AUCTORES
Globalize your Research
Research Article
*Corresponding Author: Amro Abd Al Fattah Amara, City of Scientific Research and Technological Applications, Universities and Research Center District, Egypt.
Citation: Amro A. Amara (2021) Different simple protocols for separating different types of coexisted plasmids in the same cell. J, Biotechnology and Bioprocessing 2(3); DOI: 10.31579/2766-2314/027
Copyright: © 2021, Amro Abd Fattah Amara, This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
Received: 24 January 2021 | Accepted: 27 January 2021 | Published: 19 February 2021
Keywords: plasmid, coexisted, isolation, competent cells, transformation efficacy
The existence of mixed plasmids in the same cell is tricky and there is a need for separating them from each other. However, isolating two existed plasmids might be difficult, particularly if they are same in their sizes, with same antibiotic marker, or different only in one or more mutants without different restriction cut. Two different plasmids in the same cells is a natural phenomenon as well as a normal practice in molecular biology experiments. For example during random mutagenesis experiment for a single gene (existed naturally in an operon) using a mutator strain like E. coli XL1 Red, the single mutated gene is then complemented with the other essential genes for producing certain products. Another example, during site directed mutagenesis experiment using double antibiotics selection method, in many cases, the original plasmid is existed side by side with the one carry the new mutant. There are many examples where plasmids coexisted with each other either naturally or under experimental conditions. The problem is how one could separate those plasmids particularly when they are similar in their molecular weight and have the same marker. This study introduces two main strategies; the first is based on increasing or decreasing the competent cells transformation efficacy. Where, in general harvesting competent cells either E. coli or other bacterial strains in the first 2-3 hours (or less) of their cultivation and using the enhanced protocol for competent cells preparation will improve the transformation processes. Letting cells to be more ages will reduce the transformation processes. Using four 2-3 hour grown competent Azotobacter sp enable plasmid transformation. The second strategy for separating the coexisted plasmid is based on using diluted plasmids. The antibiotic screening method is based on blind selection where growing on plat containing the first antibiotic and non growing in the second antibiotic means that the tow existed plasmids are separated. In case of existing of plasmids with the same size and the same antibiotic marker for example during the site-directed-mutagenesis protocol (mutants did not have different restriction enzymes cut), the plasmid is diluted and transformed in recombinant E. coli and each clone was cultivated alone and the mutated region is sequenced. The presence of a single base pair in the site of the mutant means presence of a single plasmids and vice versa. As a conclusion same plasmids with point mutation are usually coexisted. In some cases the coexisted plasmids are with similar antibiotic marker, no different restriction enzyme cut sites are existed, no white and blue selection or any other phenotype for selection. In such cases and similar ones diluting plasmid and transforming them in conditions enable single plasmid per cell must be controlled by the sequencer. The protocols included in this study are summarized from the experiences with random and site directed mutagenesis experiments where plasmid with a single mutant is coexisted with the wild mother plasmid or with the other coexisted different mutants.
Molecular biology tools are in need always for simplifying the used protocols. New idea could reduce time and costs. Site directed mutagenesis is one of the most perfect tools to introduce mutants in genes[1]. The random mutagenesis is another tool for mutagenesis E. coli XL1Red is a mutator strain deficient in three of the primary DNA repair pathways. The mutS (error-prone mismatch repair)[2], mutD (deficient in 3´-to 5´- exonuclease of DNA polymerase III) [3] and mutT (unable to hydrolyse 8-oxodGTP) mutations were present in strain XL1-Red (Stratagene, LaJolla, USA). This strain is able to mutate a gene carried on plasmid and transformed to it. The mutants could be accumulated. From one colony many mutants could be obtained [4]. In nature plasmids are coexisted in a single microbe. This study introduces simple solutions for separating two existed similar or different plasmids in one cell.
