AUCTORES
Globalize your Research
Research | DOI: https://doi.org/10.31579/2637-8914/055
*Corresponding Author: Hamouda Elabed, Laboratory of Contagious Diseases and Biologically Active Substances LR99-ES27, Faculty of Pharmacy of Monastir, University of Monastir, Tunisia.
Citation: Rihab Ben Slama, Hamouda Elabed, Hanene Miladi, Tarek Zmantar, Maha Mastouri, et al. (2021). Morphological Alterations and Virulence Gene Expression of Listeria Monocytogenes Cells in Response to Gamma Irradiation Treatments. J. Nutrition and Food Processing, 4(5); DOI:10.31579/2637-8914/055
Copyright: 2021, Hamouda Elabed. 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: 10 June 2021 | Accepted: 30 June 2021 | Published: 09 July 2021
Keywords: : gamma irradiation; listeria monocytogenes; gene expression; fatty acid; AFM; agro-food industries; dehydrated vegetables; IAEA; food safety; potential pathogenic bacteria; chromatographic method
Gamma irradiation is one of the most popular ray treatments in food industry. It's used to control micro-organisms proliferation in a wide range of products however the response of bacteria to low doses is still unknown. In this study we mainly focus on morphological alteration and virulence gene expression in Listeria monocytogenes after gamma irradiation treatments. The atomic force micrographs (AFM) showed that 0.5 kGy dose has no effect on the membrane morphology of L. monocytogenes. However, after 0.7 kGy treatment, the cells lost their typical shape and smooth membrane and 1 kGy dose was totally destructive. Moreover, membrane fatty acid composition was analyzed by the chromatographic method after different gamma doses. Significant modifications on fatty acids composition were detected in the irradiated strain showing a novo synthesis of membrane lipids: C12:0; C14:0; C15:0; C16:0 and C18:0. In addition, we reported an increase of the saturated fatty acid, essential for membrane adaptation under stress conditions. The expression levels of three virulence genes (hlyA, fri and prfA) were studied in the same conditions using real-time PCR technique. The analysis revealed that both prfA and fri genes were up-regulated after gamma treatment. The induction of prfA, which is a regulator gene, may affect the expression other genes controlling the adaptive form in the treated strain. This study open prospects for further researches to explain the regulatory mechanisms of the adaptive response in Listeria monocytogenes when exposed to sublethal irradiation-stress.
In the Agro-Food Industries, the irradiation is the process of applying ionizing energy to foodstuffs to improve microbiological safety and to extend the shelf life [1]. Irradiation technology is used for decontamination and/or sterilization of dehydrated vegetables, fruits, meats, poultry, fish and seafood in order to improve product safety and shelf life [2, 3]. According to the International Atomic Energy Agency (IAEA), the quantities of foods that are irradiated are increasing each year and the majority of these foods are treated by gamma irradiation. Moreover, it is expected that more food will be treated by irradiation in the future, because this technology can address food safety and security problems without significantly affecting at the nutritional attributes [4].
The treatment depended on the process objective with respect radiological safety and generally, the doses used in gamma irradiation treatment are between 0.3 and 3 kGy [5-7]. For microbial decontamination, the targets of gamma irradiation are chromosomal DNA, RNA, cytoplasmic membrane and proteins [8, 9]. Escherichia coli, Toxoplasma gonkii, Salmonella enterica and Listeria monocytogenes were controlled in food using irradiation [10]. It has been reported that the Gram-positive bacteria is more resistant to gamma irradiation than other potential pathogenic bacteria like E. coli [11].
Listeria monocytogenes is a Gram-positive bacteria, frequently associated with food borne disease outbreaks. It is also considered as a major food safety challenge in the food industry. Improved control measures and risks assessments have greatly reduced the prevalence of this pathogen in food. However, the rate of illness has remained constant during the last decade [12]. The psychotropic nature of L. monocytogenes allows replication in refrigerated ready-to-eat food products that were contaminated during processing and packaging. Consequently, L. monocytogenes is characterized by widespread distribution and relatively high mortality rates [13]. The effectiveness of gamma irradiation in inactivating the L. monocytogens on different food products was previously reported; Song et al. confirmed the efficacy of gamma irradiation to inactivate foodborne pathogens on nuts indicating that the D-value of L. monocytogenes on pistachios was 1.02 kGy, larger than those of S. enterica or E. coli [14]. Other study has shown that L. monocytogenes and S. enterica growth was inactivated in fresh orange juice at 0-4 kGy [15]. Moreover, Niemira et al. evaluated the effect of gamma irradiation (0.3 and 0.6 kGy) on L. monocytogens inoculated cut pieces of endive, thereby demonstrating that irradiation and modified atmosphere packaging can be combined to prevent the regrowth of L. monocytogenes during post-irradiation refrigerated storage [16].
