Proteinase K treatment in absence of RNA isolation classical procedures is a quick and cheaper alternative for SARS-CoV-2 molecular detection

ABSTRACT MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.

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Potential of Cell-Free Supernatant from Lactobacillus plantarum NIBR97, Including Novel Bacteriocins, as a Natural Alternative to Chemical Disinfectants

Pharmaceuticals ◽

10.3390/ph13100266 ◽

2020 ◽

Vol 13 (10) ◽

pp. 266

Author(s):

Sam Woong Kim ◽

Song I. Kang ◽

Da Hye Shin ◽

Se Yun Oh ◽

Chae Won Lee ◽

...

Keyword(s):

Lactobacillus Plantarum ◽

Influenza A ◽

Antibacterial Activities ◽

Proteinase K ◽

Bacterial Membranes ◽

Cell Free Supernatant ◽

Antiviral Activities ◽

Viral Envelopes ◽

Proteinase K Treatment ◽

Cellular Lysis

The recent pandemic of coronavirus disease 2019 (COVID-19) has increased demand for chemical disinfectants, which can be potentially hazardous to users. Here, we suggest that the cell-free supernatant from Lactobacillus plantarum NIBR97, including novel bacteriocins, has potential as a natural alternative to chemical disinfectants. It exhibits significant antibacterial activities against a broad range of pathogens, and was observed by scanning electron microscopy (SEM) to cause cellular lysis through pore formation in bacterial membranes, implying that its antibacterial activity may be mediated by peptides or proteins and supported by proteinase K treatment. It also showed significant antiviral activities against HIV-based lentivirus and influenza A/H3N2, causing lentiviral lysis through envelope collapse. Furthermore, whole-genome sequencing revealed that NIBR97 has diverse antimicrobial peptides, and among them are five novel bacteriocins, designated as plantaricin 1 to 5. Plantaricin 3 and 5 in particular showed both antibacterial and antiviral activities. SEM revealed that plantaricin 3 causes direct damage to both bacterial membranes and viral envelopes, while plantaricin 5 damaged only bacterial membranes, implying different antiviral mechanisms. Our data suggest that the cell-free supernatant from L. plantarum NIBR97, including novel bacteriocins, is potentially useful as a natural alternative to chemical disinfectants.

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Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109355059 ◽

2009 ◽

Vol 15 (1) ◽

pp. 80-85 ◽

Cited By ~ 10

Author(s):

Sean T. Glenn ◽

Karen L. Head ◽

Bin T. Teh ◽

Kenneth W. Gross ◽

Hyung L. Kim

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reverse Transcription ◽

Gene Expression Analysis ◽

Rna Isolation ◽

Proteinase K ◽

Specific Primers ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

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Cytoplasmic p53 polypeptide is associated with ribosomes.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3146 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3146-3154 ◽

Cited By ~ 39

Author(s):

B M Fontoura ◽

C A Atienza ◽

E A Sorokina ◽

T Morimoto ◽

R B Carroll

Keyword(s):

Sodium Dodecyl ◽

Proteinase K ◽

Mutant P53 ◽

Tumor Suppressor P53 ◽

Wild Type ◽

Mcf7 Cells ◽

Gapdh Mrna ◽

5.8S Rrna ◽

Proteinase K Treatment ◽

Covalently Linked

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.

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Borrelia burgdorferi Binds to, Invades, and Colonizes Native Type I Collagen Lattices

Infection and Immunity ◽

10.1128/iai.72.6.3138-3146.2004 ◽

2004 ◽

Vol 72 (6) ◽

pp. 3138-3146 ◽

Cited By ~ 42

Author(s):

Maria C. Zambrano ◽

Anastasia A. Beklemisheva ◽

Anton V. Bryksin ◽

Stuart A. Newman ◽

Felipe C. Cabello

Keyword(s):

Borrelia Burgdorferi ◽

Type I Collagen ◽

High Efficiency ◽

Immunoblot Analysis ◽

Proteinase K ◽

Type I ◽

Collagen Matrices ◽

Proteinase K Treatment ◽

Collagen Lattices

ABSTRACT Borrelia burgdorferi binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans. We investigated the interactions between B. burgdorferi cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices. B. burgdorferi cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans. Moreover, following adhesion to these matrices, B. burgdorferi grew and formed microcolonies. The collagen used in these studies was confirmed to lack decorin by immunoblot analysis; B. burgdorferi cells lacking the decorin adhesin bound readily to intact collagen matrices. B. burgdorferi also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans. Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions. Proteinase K treatment of B. burgdorferi cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of B. burgdorferi to interact with intact collagen matrices. The high efficiency of binding of B. burgdorferi strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions.