E. coli strain
XL1-Blue, recA1, endA1, gyrA96, thi-1, hsdR17 (rK-,mK+), supE44, relA1, λ-, lac [F, proAB, lacIq, (lacZ)M15, Tn10(Tcr)] [5]
Cultures containing the different respective plasmids were grown in 20 ml LB medium with the appropriate antibiotics at 37°C for overnight. 1.5 ml of culture broth in Eppendorf tube was centrifuged at 4,000 rpm for 3 min (Biofuge 13, Heraeus instrument Christ, Osterode), the supernatant was removed. 100 µl ice-cold GET solution was added to the pellet and was completely suspended, followed by addition of 200 µl fresh-prepared SDS-NaOH solution. Clear solution was obtained after gently inverting the Eppendorf tubes up and down, indicating that cell lysis was complete. The protein was precipitated by addition of 150 µl potassium acetate solution. After centrifugation at 13,000 rpm for 15 min (Biofuge A, Heraeus Christ, Osterode), the supernatant was transferred to a new Eppendorf tube, then 900 µl of ice-cold ethanol was added and the pellet was washed with 70% (v/v) ethanol. The pellet was dried either at room temperature or in the vacuum drier. The dried plasmid DNA was dissolved in 10-20 µl RNase-containing TE-buffer or distilled water and stored at -20°C. GET-solution: 25 Mm Tris/HCl, pH 8.0; 10 mM EDTA; 50 mM Glucose. SDS-NaOH solution: 200 mM NaOH; 1% (w/v) SDS. 5 M potassium acetate-solution: 29.5 ml Acetic acid; 100 ml H2Obidest; Adjust the pH to 4.8 with KOH. RNase-solution: 150 mM NaCl, pH 5.0; 1 % (w/v) RNase A (80 °C, 15 min to inactivate DNase); TE-Puffer: 10 mM Tris/HCl ; 1 mM EDTA, pH 8.0 ; TE buffer containing RNase 1µl RNase solution : 100µl TE buffer [6].
Determination of the plasmid DNA concentration
The purity and concentration of the plasmid DNA solution was determined by measuring the absorption at 260 nm and 280 nm. An extinction E260 = 1 corresponds to 50 µg dsDNA ml-1 and 38.5-40 µg ssDNA ml-1. For evaluation of pure DNA preparation, the following equation could be used: E260 nm / E280 nm = 1.8. During routine work with small volumes of plasmid DNA solutions, the concentration of DNA was estimated by following fluorescence of bands within the agarose gel that had been stained with ethidium bromide. The amount of DNA which was just visible after electrophoresis, staining and photography on the gel was set to 2 ng [6].
Preparation of competent cells by CaCl2 method
E. coli cells was grown in 20 ml LB medium at 37°C until OD600 reached 0.5-0.7. The cells were harvested by centrifugation at 4,000 rpm for 10 min. 5-10 ml CaCl2-Tris solution was added to suspend the cells, followed by incubating this suspension on ice for 20 min. The competent cells were harvested by centrifugation at 4,000 rpm for 10 min (Megafuge 1.0R, Heraeus instrument Christ, Osterode) and resuspended in 2 ml CaCl2 solution. The competent cells were then mixed with DMSO to a concentration of 7% (v/v) or with glycerol to a concentration of 10% (v/v) and aliquots of 200 µl were distributed into Eppendorf tubes. The competent cells were then stored at –70°C. CaCl2 - Tris solution: 50 mM CaCl2, 10 mM Tris/HCI, pH 8.0
Preparation of competent cells for long term storage [7, 8]
The Xl1Blue E. coli strain was cultivated in 50 ml LB medium at 37°C until OD600 reached 0.3. After 10 to 15 min incubation on ice, the cells were harvested by centrifugation (15 min; 4,000 rpm). The cell pellet was then suspended in 18 ml RF1 solution. After incubation on ice for 30 min, the cells were again centrifuged for 15 min at 4,000 rpm (Megafuge 1.0R, Heraeus Christ, Osterode) and suspended in 4 ml RF2 solution. Competent cells were distributed to Eppendorf tubes (200 µl per tube) and stored at –70°C. RF1 solution: 100 mM RbCl, 50 mM MnCl2, 30 mM Potassium acetate, 10 mM CaCl2 . 6 H2O, Adjust the pH to 5.8 with acetic acid. RF2 solution: 10 mM RbCl, 10 mM MOPS, 75 mM CaCl2 . 6 H2O, 15 % (v/v) Glycerol, Adjust the pH to 8.5 with NaOH [7, 8].