Listeria treatments using gamma rays have been widely applied for different goals; however, none of this research had studied the problems related to radio-resistant L. monocytogenes cells. Therefore, a better understanding of resistant cells is required to reduce listeriosis incidence. Adaptive response, morphological alteration and gene regulation should be key elements to study bacteria behavior. One of the most important issues related to the microbiological safety of irradiated foods is to know if the sub-lethal doses would make surviving bacterial cells more pathogenic.
The aim of this work was to study the responses elicited in Listeria monocytogenes following different gamma irradiation doses, by analyzing: (i) The morphological alteration using atomic force microscopy (ii) The membrane fatty acid profiles and (iii) The expression levels of three virulence genes.
1. Strains and culture conditions
The American Type Culture Collection Listeria monocytogenes ATCC 19115, serotype 4b [17] was used in this study. Cells were grown in tryptic soy broth with 0.6% yeast extract (TSB-YE) (Pronadisa, Spain) at 37°C for 20h. Cultures were centrifuged at 2,862 g for 10 min and resuspended in 0.1% peptone. The optical density of each suspension was adjusted to 0.5 (108 CFU/ml) at 600 nm using a spectrophotometer (Spectro UV-Vis; Model UVD-2960, Labomed, Inc., CA). Suspensions were divided into 1ml aliquots, and were subjected in triplicate, on ice to different gamma irradiation doses using 60Co Gamma-irradiator (point source, AECL, IR-79; Technopole, Sidi Thabet, Tunisia). The applied doses were 0.5, 0.7 and 1 kGy. The irradiated cultures were diluted 50-fold using 50 ml of fresh TSB-YE medium and then harvested after 24h of incubation at 37°C with shaking.
2. Determination of morphological changes by AFM
In order to visualize the effect of Gamma-rays on cytoplasmic membrane, L. monocytogenes cells were examined, in triplicate, by atomic force microscopy (AFM) (Nanoscope IIIA, Digital Instrument, VEECO) after 0.5, 0.7 and 1kGy gamma irradiation doses. For this experiment, the cells were collected, washed three times with PBS, and centrifuged. The final pellet was resuspended in PBS, placed on a round microscope cover slide and was simply dried in air according to the method previously described by Braga and Ricci [18].
3. Analysis of fatty acids
To analyze the total cellular fatty acids, Listeria strains were incubated after irradiation treatment in buffered peptone water (Biolife Italiana) for 24 h at 37 °C. Each experiment was performed in triplicate by using three independently grown cultures. Cells recovered from 10 ml suspension were pretreated following the Microbial Identification Inc (MIDI) protocols [19]. All reagents for saponification, methylation, extraction, and washing were dispensed with autopipets into the same tube, making the hands-on time minimal. Next, the final extracts were analyzed by gas chromatography (column: 30 m × 0.25 mm HP-Innowax; flame ionization detect temperature at 280°C; carrier gas N2 at 1 ml/min; injector temperature 270°C; oven temperature programmed from 130 to 230°C) using a Hewlett-Packard HP 5890 capillary gas chromatograph linked to an HP Chemstation integrator. The identification of fatty acid methyl esters was performed by external standards (all purchased from Sigma Chemical Co) submitted to the same processes of manipulation as the biological samples analyzed. Total saturated fatty acids (SFA), total unsaturated fatty acids (UFA) were used to determine the differences among membrane fatty acids of L. monocytogenes cells, and they were examined under the different conditions. The UFA/SFA ratio was used as an indirect indicator of the membrane fluidity.
4. DNA extraction and polymerase chain reaction
Genomic DNA of treated and untreated L. monocytogenes cells was extracted using the bacteria DNA preparation kit (JenaBioscience) as manufacturer’s instructions. The concentration and the quality of the DNA were determined by measuring the OD260nm and the agarose gel electrophoresis (0.8%) containing ethidium bromide. Primers used in both PCR and qRT-PCR are listed in Table 1.
These primers were used in our last studies and by Miladi et al. [13, 39]. Polymerase chain reaction (PCR) was performed in a reaction volume of 25 μl containing 2.5 µl 10X DreamTaq Green Buffer (Thermo Scientific), 100 µM dNTP Mix (JenaBioscience), 0.2 µM of both forward and reverse primers, 1 unit of DreamTaq DNA Polymerase (Thermo Scientific), and approximately 50 ng of DNA template.
