 457

Author(s):

Elżbieta Gniazdowska ◽

Wojciech Goch ◽

Joanna Giebułtowicz ◽

Piotr J. Rudzki

Keyword(s):

Sample Size ◽

Confidence Intervals ◽

Method Validation ◽

Drug Stability ◽

Storage Conditions ◽

Stability Testing ◽

Bioanalytical Method Validation ◽

Bioanalytical Method ◽

High Concentrations ◽

The Stability

Background: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. Methods: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes—tramadol and its major metabolite (O-desmethyl-tramadol)—in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). Results: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85–115% acceptance criteria. Conclusions: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

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Influence of Storage Conditions on the Quality, Metabolites, and Biological Activity of Soursop (Annona muricata. L.) Kombucha

Frontiers in Microbiology ◽

10.3389/fmicb.2020.603481 ◽

2020 ◽

Vol 11 ◽

Author(s):

Wee Ching Tan ◽

Belal J. Muhialdin ◽

Anis Shobirin Meor Hussin

Keyword(s):

Antioxidant Activity ◽

Biological Activity ◽

Alcoholic Beverage ◽

Black Tea ◽

Storage Conditions ◽

Total Phenolic ◽

Prolonged Storage ◽

The Novel ◽

Surface Method ◽

Optimum Production

Kombucha is a slightly alcoholic beverage produced using sugared tea via fermentation using the symbiotic culture of bacteria and yeast (SCOBY). This study aimed to optimize the production of soursop kombucha and determine the effects of different storage conditions on the quality, metabolites, and biological activity. The response surface method (RSM) results demonstrated that the optimum production parameters were 300 ml soursop juice, 700 ml black tea, and 150 g sugar and 14 days fermentation at 28°C. The storage conditions showed significant (P < 0.05) effects on the antioxidant activity including the highest antioxidant activity for the sample stored for 14 days at 25°C in light and the highest total phenolic content (TPC) for the sample stored for 7 days at 4°C in the dark. No significant effects were observed on the antimicrobial activity of soursop kombucha toward Escherichia coli and Staphylococcus aureus. The microbial population was reduced from the average of 106 CFU/ml before the storage to 104 CFU/ml after the storage at 4 and 25°C in dark and light conditions. The metabolites profiling demonstrated significant decline for the sucrose, acetic acid, gluconic acid, and ethanol, while glucose was significantly increased. The storage conditions for 21 days at 25°C in the dark reduced 98% of ethanol content. The novel findings of this study revealed that prolonged storage conditions have high potential to improve the quality, metabolites content, biological activity, and the Halal status of soursop kombucha.

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Microencapsulation Preservation of the Stability and Efficacy of Citrus Grandis Oil-Based Repellent Formulation against Aedes aegypti during Storage

Molecules ◽

10.3390/molecules26123599 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3599

Author(s):

Norashiqin Misni ◽

Zurainee Mohamed Nor ◽

Rohani Ahmad ◽

Nur Raihana Ithnin ◽

Ngah Zasmy Unyah

Keyword(s):

Relative Humidity ◽

Essential Oil ◽

Zeta Potential ◽

Aedes Aegypti ◽

Essential Oils ◽

Ph Value ◽

Physical Stability ◽

Storage Conditions ◽

Mosquito Repellent ◽

The Stability

Essential oils have been widely used as an active ingredient in mosquito repellent products. However, essential oils are highly unstable and prone to degradation when exposed to the environment during storage. Microencapsulation techniques help to maintain the stability of molecules in essential oils that are sensitive to environmental stress, and therefore improve shelf life. In this study, the physical stability and efficacy of a repellent formulation consisting of encapsulated Citrus grandis essential oil (CGEO) were evaluated under different storage conditions over a 12-month period by comparing the formulation with a non-encapsulated formulation. The formulations were both stored under two different storage conditions, i.e., 25 ± 2 °C/60% ± 5% relative humidity (RH) and 40 ± 2 °C/75% RH ± 5%, for 12 months. Droplet size, zeta potential, and pH value were measured after 1, 6, and 12 months of storage to determine their stability. For the study of efficacy, each formulation was tested against Aedes aegypti under laboratory conditions. We found that the microencapsulated formulation’s physical characteristics showed insignificant changes as compared with the non-encapsulated formulation during storage. The microencapsulated formulation demonstrated better repellent effects, sustaining high protection (>80%) for 4 more hours of exposure after 12 months of storage as compared with the non-encapsulated formulation that demonstrated high protection for only an hour post application. Microencapsulation helped to preserve the stability of the formulation, which resulted in high protection being maintained for over 12 months of storage.