Transformation of E. coli cells
200 µl competent cells were mixed thoroughly with 1 µl plasmid DNA to be transferred and incubated for 30 min in an ice bath. During this incubation step, the DNA was adsorbed at the surface of the competent cells. For the uptake of the adsorbed DNA, the cells were heated at 42°C for exactly 90 sec and subsequently incubated on ice for 3-5 min. For regeneration of the cells and for expression of the plasmid encoded antibiotic resistance, 800 µl LB medium was added and the cells were subsequently incubated at 37°C for at least 30 min. For isolation of the recombinant clones, 100 µl aliquots were spread on selective solid medium containing the selective antibiotic and the plates were incubated overnight at 37°C.
DNA sequencing and mutant detection
Standard criteria were used to sequence the targeted gene (the gene data not shown) using LI-COR 4000L (LI-COR, Alabama, USA). The changed base pairs were observed manually.
Separation of coexisted plasmids with different antibiotic markers and similar size
Inoculums from Xl1Blue E. coli which contain two plasmids similar in size but with different antibiotic markers added to 25 ml LB medium in 100 ml flask (2 flasks). For each flask one antibiotic is added with the proper amount. Plasmids were collected from the overnight cultivated E. coli strain at 37oC using the above described alkaline lysis protocol for plasmid isolation where one ml of the overnight culture were centrifuged at 4000 rpm and the pellet is used for the plasmid isolation using the above described alkaline lysis protocol for plasmid isolation [6]. The plasmid pellets after isolation and drying at 37oC were re-suspended in 50 µl of sterile bi-distilled water and then 5 µl were diluted each in one ml of sterile distilled water. 10 µl was used to transform the recombinant E. coli XL1Blue (modified from the above described protocol). Further steps were followed as described above. The cells left to grow for six hr and then 50µl of the culture is spread on LB medium with the first antibiotic LB medium plats. The plates are incubated overnight. Colonies from each plat were selected and re-cultivated in the second antibiotic LB medium plates. The growth on the first antibiotic and the non growth on the second antibiotic mean that plasmids are separated from each other (Figure 1) and vice versa.
Separation of coexisted similar plasmids with mutated and non mutated genes with similar antibiotic marker
Inoculum from E. coli which contain two plasmids similar in size and in the antibiotic marker but different due to the presence of mutant in the gene is added to 25 ml LB medium in 100 ml flask (one flask). Plasmids were collected from the overnight cultivated E. coli strain at 37oC using the above described alkaline lysis protocol for plasmid isolation [6] where one ml of the overnight culture was centrifuged at 4000 rpm and the pellet is used for the plasmids isolation. The isolated plasmids pellet after drying was re-suspended in 50 µl of sterile bi-distilled water and then 5 µl was further diluted in one ml of sterile distilled water. 10 µl then used to transform the recombinant E. coli XL1Blue. The cells left to grow for six hr and then 50µl of the culture is spread on LB medium with the proper antibiotic plat. Colonies were selected, cultivated and the isolated plasmids from each clone were investigated either by restriction cut if the mutants have different restriction sites to prove the plasmid separation or by using the sequencer to confirm the differences.
Separation of coexisted identical different plasmids with mutants and similar antibiotic marker without restriction cuts
In case of absence of different restriction cut sites, the same process will be followed but instead of using the restriction cut, the sequencing of the mutation region will prove either that plasmid still existed with the wild type or it is already separated. In case of the coexistence of both plasmids the changed base pair will appear in the same line in the sequence image with the original base pair as in Figure 2.
Separation of coexisted plasmids with different antibiotic markers and different sizes
It is recommended to use the recently published fast slot lysis protocol for plasmid isolation as described by Amara (2018) [9]. One can cut the agarose band of each plasmid and use the double Eppendorf tubes centrifugation method for isolating DNA and Plasmids as described by Amara 2018 and 2020 [8, 10].