PCR reactions were conducted in a MultiGene gradient thermal cycler (Labnet International) using the following program: 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 57°C (annealing) for 30 seconds, 72°C for 30 seconds, and finally 72°C for 10 minutes. PCR products were analyzed by electrophoresis on 1.5% agarose gel ethidium bromide stained.
5. Isolation of total RNA
Total RNA was isolated from Gamma irradiated and control L. monocytogenes cells using the SV total RNA isolation system, following the manufacturer’s instructions (Promega, Madison, WI, USA). The RNA was treated with one unit DNase I enzyme (Thermo Scientific) to eliminate residual genomic DNA. Samples were electrophoresed in a formaldehyde agarose gel to check the integrity of RNA.
6. First strand cDNA synthesis
For the reverse transcription (RT) reactions, a mixture of 500 ng of total RNA, 1 µl of 10 µM reverse gene-specific primer, 0.6µl of 100 mM dNTP Mix (Jena Bioscience), 4 μl of MMLV Reverse Transcriptase 5X reaction buffer (Promega), and 200 units of Moloney Murine Leukemia Virus (MMLV) Reverse Transcriptase (Promega) and the final volume was adjusted to 20 μl with Nuclease-free water. The synthesis reaction was carried out for 45 min at 40°C and stopped by incubation at 75°C for 5 min. To avoid detecting genomic DNA contamination in RT-PCR, a control RNA sample without MMLV reverse was used.
7. Real-time PCR assays for relative quantification of gene expression
Real-time PCR was performed using a Chromo4 real-time PCR detection system with Opticon Monitor 3 software (Bio-Rad, Hercules, USA). Amplification and detection were carried out in 96-wells plate with qPCR GreenMaster containing EvaGreen as the fluorescent DNA stain (JenaBioscience). The primers used to perform the quantitative PCR were the same as those used previously for PCR optimization (Table 1). Each reaction was done in triplicate in 20 μl of final volume: 10µl qPCR Green Master, 1 µl of each forward and reverse primers (10 µM), 4µl reverse transcription product as a template and 4 µl PCR-grade water (Jena Bioscience). Amplifications were carried out as follows: 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 57°C for 20 seconds and 72°C for 30 seconds. To assess the PCR specificity, the melting curve analysis was performed by increasing the temperature from 50 to 95°C, with a raise in temperature by 1°C every 10 sec with a plate scan step to read the fluorescent signal. The method of Pfaffl et al., 2002 was used to analyze the relative gene expression from real-time quantitative PCR experiments [20].
8. Statistical analysis
Statistical analysis was performed using the S.P.S.S. 13.0 statistics package for Windows. The differences in UFA/SFA ratio were examined by the Friedman test, followed by the Wilcoxon signed ranks test. P-values < 0>
1. Morphological alterations of irradiated Listeria monocytogenes
Cells were scanned after Gamma irradiation treatment (Figure 1). Like the control L. monocytogenes, the 0.5 kGy irradiated Listeria strains have a normal rod shape with a smooth surface (Figure1 A-B); whereas the treated cells with 0.7 kGy gamma irradiation dose present irregular shape and rough surface characterized by dispersed lumps (Figure1 C-D). However, after 1kGy gamma treatment the membrane was destroyed and cell components were scattered around (Figure1 E-F).
2. Membrane fatty acids composition of irradiated Listeria monocytogenes
The membrane fatty acid composition of L. monocytogenes cultured under the different growth conditions (irradiated and normal cells) was determined using a chromatographic method (figure 2).
The X-axis represents the 16 detected fatty acids in the studied strain and the Y-axis represents the relative fatty acid abundance (Percentage of total fatty acids).
Sixteen fatty acids were found in control and stressed cells. The five main peaks were identified as Tetradecanoic acid (iso-C14:0), Pentadecanoic acid (iso-C15:0 and anteiso-C15:0) and Heptadecanoic acid (iso-C17:0 and anteiso-C17:0). Their relative percentages were between 1.68% for iso-C14:0 and 35.02 % for anteiso-C15:0. As expected, the irradiation of L. monocytogenes resulted in differences in membrane fatty acid composition. Indeed, a significant increase in SFA accompanied by a significantly decrease in the UFA was observed (P < 0>
It has been previously reported that membranes with high UFA/SFA ratio show a high fluidity [21, 22].
