scholarly journals Optimizing the synthesis and purification of MS2 virus like particles

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Khadijeh Hashemi ◽  
Mohammad Mahdi Ghahramani Seno ◽  
Mohammad Reza Ahmadian ◽  
Bizhan Malaekeh-Nikouei ◽  
Mohammad Reza Bassami ◽  
...  
Keyword(s):  
Size Range ◽  
Therapeutic Agents ◽  
Precipitation Method ◽  
Rna Sequences ◽  
Virus Like Particles ◽  
Bacteriophage Ms2 ◽  
Effects Of Ph ◽  
Peg Precipitation Method ◽  
The Stability

AbstractIntroducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.

scholarly journals MS2 Virus-like Particle as an Efficient Drug Delivery Tool: Optimizing the Synthesis and Purification Protocol

2021 ◽  
Author(s):  
Khadijeh Hashemi ◽  
Mohammad Mahdi Ghahramani Seno ◽  
Mohammad Reza Ahmadian ◽  
Bizhan Malaekeh-Nikouei ◽  
Mohammad Reza Bassami ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Precipitation Method ◽  
Rna Sequences ◽  
Bacteriophage Ms2 ◽  
Virus Like Particle ◽  
Effects Of Ph ◽  
Peg Precipitation Method ◽  
The Stability ◽  
Purification Protocol

Abstract Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated.In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30nm and suitable homogeneity with minimum 12-month stability at 4◦C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 hours in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.

scholarly journals Stability of Secondary and Tertiary Structures of Virus-Like Particles Representing Noroviruses: Effects of pH, Ionic Strength, and Temperature and Implications for Adhesion to Surfaces

2015 ◽  
Vol 81 (22) ◽  
pp. 7680-7686 ◽  
Author(s):  
Idrissa Samandoulgou ◽  
Riadh Hammami ◽  
Rocio Morales Rayas ◽  
Ismail Fliss ◽  
Julie Jean
Keyword(s):  
Ionic Strength ◽  
Structural Rearrangement ◽  
Tertiary Structures ◽  
Hydrophobic Residues ◽  
Virus Like Particles ◽  
Nacl Concentration ◽  
Effects Of Ph ◽  
The Stability ◽  
Α Helix ◽  
Fresh Foods

ABSTRACTLoss of ordered molecular structure in proteins is known to increase their adhesion to surfaces. The aim of this work was to study the stability of norovirus secondary and tertiary structures and its implications for viral adhesion to fresh foods and agrifood surfaces. The pH, ionic strength, and temperature conditions studied correspond to those prevalent in the principal vehicles of viral transmission (vomit and feces) and in the food processing and handling environment (pasteurization and refrigeration). The structures of virus-like particles representing GI.1, GII.4, and feline calicivirus (FCV) were studied using circular dichroism and intrinsic UV fluorescence. The particles were remarkably stable under most of the conditions. However, heating to 65°C caused losses of β-strand structure, notably in GI.1 and FCV, while at 75°C the α-helix content of GII.4 and FCV decreased and tertiary structures unfolded in all three cases. Combining temperature with pH or ionic strength caused variable losses of structure depending on the particle type. Regardless of pH, heating to pasteurization temperatures or higher would be required to increase GII.4 and FCV adhesion, while either low or high temperatures would favor GI.1 adhesion. Regardless of temperature, increased ionic strength would increase GII.4 adhesion but would decrease GI.1 adhesion. FCV adsorption would be greater at refrigeration, pasteurization, or high temperature combined with a low salt concentration or at a higher NaCl concentration regardless of temperature. Norovirus adhesion mediated by hydrophobic interaction may depend on hydrophobic residues normally exposed on the capsid surface at pH 3, pH 8, physiological ionic strength, and low temperature, while at pasteurization temperatures it may rely more on buried hydrophobic residues exposed upon structural rearrangement.

scholarly journals ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS

1969 ◽  
Vol 41 (1) ◽  
pp. 167-176 ◽  
Author(s):  
S. L. Howell ◽  
D. A. Young ◽  
P. E. Lacy
Keyword(s):  
Islets Of Langerhans ◽  
Secretory Granules ◽  
Sodium Deoxycholate ◽  
Rat Islets ◽  
The Matrix ◽  
Effects Of Ph ◽  
The Stability ◽  
Rat Islets Of Langerhans ◽  
Specific Insulin

