AUCTORES
Globalize your Research
Research Article | DOI: https://doi.org/10.31579/2692-9406/008
*Corresponding Author: Samad Farashi Bonab, Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Citation: Samad Farashi B, Maryam M. (2020) Differential expression of apoptosis related genes in the peripheral blood mononuclear cells of acute lymphoblastic/lymphocytic leukemia (ALL) patients. Biomedical Research and Clinical Reviews. 1(4); DOI: 10.31579/2692-9406/008
Copyright: © 2020 Samad Farashi Bonab, This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 11 June 2020 | Accepted: 04 November 2020 | Published: 09 November 2020
Keywords: acute lymphoblastic/lymphocytic leukemia; pbmc, apoptosis; gene expression
Apoptosis is the cell’s intrinsic death program which plays a crucial role in the regulation of many normal physiological processes in the body’s tissues. In leukemia patients, the extent of cancer cell susceptibility to apoptosis is correlated with clinical responses to chemotherapy and disease prognosis. The aim of this study was evaluation of the expression of apoptosis related genes in the peripheral blood mononuclear cells (PBMCs) of acute lymphoblastic/lymphocytic leukemia (ALL) patients. Peripheral blood samples were obtained from 20 patients with ALL and 20 healthy individuals. PBMCs were isolated using Ficoll-Paque density gradient centrifugation method. After RNA extraction and cDNA synthesis, gene expression levels of apoptosis related genes including caspase-3, 8, 9, BAX, and BAK genes were measured by real-time RT-PCR technique. Gene expression analysis showed that the expression levels of the initiator caspases-8 and -9 are increased in the PBMCs of adult ALL patients when compared with that in PBMCs of healthy individuals. Increased gene expression levels of the proapoptotic protein BAK was also detected in PBMCs of ALL patients. In contrast, decreased expression levels of the proapoptotic BAX and the executioner caspase-3 were observed in the PBMCs of ALL patients. These results suggest that the expression of genes involved in both extrinsic and intrinsic signaling pathways of apoptosis are induced in PBMCs of ALL patients while the gene expression of other proapoptotic molecule, BAK, and the executioner caspase-3 diminished in PBMCs cells of ALL patients. This findings indicate that resistance to apoptosis may be one of the hallmarks of ALL cells.
Acute lymphoblastic/lymphocytic leukemia (ALL) is a malignancy of B or T lymphoblasts characterized by uncontrolled proliferation of abnormal, immature lymphocytes in the bone marrow, blood and extramedullary sites. Approximately 85% of childhood ALL cases and 75% of adult ALL cases develop from B cell precursors, while the remaining ALL cases develop from T cell precursors [1]. Disease symptoms arise from various degrees of anemia, neutropenia, and thrombocytopenia or infiltration of ALL cells into tissues. Leukemia cells in the peripheral blood can enter various organs such as the lymph nodes, spleen, liver, central nervous system, and skin [2]. ALL is the most common type of childhood leukemia, accounts for 75% of childhood leukemias [3] and makes up 25% of all childhood cancers. About 54% of ALL cases are diagnosed in patients aged younger than 20 years [4]. In adults, ALL is accounts for 20% of adult leukemia [5], and makes up 0.4% of all adult cancers. Adult ALL often occurs around the age 50, especially in those with history of chemotherapy, exposure to ionizing radiation, pesticides, certain chemicals or viruses [1]. In 2020, the estimated numbers of new cases of ALL and its related deaths in the United States are 6,150 and 1,520, respectively [6].Intensive chemotherapy can induce complete responses in ALL patients. However, survival rate for older patients achieving a remission is relatively low [7]. The 5‐year relative survival rate is 89% for ages less than 20 years, 47% for ages 20-49 years, 28% for ages 50-64 years, and 17% for ages 65 years and older [8].
It has been shown that the extent of cancer cell susceptibility to apoptosis is correlated with clinical responses to chemotherapy and disease prognosis [9, 10]. Apoptosis is a major form of cell death, characterized by a series of distinct morphological changes including DNA fragmentation, nuclear condensation and fragmentation, fragmentation of cytoplasmic organelles (the Golgi apparatus, endoplasmic reticulum and mitochondrial network), cytoskeletal rearrangement, plasma membrane blebbing, and cell shrinkage [11, 12]. These events are mainly orchestrated by members of a family of cysteine proteases known as caspases [13]. Caspase-dependent proteolysis leads to detachment of the apoptotic cell from the extracellular matrix and fragmentation of the genomic DNA, Golgi, endoplasmic reticulum and mitochondrial networks. Finally, the cell breaks into small membrane-surrounded fragments, known as apoptotic bodies, which are rapidly removed by phagocytes, a process that minimizes damage and disruption to neighboring cells and avoids activation of immune responses.