3. Virulence genes expression
Relative [removed]RE) of prfA, fri and hlyA genes were determined after exposition of Listeria monocytogenes cells to low-dose gamma irradiation (0.5 kGy and 0.7 kGy). To measure transcripts levels of these virulence genes, various steps in the qRT-PCR procedure were optimized. The RNA sample quality remained stable over the period of analysis and showed no differential transcript degradation. Primers for individual genes were chosen to be specific for L. monocytogenes. In addition, the melting curve was carried out to detect any no specific amplification.
The optimization of the real-time PCR was evaluated based on the amount and the quality of total RNA template, specificity and efficiency of the reaction. The observed results suggest that the conditions used for RNA template preparation, reverse transcription and real-time PCR were compatible with quantitative analysis of RNA transcripts.
Expression levels were evaluated after the comparison of the two conditions (control and gamma treated cells).
The analysis of virulence gene expression in L. monocytognes using Real-Time PCR revealed that both genes prfA and fri were upregulated after low doses Gamma exposition. While hlyA gene was mostly down-regulated (Figure 3).
In addition, the expression levels were more pronounced after gamma treatment with 0.7 kGy dose. Indeed, the fold changes expression of prfA in treated L. monocytognes cells were 3.22 and 59.79 after 0.5 kGy and 0.7 kGy gamma doses, respectively. Similarly, fri gene was overexpressed after 0.5 kGy and 0.7 kGy in L. monocytognes (2.07 and 10.06 fold change, respectively). On the other hand, the hlyA gene was down-regulated in treated L. monocytognes cells.
Thus, the relative expression of prfA, which is a regulator gene may depend on gamma radiation dose and influences the expression of the two other studied genes.
In this study, three low radiation doses were chosen which are generally tolerated in food irradiation to eliminate pathogenic bacteria. Various factors, such as chemical composition of foods may affect the radiation resistance and sensitivity [5, 7]. Thus, as a try to reduce this problem, gamma treatment was applied for bacteria incubated in TSB-YE.
The effectiveness of gamma irradiation in inactivating pathogenic bacteria at low gamma doses was previously reported; E. coli 0157:H7 and aerobic mesophiles in lettuce at 0-0.55 kGy [23], L. monocytogenes and S. enterica growth was inactivated in fresh orange juice at 0-4 kGy [15], S. typhimurium and E. coli in carrot and kale juice at 0-3 kGy [7] and total aerobic and coliform bacteria in carrot and kale juice were inactivated at 0-5 kGy [5].
Some radiation-resistant bacteria can recuperate their growth potency when the environmental conditions are suitable for bacteria development. In our study L. monocytogenes strain was resistant to 0.5 and 0.7 kGy gamma irradiation dose but not to 1kGy.
The 0.5 kGy dose did not significantly affect neither the shape nor the smooth of the bacteria.
Bacterial cytoplasmic membranes define cells from the external environment. It represents an additional location for stress sensing that is capable to transduce signals affecting the expression of genes involved in defense [24]. It has been proposed that under some stress conditions like starvation, member of L. monocytogenes can undergo a number of morphological and physiological changes to adapt to such severe condition [25]. According to the obtained results, important modifications seemed to take place after gamma irradiation treatment with 0.5, 0.7 and 1 kGy dose for L. monocytogenes. This sensitivity implies that membrane lipids play an important role in microbial adaptation to the destructive effect of gamma rays. We also observed that the bacterial membrane resistance was associated with the alteration of virulence gene expression. According to Russel [26], a change in membrane fluidity is often caused by a change in the lipid composition of the membranes to keep an adequate proportion of liquid-crystalline lipid in the membrane and to maintain the bilayer phase. In the same way, treatment of E. coli with non-lethal doses of heat or benzyl alcohol causes transient membrane fluidization and permeability, and induces the rapid transcription of heat-shock genes in a σ32-dependent manner [27].
Total saturated and unsaturated fatty acids were used to determine the differences among membrane fatty acids of L. monocytogenes cells grown under the different stress conditions. The UFA/SFA ratio was used as an indirect indicator of the membrane fluidity. Significant modifications on fatty acid (FA) composition (P ≤ 0.05) seemed to take place after Gamma-irradiation treatment with a 0.5 kGy and 0.7 kGy doses for studied strain. So, a significant (P ≤ 0.05) decrease in the UFA/SFA ratio was observed with the two doses. Moreover, our results showed a novo synthesis of membrane lipids: C12:0; C14:0; C15:0; C16:0 and C18:0. A wide range of bacteria are able to change their membrane composition by novo synthesis of membrane lipids during growth [28]. Bacteria regulate their lipid composition to achieve a degree of fluidity compatible for life. However, little is known on the influence of these modifications in membrane composition on the bacterial resistance to subsequent stresses. One of the most important consequences of membrane fatty acid changes in microorganisms is to modulate the activity of intrinsic proteins that perform functions such as ion pumping and nutrient uptake [29]. The membrane lipid plays an important role in microbial adaptation to the destructive effect of the reactive oxygen species. This adaptation was related to the increase of SFA production. Therefore, it’s supposed that the modification of SFA proportions constituted an adaptive response to Gamma-irradiation stress in L. monocytogenes. It is important to note that several studies have suggested that an increase in SFA content could cause a decrease in membrane fluidity [21, 22].