Using young and old competent cells
Young (usually 2-3h) OD600 reached 0.2-0.3 or old competent cells (usually 7-10h) OD600 reached 0.5-0.7 either from E. coli or other wild bacterial strains such as Azotobacter sp strain can be prepared for increasing (in case of the young cells) or decreasing of the transformation percentage. Young or old competent cells can be used in separating coexisted plasmid beside the above described tools (Table 1).
This study is a collection of experiences concerning conducting different mutagenesis protocols with different plasmids. In case of random mutagenesis [4] or the site directed mutagenesis [1] using double mutation protocol (where the first mutant is the needed one and the other is caused a second antibiotic resistance) and the selection will be by using a second antibiotic. In some cases different plasmid could be coexisted in the same cells which will be so tricky. One could find two base pairs in the same line in the sequencer image or one could sequence plasmids with (for example) three mutant in the targeted gene and after that one mutant is lost. Those examples represent that different plasmids are coexisted in the same cells. Additionally, in case of sub cloning of a gene in another plasmid similar to the original one or cloning a new gene by eliminating the original gene in the same plasmid or using plasmid to perform PCR amplification for sub-cloning or ligating the amplified gene in new plasmid; in those all cases and more the original plasmid is still existed in the background with few amount but enough to disturb the results. It is recommended to use those described plasmid re-isolation steps to be sure that only one type of plasmid is existed in the used cells.
Separation of coexisted plasmids with different antibiotic markers and similar size
The described method will enable the plasmids separation. The method could be enhanced by adding 25 percentage of the proper antibiotic just after the first hr of the transformation and cultivation steps. That will improve the separation and the selection on the LB medium with the expected antibiotic (data not shown). That also could be reconfirmed by the result of the recultivation on the second antibiotic plat (Figure 1). The growth in the first plate (contain the first antibiotic) and the absence in second one (containing the second antibiotic) using the same colony prove that it contains a single plasmid and vice versa. There is no need for further prove (Figure 1).
Separation of coexisted plasmids with different genes equal in size and similar antibiotic marker
In case that a restriction cut site is existed, cutting with the proper restriction enzyme will prove the presence of a single or different plasmids.
Separation of coexisted identical plasmids with mutants and similar antibiotic marker without restriction cut
In this case the described method will lead to separate the coexisted plasmids but there is a need for sequencing the region where the mutant is designed (in case of the site-directed-mutagenesis). And the whole gene must be sequenced in case of the random mutagenesis. One should remark that to prove the presence of a correct mutant the whole gene must be sequenced from its forwarded and reverse sites.
Separation of coexisted plasmids with different antibiotic markers and different sizes
This might be tricky because the existence of different antibiotic marker will not guarantee separation in case of using a single antibiotic. One antibiotic could cause selection for tranforments containing the different plasmids. However, one could separate the plasmids based on their sizes using the fast slot lysis protocol as described by Amara (2018) [9]. And the plasmids must be separated using the agarose gel electrophoresis. One can use the double Eppendorf tubes centrifugation method to isolate the plasmid from the cut agarose gel bands [10]. The presence of the plasmids could be measured spectrophotometrically as described by Amara (2020) [6, 10].
Using young and old competent cells
Using young competent cells is a wonderful tool for transforming different microbes and can be used for some extend as an alternative to the Electroporation. Young (cells 2-3 hr cultivation OD600 reached 0.2-0.3); was used successfully to transform Azotobacter sp with certain plasmid [data not shown]. Young competent cells of bacteria (other than E. coli) show increase in the number of transforments based on the type of the original cells. In contrast old competent cells show decrease in the number of the transforments and can be used to isolate different coexisted plasmid if diluted. Based on the used E. coli competent cells or other bacteria prepared as competent cells, their age, the competent cells preparation methods and the plasmid purity the percentage of the transformation could be different in each different case (Table 1).