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Effect of Storage Temperature and Time on Stability of Liver Enzymes in Blood Serum

Archives of Iranian Medicine ◽

10.34172/aim.2020.18 ◽

2020 ◽

Vol 23 (5) ◽

pp. 296-301

Author(s):

Shadi Kolahdoozan ◽

Sadaf G. Sepanlou ◽

Maryam Sharafkhah ◽

Elaheh Shaker ◽

Ameneh Shayanrad ◽

...

Keyword(s):

Linear Models ◽

Liver Enzymes ◽

Storage Temperature ◽

Linear Regression Analysis ◽

Storage Conditions ◽

Prolonged Storage ◽

Serum Samples ◽

Long Term Storage ◽

The Stability ◽

Almost All

Background: It is increasingly common to collect and store specimens for future unspecified research. However, the effects of prolonged storage on the stability and quality of analytes in serum have not been well investigated. We aimed to determine whether the stability of liver enzymes extracted from frozen bio-samples stored at the baseline is affected by storage conditions. Methods: A total of four liver enzymes in the sera of 400 patients were examined following storage. After deter-mining the baseline measurements, the serum of each patient was aliquoted and stored at −70°C for three and six months, as well as one, two, and five years after collecting the original sample. The percent change from baseline measurements was calculated both statistically and clinically. Linear models were also used to correct the results of the samples based on the time they were frozen. Results: In almost all samples, liver enzymes were detectable until two years after the baseline, while in a signifi-cant proportion of samples, enzymes were not ultimately detectable five years after the baseline. Linear regression analysis on log-transformed levels of enzymes shows that the performance is acceptable until one year after the baseline. The performance of the prediction model declines substantially two and five years after the baseline, except for GGT. Conclusion: Long-term storage of serum samples significantly decreases the concentration of the liver enzymes from the baseline, except for GGT. It is not recommended to store samples for more than two years, as liver en-zymes are not detectable afterwards.

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Study of the Stability of Various Biochemical Analytes in Samples Stored at Different Predefined Storage Conditions at an Accredited Laboratory of India

Journal of Laboratory Physicians ◽

10.4103/0974-2727.187928 ◽

2017 ◽

Vol 9 (01) ◽

pp. 011-015 ◽

Cited By ~ 12

Author(s):

Kamal Kachhawa ◽

Poonam Kachhawa ◽

Meena Varma ◽

Rasmirekha Behera ◽

Divya Agrawal ◽

...

Keyword(s):

Total Bilirubin ◽

Serum Amylase ◽

Storage Conditions ◽

Prolonged Storage ◽

Blood Products ◽

Sodium Potassium ◽

Calcium Phosphorus ◽

Accredited Laboratory ◽

Technical Issues ◽

The Stability

ABSTRACT Background: Storage of serum and other blood products is often necessary in laboratories because of technical issues or to preserve samples for subsequent research purposes. The aim of this study was to determine whether the stability of biochemical analytes is affected by storage conditions. Materials and Methods: A total of 17 biochemical analytes in the sera of ten patients were examined following storage. Subsequent to determining the baseline measurements, the serum of each patient was aliquoted and stored at −20°C for 7, 15, and 30 days and then analyzed for stability. The results were compared with the initial analysis measurements obtained from fresh samples. Mean changes compared to baseline (T0) concentrations were evaluated both statistically and clinically. Results: Our results show that sodium, potassium, urea, creatinine, uric acid, total calcium, phosphorus, direct bilirubin, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, cholesterol, and triglyceride levels were stable under all conditions. Serum amylase was the only analyte demonstrating instability following prolonged storage; amylase levels changed significantly (both statistically and clinically) at 7, 15, and 30 days (P < 0.05). Conclusion: Most common biochemical analytes, except for amylase, showed adequate stability in serum following 30 days of storage at −20°C. Serum amylase analysis should be conducted on the same day that the sample is received in the laboratory.

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FORMULATION AND DEVELOPMENT OF FAST DISSOLVING TABLET OF METOCLOPRAMIDE: AN ANTI-EMETIC DRUG

Journal of Biomedical and Pharmaceutical Research ◽

10.32553/jbpr.v8i6.699 ◽

2019 ◽

Vol 8 (6) ◽

Author(s):

Avilash Carpenter ◽

M.K. Gupta ◽

Neetesh Kumar Jain ◽

Urvashi Sharma ◽

Rahul Sisodiya

Keyword(s):