A partially purified secretory granule fraction, isolated from rat islets of Langerhans by differential centrifugation, was used for investigating the stability of the beta granules during incubation in various conditions. Effects of pH, temperature, and time were studied; the granules possessed optimal stability at 4° and pH 6.0, and could be solubilized at pH 4.0 or 8.5, or in the presence of sodium deoxycholate, but not by phospholipase c, ouabain, or alloxan. Incubation with glucose or some of its metabolites, or with tolbutamide, ATP, or cyclic 3',5'-AMP did not alter the stability of the beta granules Exogenous insulin-131I was not bound by the isolated granules under the conditions used; no specific insulin-degrading activity could be detected in subcellular fractions of the islets. These findings indicate that intracellular solubilization of the granules with subsequent diffusion of the insulin into the extracellular space is not a likely mode of insulin secretion in vivo, and suggest that a crystalline zinc-insulin complex may exist in the matrix of the beta granules.

Antisense strategies and therapeutic applications

1996 ◽  
Vol 53 (2) ◽  
pp. 151-160 ◽  
Author(s):  
Putnam David A.
Keyword(s):  
Virus Infection ◽  
Messenger Rna ◽  
Therapeutic Agents ◽  
Water Soluble ◽  
Rna Sequences ◽  
Potential Applications ◽  
Antisense Approach ◽  
Simplex Virus

The concepts underlying the antisense approach to disease therapy are discussed, and potential applications are examined. Antisense therapeutic agents bind to DNA or RNA sequences, biocking the synthesis of cellular proteins with unparalleled specificity. Transcription and translation are the two processes with which the agents interfere. There are three major classes of antisense agents: antisense sequences, commonly called antisense oligonucleotides; antigene sequences; and ribozymes. Antisense sequences are derivatives of nucleic acids that hybridize cytosolic messenger RNA (mRNA) sense strands through hydrogen bonding to complementary nucleic acid bases. Antigene sequences hybridize double-stranded DNA in the nucleus, forming triple helixes. Ribozymes, rather than inhibiting protein synthesis simply by binding to a single targeted mRNA. combine enzymatic processes with the specificity of antisense Iwse pairing, creating a molecule that can incapacitate multiple targeted niRNAs. Anti-sense therapeutic agents are being investigated in vitro and in vivo for use in treating human immunodeficiency virus infection, hepatitis B virus infection, herpes simplex virus infection, papillomavirus infection, cancer, restenosis, rheumatoid arthritis, and allergic disorders. Although many results are preliminary, some are promising and has e led to clinical trials. A major goal in developing methods of delivering antisense agents is to reduce their susceptibility to nucleases while retaining their ability to bind to targeted sites. Modification of the phosphodiester linkages in oligonucleotides can lend the sequences enzymatic stability without affecting their binding capacities. Carrier systems designed to protect the antisense structure and improve passage through the cell membrane include liposomes, water-soluble polyrners, and nanoparticles. The pharmacokinetics of anti-sense agents are under investigation. Antisense therapeutic agents have the potential to become an integral part of medicinal regimens.

scholarly journals Biomimetic Virus-like Particles as SARS-CoV-2 Positive Controls for RT-PCR Diagnostics

2020 ◽  
Author(s):  
Soo Khim Chan ◽  
Pinyi Du ◽  
Karole Ignacio ◽  
Sanjay Metha ◽  
Isabel G. Newton ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Cold Chain ◽  
Coat Proteins ◽  
Rt Pcr ◽  
Rna Sequences ◽  
Virus Like Particles ◽  
Chimeric Vlps ◽  
Positive Controls

ABSTRACTCoronavirus disease 2019 (COVID-19) is a highly transmissible disease that has affected more than 90% of the countries worldwide. At least 17 million individuals have been infected, and some countries are still battling first or second waves of the pandemic. Nucleic acid tests, especially reverse-transcription polymerase chain reaction (RT-PCR), have become the workhorse for early detection of COVID-19 infection. Positive controls for the molecular assays have been developed to validate each test and to provide high accuracy. However, most available positive controls require cold-chain distribution and cannot serve as full-process control. To overcome these shortcomings, we report the production of biomimetic virus-like particles (VLPs) as SARS-CoV-2 positive controls. A SARS-CoV-2 detection module for RT-PCR was encapsidated into VLPs from a bacteriophage and a plant virus. The chimeric VLPs were obtained either by in vivo reconstitution and co-expression of the target detection module and coat proteins or by in vitro assembly of purified detection module RNA sequences and coat proteins. These VLP-based positive controls mimic SARS-CoV-2 packaged RNA while being non-infectious. Most importantly, we demonstrated that the positive controls are scalable, stable, and can serve broadly as controls, from RNA extraction to PCR in clinical settings.Table of contents graphic

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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