Using different competent cells preparation protocols
Beside the above described tools the type of the method used for competent E. coli preparation will give different results. Using competent cells prepared by CaCl2 method following Sambrook et al. (1989) [6] is recommended due to its low cost and easy handling as described above. Another method proves to be efficient is the competent cells prepared for long term storage as described by Hanahan, (1983) and Hanahan, (1985). It is recommended to use highly transformed protocol [7, 8] in case of very rare plasmids such as in case of the site directed mutagenesis plasmids to guarantee that the correct targeted plasmids are transformed. Then it is recommended to allow the harboring cells (e.g. E. coli) to grow first then start to investigate your clone or separate coexisted plasmid. One should remark that the decrease in the number of colonies in case of XL1Blue at OD600 reached 0.2-0.3 did not mean low transformation efficacy but that due to the presence of lower number of the cells used in the transformation step. However, in case of the Azotobacter sp only at OD600 reached 0.2-0.3 show successful transformation. One should observe the difference effect of the competent cells prepared as young have high transformation % but their low number reduce the total number of the transformants. That might be clearer in case of using Azotobacter sp., where large number but old cells show no transformation as in Table 1.
This study introduces different tools for isolating coexisted plasmids either totally similar such as in case of using plasmids in performing site directed mutagenesis and random mutagenesis without any existence of restriction cut in the new mutated gene. In many cases the sequencer proves that the wild type still existed where the new mutant appear in the same line of the wild type gene. The original base pair and the mutated one appear in the same line. In such cases and the other described ones in this study or those non described there is a real need to separate the coexisted plasmids without losing them. The different experiments introduced in this study will enable the isolation of both of the similar or the different plasmids using two main strategies, the first by reducing the transformation efficacy and the other by diluting the plasmids before their transformation and conducting a transformation experiments guarantee transforming one plasmid/cell.
Clearly Auctoresonline and particularly Psychology and Mental Health Care Journal is dedicated to improving health care services for individuals and populations. The editorial boards' ability to efficiently recognize and share the global importance of health literacy with a variety of stakeholders. Auctoresonline publishing platform can be used to facilitate of optimal client-based services and should be added to health care professionals' repertoire of evidence-based health care resources.
Journal of Clinical Cardiology and Cardiovascular Intervention The submission and review process was adequate. However I think that the publication total value should have been enlightened in early fases. Thank you for all.
Journal of Women Health Care and Issues By the present mail, I want to say thank to you and tour colleagues for facilitating my published article. Specially thank you for the peer review process, support from the editorial office. I appreciate positively the quality of your journal.
Journal of Clinical Research and Reports I would be very delighted to submit my testimonial regarding the reviewer board and the editorial office. The reviewer board were accurate and helpful regarding any modifications for my manuscript. And the editorial office were very helpful and supportive in contacting and monitoring with any update and offering help. It was my pleasure to contribute with your promising Journal and I am looking forward for more collaboration.
We would like to thank the Journal of Thoracic Disease and Cardiothoracic Surgery because of the services they provided us for our articles. The peer-review process was done in a very excellent time manner, and the opinions of the reviewers helped us to improve our manuscript further. The editorial office had an outstanding correspondence with us and guided us in many ways. During a hard time of the pandemic that is affecting every one of us tremendously, the editorial office helped us make everything easier for publishing scientific work. Hope for a more scientific relationship with your Journal.
The peer-review process which consisted high quality queries on the paper. I did answer six reviewers’ questions and comments before the paper was accepted. The support from the editorial office is excellent.
Journal of Neuroscience and Neurological Surgery. I had the experience of publishing a research article recently. The whole process was simple from submission to publication. The reviewers made specific and valuable recommendations and corrections that improved the quality of my publication. I strongly recommend this Journal.
Dr. Katarzyna Byczkowska My testimonial covering: "The peer review process is quick and effective. The support from the editorial office is very professional and friendly. Quality of the Clinical Cardiology and Cardiovascular Interventions is scientific and publishes ground-breaking research on cardiology that is useful for other professionals in the field.
Thank you most sincerely, with regard to the support you have given in relation to the reviewing process and the processing of my article entitled "Large Cell Neuroendocrine Carcinoma of The Prostate Gland: A Review and Update" for publication in your esteemed Journal, Journal of Cancer Research and Cellular Therapeutics". The editorial team has been very supportive.
Testimony of Journal of Clinical Otorhinolaryngology: work with your Reviews has been a educational and constructive experience. The editorial office were very helpful and supportive. It was a pleasure to contribute to your Journal.