Mechanical Strength ◽

Standard Procedure ◽

Flow Property ◽

Storage Conditions ◽

Angle Of Repose ◽

Dissolution Time ◽

Disintegration Time ◽

Powder Blend ◽

Standard Curve ◽

The Stability

Aim: The main of the study is to formulate and develop orally disintegrating fast dissolving tablet of Metoclopramide hydrochloride. Material & Methods: Before formulation and development of selected drug, the standard curve in buffer was prepared and absorbance at selected maxima was taken. Then two different disintegrating agents were selected and drug was mixed with disintegrating agents in different ratio. Various Preformulation parameters and evaluation of tablet i.e. disintegration time, dissolution time, friability, hardness, thickness were measured by standard procedure. Result & Discussion: The angle of repose for all the batches prepared. The values were found to be in the range of 30.46 to 36.45, which indicates good flow property for the powder blend according to the USP. The bulk density and tapped density for all the batches varied from 0.49 to 0.54 g/mL and 0.66 to 0.73, respectively. Carr’s index values were found to be in the range of 23.33 to 25.88, which is satisfactory for the powders as well as implies that the blends have good compressibility. Hausner ratio values obtained were in the range of 1.22 to 1.36, which shows a passable flow property for the powder blend based on the USP. The results for tablet thickness and height for all batches was found to range from 4.45 to 4.72 mm and 3.67 to 3.69 mm, respectively. Hardness or breaking force of tablets for all batches was found to range from 32.8 to 36.2 N. Tablet formulations must show good mechanical strength with sufficient hardness in order to handle shipping and transportation. Friability values for all the formulations were found to be in the range of 0.22 % to 0.30 %. Conclusion: Orally disintegrating tablets were compressed in order to have sufficient mechanical strength and integrity to withstand handling, shipping and transportation. The formulation was shown to have a rapid disintegration time that complied with the USP (less than one minute). The data obtained from the stability studies indicated that the orally disintegrating mini-tablets of MTH were stable under different environmental storage conditions. Keywords: Formulation & Development, Fast Dissolving Tablet, Metoclopramide, Anti-Emetic Drug, Oral Disintegrating Tablet

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The Effects of Storage Conditions on the Stability of House Dust Mite Extracts

Allergy Asthma and Immunology Research ◽

10.4168/aair.2013.5.6.397 ◽

2013 ◽

Vol 5 (6) ◽

pp. 397 ◽

Cited By ~ 11

Author(s):

Kyoung Yong Jeong ◽

Soo-Young Choi ◽

In-Soo Han ◽

Jae-Hyun Lee ◽

Joo-Shil Lee ◽

...

Keyword(s):

House Dust ◽

House Dust Mite ◽

Storage Conditions ◽

Dust Mite ◽

The Stability

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Insufficient Stability of Clavulanic Acid in Widely Used Child-Appropriate Formulations

Antibiotics ◽

10.3390/antibiotics10020225 ◽

2021 ◽

Vol 10 (2) ◽

pp. 225

Author(s):

Ines Mack ◽

Mike Sharland ◽

Janneke M. Brussee ◽

Sophia Rehm ◽

Katharina Rentsch ◽

...

Keyword(s):

Ambient Temperature ◽

Clavulanic Acid ◽

Storage Conditions ◽

Essential Medicines ◽

Active Components ◽

Middle Income ◽

Ambient Temperatures ◽

Essential Medicines List ◽

Dispersible Tablets ◽

The Stability

Amoxicillin-clavulanic acid (AMC) belongs to the WHO Essential Medicines List for children, but for optimal antimicrobial effectiveness, reconstituted dry powder suspensions need to be stored in a refrigerated environment. Many patients in low- and middle-income countries who are sold AMC suspensions would be expected not to keep to the specified storage conditions. We aimed to assess the stability of both ingredients in liquid formulations and dispersible tablets, combined with nationally representative data on access to appropriate storage. Degradation of amoxicillin (AMX) and clavulanic-acid (CLA) was measured in suspensions and dispersible tablets commercially available in Switzerland at different ambient temperatures (8 °C vs. 28 °C over 7 days, and 23 °C vs. 28 °C over 24 h, respectively). Data on access to refrigeration and electricity were assessed from the USAID-funded Demographic and Health Survey program. In suspensions, CLA degraded to a maximum of 12.9% (95% CI −55.7%, +29.9%) at 8°C and 72.3% (95% CI −82.8%, −61.8%) at a 28 °C ambient temperature during an observation period of 7 days. Dispersible tablets were observed during 24 h and CLA degraded to 15.4% (95% CI −51.9%, +21.2%) at 23 °C and 21.7% (−28.2%, −15.1%) at a 28 °C ambient temperature. There is relevant degradation of CLA in suspensions during a 7-day course. To overcome the stability challenges for all active components, durable child-appropriate formulations are needed. Until then, prescribers of AMC suspensions or pharmacists who sell the drug need to create awareness for the importance of proper storage conditions regarding effectiveness of both antibiotics and this recommendation should be reflected in the WHO Essential Medicines List for children.

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