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Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment

Prion ◽

10.1080/19336896.2016.1189053 ◽

2016 ◽

Vol 10 (4) ◽

pp. 305-318 ◽

Cited By ~ 5

Author(s):

Zhifu Shan ◽

Takeshi Yamasaki ◽

Akio Suzuki ◽

Rie Hasebe ◽

Motohiro Horiuchi

Keyword(s):

Prion Protein ◽

Direct Detection ◽

Cell Lysis ◽

Proteinase K ◽

Simple Cell ◽

Infected Cells ◽

Proteinase K Treatment

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Isolation, characterization, and molecular identification of soil bacteria showing antibacterial activity against human pathogenic bacteria

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00219-x ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

R. Prashanthi ◽

Shreevatsa G.K. ◽

Krupalini S. ◽

Manoj L.

Keyword(s):

Antibacterial Activity ◽

Soil Bacteria ◽

Pathogenic Bacteria ◽

Nitrogen Sources ◽

Proteinase K ◽

Human Pathogens ◽

E Coli ◽

Carbon And Nitrogen ◽

Culture Filtrates ◽

Proteinase K Treatment

Abstract Background The present study dealt with the screening of soil bacteria with antibacterial activity from different locations in Bangalore, India. Antibiotics play the role of self-defense mechanism for the bacteria and are produced as secondary metabolites to protect themselves from other competitive microorganisms. The need for new antibiotics arose as the pathogenic bacteria acquire resistance to various antibiotics meant for treating human diseases. Given the importance of antibiotics of bacterial origin, standard techniques have been used to isolate and characterize the soil bacteria which showed antibacterial activity. Results The isolated bacteria were tested against human pathogenic bacteria like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae by primary and secondary screening methods. The isolates PR1, PR2, and PR3 were confirmed to have antibacterial activity against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae by both methods. Studies on the effect of filter sterilization, autoclaving, and proteinase K treatment on culture filtrates showed filter sterilization as the best method. The effect of different carbon and nitrogen sources on the antibacterial activity showed that preference by each isolate differed for carbon and nitrogen requirements. The isolates PR1, PR2, and PR3 were identified as Bacillus aryabhattai strain PR-D07, Arthrobacter humicola strain PR-F07, and Neomicrococcus lactis strain PR-F11 through 16S rRNA sequencing. Conclusion Findings from this research work are encouraging and could proceed further to applied aspects. Only 3 bacterial isolates out of 263 isolates from soil samples displayed antibacterial activity against human pathogens S. aureus, E. coli, P. aeruginosa, and K. pneumoniae. They were identified as B. aryabhattai, A. humicola, and N. lactis by 16S rRNA studies and all of them are Gram-positive. Each isolate preferred different carbon and nitrogen sources for their enhanced antibacterial activity. Efficacy of the culture filtrates of these isolates was tested by filter sterilization, autoclaving, and proteinase K treatment. Filter-sterilized culture filtrates showed higher antibacterial activity than other treatments. A comparison of the antibacterial activity of culture filtrates and antibiotic streptomycin produced an inhibition zone of 18.5 mm and 15.5 mm respectively. This is the first report on the antibacterial activity of all the 3 bacterial strains (B. aryabhattai strain PR-D07, A. humicola strain PR-F07, and N. lactis strain PR-F11), against all the human pathogens, mentioned earlier. It is also found that the antibiotic factor is proteinaceous as proteinase K considerably reduced the antibacterial activity of the culture filtrates. With the above significant results, these 3 bacteria are considered to be promising candidates for the isolation of new antibacterial agents.

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A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock

PLoS ONE ◽

10.1371/journal.pone.0248885 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248885

Author(s):

Adolfo Marcelo Ñique ◽

Fiorella Coronado-Marquina ◽

Jairo Andrés Mendez Rico ◽

María Paquita García Mendoza ◽

Nancy Rojas-Serrano ◽

...

Keyword(s):

Thermal Shock ◽

Real Time ◽

Internal Control ◽

Rna Extraction ◽

Cost Effective ◽

Proteinase K ◽

Rt Pcr ◽

Critical Material ◽

Proteinase K Treatment ◽

Commercial Kit

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol–samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.

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An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2460-2466.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2460-2466 ◽

Cited By ~ 2

Author(s):

G Brewer

Keyword(s):

Cellular Response ◽

Mrna Decay ◽

Rna Binding ◽

Binding Capacity ◽

Proteinase K ◽

Rapid Turnover ◽

A Cell ◽

Degradation Activity ◽

Proteinase K Treatment

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.

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