Dr. Bernard Terkimbi Utoo, I am happy to publish my scientific work in Journal of Women Health Care and Issues (JWHCI). The manuscript submission was seamless and peer review process was top notch. I was amazed that 4 reviewers worked on the manuscript which made it a highly technical, standard and excellent quality paper. I appreciate the format and consideration for the APC as well as the speed of publication. It is my pleasure to continue with this scientific relationship with the esteem JWHCI.
This is an acknowledgment for peer reviewers, editorial board of Journal of Clinical Research and Reports. They show a lot of consideration for us as publishers for our research article “Evaluation of the different factors associated with side effects of COVID-19 vaccination on medical students, Mutah university, Al-Karak, Jordan”, in a very professional and easy way. This journal is one of outstanding medical journal.
Dear Hao Jiang, to Journal of Nutrition and Food Processing We greatly appreciate the efficient, professional and rapid processing of our paper by your team. If there is anything else we should do, please do not hesitate to let us know. On behalf of my co-authors, we would like to express our great appreciation to editor and reviewers.
As an author who has recently published in the journal "Brain and Neurological Disorders". I am delighted to provide a testimonial on the peer review process, editorial office support, and the overall quality of the journal. The peer review process at Brain and Neurological Disorders is rigorous and meticulous, ensuring that only high-quality, evidence-based research is published. The reviewers are experts in their fields, and their comments and suggestions were constructive and helped improve the quality of my manuscript. The review process was timely and efficient, with clear communication from the editorial office at each stage. The support from the editorial office was exceptional throughout the entire process. The editorial staff was responsive, professional, and always willing to help. They provided valuable guidance on formatting, structure, and ethical considerations, making the submission process seamless. Moreover, they kept me informed about the status of my manuscript and provided timely updates, which made the process less stressful. The journal Brain and Neurological Disorders is of the highest quality, with a strong focus on publishing cutting-edge research in the field of neurology. The articles published in this journal are well-researched, rigorously peer-reviewed, and written by experts in the field. The journal maintains high standards, ensuring that readers are provided with the most up-to-date and reliable information on brain and neurological disorders. In conclusion, I had a wonderful experience publishing in Brain and Neurological Disorders. The peer review process was thorough, the editorial office provided exceptional support, and the journal's quality is second to none. I would highly recommend this journal to any researcher working in the field of neurology and brain disorders.
Dear Agrippa Hilda, Journal of Neuroscience and Neurological Surgery, Editorial Coordinator, I trust this message finds you well. I want to extend my appreciation for considering my article for publication in your esteemed journal. I am pleased to provide a testimonial regarding the peer review process and the support received from your editorial office. The peer review process for my paper was carried out in a highly professional and thorough manner. The feedback and comments provided by the authors were constructive and very useful in improving the quality of the manuscript. This rigorous assessment process undoubtedly contributes to the high standards maintained by your journal.
International Journal of Clinical Case Reports and Reviews. I strongly recommend to consider submitting your work to this high-quality journal. The support and availability of the Editorial staff is outstanding and the review process was both efficient and rigorous.
Thank you very much for publishing my Research Article titled “Comparing Treatment Outcome Of Allergic Rhinitis Patients After Using Fluticasone Nasal Spray And Nasal Douching" in the Journal of Clinical Otorhinolaryngology. As Medical Professionals we are immensely benefited from study of various informative Articles and Papers published in this high quality Journal. I look forward to enriching my knowledge by regular study of the Journal and contribute my future work in the field of ENT through the Journal for use by the medical fraternity. The support from the Editorial office was excellent and very prompt. I also welcome the comments received from the readers of my Research Article.
Dear Erica Kelsey, Editorial Coordinator of Cancer Research and Cellular Therapeutics Our team is very satisfied with the processing of our paper by your journal. That was fast, efficient, rigorous, but without unnecessary complications. We appreciated the very short time between the submission of the paper and its publication on line on your site.
I am very glad to say that the peer review process is very successful and fast and support from the Editorial Office. Therefore, I would like to continue our scientific relationship for a long time. And I especially thank you for your kindly attention towards my article. Have a good day!