Listeria monocytogenes responds to environmental stress by modifying the rate of synthesis of certain proteins [30]. This study showed modification on relative expression of Listeria monocytogenes genes after its treatments with Gamma rays. The mechanism of microbial inactivation by gamma ray may damage DNA and/or some protein denaturation [31]. For a living cell, the slightest damage to DNA can affect its development and survival. Thus, gamma lesions impinge on vital cellular functions, including transcription, DNA replication and cell cycle progression. Their persistence in the genetic material also increases the chance of fixation into mutations [32]. These damages may affect directly microbial DNA by making strand breaks of nucleic acids or indirectly via hydroxyl radicals originating from radiolysis of water [33]. In our study, the expression of pathogenic genes was affected after 0.5 and 0.7 kGy gamma irradiation (figure 3). The qRT-PCR analysis demonstrated that prfA and fri genes were up-regulated after 0.5 kGy treatment and were strongly induced after 0.7 kGy dose. However, the hlyA gene was down regulated. Other genes could be affected by this treatment and their expression could be influenced by the product of prfA expression. It has been previously demonstrated that the expression of 145 L. monocytogenes genes is influenced by PrfA according to transcriptomic profiling and proteomic analyses [34-36]. These indirectly regulated genes include transporters, metabolic enzymes, regulators, proteins of different functions, together with many members of stress response regulation [34, 35]. Indeed, PrfA is a central virulence regulator and may potentially exert many general effects on L. monocytogenes homeostasis. Although loss of expression from the P2prfA promoter affects in vitro virulence phenotypes, such as hemolysin production [37].
It has been reported that gamma irradiation could induce gene mutation. But, there has been no evidence of irradiation-induced enhanced pathogenicity of food borne microorganisms. However, it should not be ignored that there were no involved mechanisms of post-irradiation DNA repair.
Lim et al. [38] reported that after exposure to a dose of 0.5 or 1 kGy, the transcriptional changes of vvhA, ctxA, and tdh genes respectively in V. vulnificus, V. cholerae, and V. parahaemolyticus ranged from 0.3- to 0.001-fold. He also indicated in his study that gamma radiation reduces the virulence gene expression of surviving pathogens.
Several genes contributing to L. monocytogenes virulence have been well identified and characterized. The majority of these virulence genes, lies in a single cluster on the L. monocytogenes chromosome, are controlled by the transcriptional activator PrfA [39, 40]. This is the first report mentioning the effect of gamma irradiation on L. monocytogenes gene expression.
Beauchamp [11] has compared the natural resistance of the genome of Gram-positive and Gram-negative bacterium against irradiation solution of pure DNA and bacterial strains such as Listeria monocytogenes and Escherichia coli which were irradiated using gamma rays. The authors demonstrated that gamma rays induce the formation of cyclobutane pyrimidine dimers and 6-4 photoproducts in DNA in L. monocytogenes genome between 0.1 and 0.7 kGy gamma irradiation doses. In the other hand, it’s supposed that the combined effects of both stresses (freezing and ionization) disrupted the expression of the studied genes. In previous study, it was demonstrated that simultaneous exposure to most multiple stresses (e.g., osmolarity, acidity and temperature) resulted in higher resistance of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially [41]. Similar results were found with a combination of acid , water activity (aw) and temperature stress [42]. These results strongly support our expectations.
The main aim of this paper was to demonstrate whether the gamma rays can affect Listeria monocytogenes cells. The first observation was that this treatment leads to in typical form modification of Listeria. Then, by measuring the relative expression of three virulence genes it was demonstrated that low gamma doses are able to induce pathogenic gene expressions. Moreover, prfA expression was very sensitive to gamma rays and its induction was strongly related to other virulence genes expression.
Further studies are needed to explain mechanisms for regulating expression of irradiation-stress response and determining the effect of such treatment on food products in order to limit their application in food industry.
This research was supported by the National Center for Nuclear Sciences and Technologies (CNSTN), Tunis Cedex, 2020, of Tunisia. The authors thank Pr. Mokhtar Hamdi for the irradiation essays.
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.