"We recently published an article entitled “Influence of beta-Cyclodextrins upon the Degradation of Carbofuran Derivatives under Alkaline Conditions" in the Journal of “Pesticides and Biofertilizers” to show that the cyclodextrins protect the carbamates increasing their half-life time in the presence of basic conditions This will be very helpful to understand carbofuran behaviour in the analytical, agro-environmental and food areas. We greatly appreciated the interaction with the editor and the editorial team; we were particularly well accompanied during the course of the revision process, since all various steps towards publication were short and without delay".
I would like to express my gratitude towards you process of article review and submission. I found this to be very fair and expedient. Your follow up has been excellent. I have many publications in national and international journal and your process has been one of the best so far. Keep up the great work.
We are grateful for this opportunity to provide a glowing recommendation to the Journal of Psychiatry and Psychotherapy. We found that the editorial team were very supportive, helpful, kept us abreast of timelines and over all very professional in nature. The peer review process was rigorous, efficient and constructive that really enhanced our article submission. The experience with this journal remains one of our best ever and we look forward to providing future submissions in the near future.
I am very pleased to serve as EBM of the journal, I hope many years of my experience in stem cells can help the journal from one way or another. As we know, stem cells hold great potential for regenerative medicine, which are mostly used to promote the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. I think Stem Cell Research and Therapeutics International is a great platform to publish and share the understanding towards the biology and translational or clinical application of stem cells.
I would like to give my testimony in the support I have got by the peer review process and to support the editorial office where they were of asset to support young author like me to be encouraged to publish their work in your respected journal and globalize and share knowledge across the globe. I really give my great gratitude to your journal and the peer review including the editorial office.
I am delighted to publish our manuscript entitled "A Perspective on Cocaine Induced Stroke - Its Mechanisms and Management" in the Journal of Neuroscience and Neurological Surgery. The peer review process, support from the editorial office, and quality of the journal are excellent. The manuscripts published are of high quality and of excellent scientific value. I recommend this journal very much to colleagues.
Dr.Tania Muñoz, My experience as researcher and author of a review article in The Journal Clinical Cardiology and Interventions has been very enriching and stimulating. The editorial team is excellent, performs its work with absolute responsibility and delivery. They are proactive, dynamic and receptive to all proposals. Supporting at all times the vast universe of authors who choose them as an option for publication. The team of review specialists, members of the editorial board, are brilliant professionals, with remarkable performance in medical research and scientific methodology. Together they form a frontline team that consolidates the JCCI as a magnificent option for the publication and review of high-level medical articles and broad collective interest. I am honored to be able to share my review article and open to receive all your comments.
“The peer review process of JPMHC is quick and effective. Authors are benefited by good and professional reviewers with huge experience in the field of psychology and mental health. The support from the editorial office is very professional. People to contact to are friendly and happy to help and assist any query authors might have. Quality of the Journal is scientific and publishes ground-breaking research on mental health that is useful for other professionals in the field”.
Dear editorial department: On behalf of our team, I hereby certify the reliability and superiority of the International Journal of Clinical Case Reports and Reviews in the peer review process, editorial support, and journal quality. Firstly, the peer review process of the International Journal of Clinical Case Reports and Reviews is rigorous, fair, transparent, fast, and of high quality. The editorial department invites experts from relevant fields as anonymous reviewers to review all submitted manuscripts. These experts have rich academic backgrounds and experience, and can accurately evaluate the academic quality, originality, and suitability of manuscripts. The editorial department is committed to ensuring the rigor of the peer review process, while also making every effort to ensure a fast review cycle to meet the needs of authors and the academic community. Secondly, the editorial team of the International Journal of Clinical Case Reports and Reviews is composed of a group of senior scholars and professionals with rich experience and professional knowledge in related fields. The editorial department is committed to assisting authors in improving their manuscripts, ensuring their academic accuracy, clarity, and completeness. Editors actively collaborate with authors, providing useful suggestions and feedback to promote the improvement and development of the manuscript. We believe that the support of the editorial department is one of the key factors in ensuring the quality of the journal. Finally, the International Journal of Clinical Case Reports and Reviews is renowned for its high- quality articles and strict academic standards. The editorial department is committed to publishing innovative and academically valuable research results to promote the development and progress of related fields. The International Journal of Clinical Case Reports and Reviews is reasonably priced and ensures excellent service and quality ratio, allowing authors to obtain high-level academic publishing opportunities in an affordable manner. I hereby solemnly declare that the International Journal of Clinical Case Reports and Reviews has a high level of credibility and superiority in terms of peer review process, editorial support, reasonable fees, and journal quality. Sincerely, Rui Tao.
Clinical Cardiology and Cardiovascular Interventions I testity the covering of the peer review process, support from the editorial office, and quality of the journal.
Clinical Cardiology and Cardiovascular Interventions, we deeply appreciate the interest shown in our work and its publication. It has been a true pleasure to collaborate with you. The peer review process, as well as the support provided by the editorial office, have been exceptional, and the quality of the journal is very high, which was a determining factor in our decision to publish with you.
The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews journal clinically in the future time.
Clinical Cardiology and Cardiovascular Interventions, I would like to express my sincerest gratitude for the trust placed in our team for the publication in your journal. It has been a true pleasure to collaborate with you on this project. I am pleased to inform you that both the peer review process and the attention from the editorial coordination have been excellent. Your team has worked with dedication and professionalism to ensure that your publication meets the highest standards of quality. We are confident that this collaboration will result in mutual success, and we are eager to see the fruits of this shared effort.
Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, I hope this message finds you well. I want to express my utmost gratitude for your excellent work and for the dedication and speed in the publication process of my article titled "Navigating Innovation: Qualitative Insights on Using Technology for Health Education in Acute Coronary Syndrome Patients." I am very satisfied with the peer review process, the support from the editorial office, and the quality of the journal. I hope we can maintain our scientific relationship in the long term.
Dear Monica Gissare, - Editorial Coordinator of Nutrition and Food Processing. ¨My testimony with you is truly professional, with a positive response regarding the follow-up of the article and its review, you took into account my qualities and the importance of the topic¨.
Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, The review process for the article “The Handling of Anti-aggregants and Anticoagulants in the Oncologic Heart Patient Submitted to Surgery” was extremely rigorous and detailed. From the initial submission to the final acceptance, the editorial team at the “Journal of Clinical Cardiology and Cardiovascular Interventions” demonstrated a high level of professionalism and dedication. The reviewers provided constructive and detailed feedback, which was essential for improving the quality of our work. Communication was always clear and efficient, ensuring that all our questions were promptly addressed. The quality of the “Journal of Clinical Cardiology and Cardiovascular Interventions” is undeniable. It is a peer-reviewed, open-access publication dedicated exclusively to disseminating high-quality research in the field of clinical cardiology and cardiovascular interventions. The journal's impact factor is currently under evaluation, and it is indexed in reputable databases, which further reinforces its credibility and relevance in the scientific field. I highly recommend this journal to researchers looking for a reputable platform to publish their studies.
Dear Editorial Coordinator of the Journal of Nutrition and Food Processing! "I would like to thank the Journal of Nutrition and Food Processing for including and publishing my article. The peer review process was very quick, movement and precise. The Editorial Board has done an extremely conscientious job with much help, valuable comments and advices. I find the journal very valuable from a professional point of view, thank you very much for allowing me to be part of it and I would like to participate in the future!”
Dealing with The Journal of Neurology and Neurological Surgery was very smooth and comprehensive. The office staff took time to address my needs and the response from editors and the office was prompt and fair. I certainly hope to publish with this journal again.Their professionalism is apparent and more than satisfactory. Susan Weiner
My Testimonial Covering as fellowing: Lin-Show Chin. The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews.
My experience publishing in Psychology and Mental Health Care was exceptional. The peer review process was rigorous and constructive, with reviewers providing valuable insights that helped enhance the quality of our work. The editorial team was highly supportive and responsive, making the submission process smooth and efficient. The journal's commitment to high standards and academic rigor makes it a respected platform for quality research. I am grateful for the opportunity to publish in such a reputable journal.
My experience publishing in International Journal of Clinical Case Reports and Reviews was exceptional. I Come forth to Provide a Testimonial Covering the Peer Review Process and the editorial office for the Professional and Impartial Evaluation of the Manuscript.