Caspases exist in an inactive form in the absence of an activation signal. Three major apoptotic pathways by which caspases can be activated have been identified, including extrinsic (or death receptor) pathway, intrinsic (or mitochondrial-mediated) pathway, and granzyme B pathway. Caspase-8 and -9 are the initiator (or apical) caspases in the extrinsic and intrinsic pathways, respectively. These caspases possess a protein interaction prodomain, which plays a role in their recruitment and subsequent activation within cellular complexes known as the death-inducing signaling complex (DISC) and the Apaf-1 apoptosome in the extrinsic and intrinsic pathways, respectively. Activated caspase-8 and -9 then process and activate effector caspases (also called executioner caspases) including caspase-3, -6, and-7. Granzyme B, a protease released from cytotoxic T cells and natural killer cells, initiates apoptosis by cleaving the proapoptotic BID protein as well as caspase-3 and -7 in the target cells [12]. Caspase-3 is the major effector caspase in both the extrinsic and intrinsic pathway as well as in the granzyme B pathway and is responsible for cleavage of multiple cellular substrates during apoptotic cell death.
The intrinsic pathway of apoptosis can be induced in cancer cells by various stimuli, including increase in the levels of pore-forming proapoptotic BCL-2 (B cell lymphoma-2) family proteins leading to pro-caspase-9 activation [14]. Mitochondrial outer membrane permeabilization (MOMP) is involved in this pathway of apoptosis. MOMP is controlled by members of the BCL-2 protein family. Members of this protein family are divided into three classes based on their sequence homology and function. The first class includes the antiapoptotic proteins BCL-2, BCL-XL, BCL-w, BCL-B, BCLA1, and MCL-1 which contain all BCL-2 homology domains (BH1-4). The second class includes the proapoptotic BH3-only proteins BID, BIM, BAD, BIK, NOXA, PUMA, HRK, and BMF. The third class contains proapoptotic effector proteins BAX, BAK, and BOK/MTD. If the proapoptotic members of the BCL-2 family overwhelm the antiapoptotic members, the mitochondrial-mediated pathway of apoptosis triggers and the cell dies. BAX (BCL-2-associated X protein) and BAK (BCL-2 antagonist/killer 1) are core regulators of the mitochondrial pathway of apoptosis. Once activated, BAX and BAK proteins homo-oligomerize and directly cause MOMP (by making pores in mitochondrial outer membranes) which results in efflux of mitochondrial intermembrane space proteins, such as cytochrome c, into the cytosol. Cytochrome c in turn seeds the multimolecular apoptosome complex assembly and activation of caspase-9.
The ability of cancer cells to evade apoptosis is one of the hallmarks of cancer [15]. Expression of apoptosis-related genes may affect cancer outcome and prognosis. The aim of this study was evaluation of the expression profile of some apoptosis-related genes in the peripheral blood mononuclear cells of ALL patients.
Sample collection from patients with leukemia
Peripheral blood samples were obtained during routine clinical assessment of 20 patients with ALL between 2017 and 2018. All the patients were referred to the Department of Hematology and Oncology, Shariati's Hospital of Tehran University of Medical Sciences, Tehran, Iran, for verifying the diagnosis. Diagnosis was based on standard clinical, morphological and immunophenotyping criteria. The general information of the patients was summarized in Table 1. Additionally, peripheral blood samples were obtained from 20 healthy donors without a history of any malignancies as controls. Informed consent was obtained from all subjects who participated in this study.
Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Ficoll-Paque solution (Biosera, UK). Briefly, blood samples were diluted 1:1 with phosphate-buffered saline (PBS; Ca and Mg-free; Biosera, UK) and were subsequently layered onto the Ficoll-Paque in a centrifuge tube and centrifuged at 400 g for 30 min at 20°C. The PBMC layer was harvested and washed twice with PBS. Isolated PBMCs immediately subjected for the extraction of total RNA.
RNA extraction
For RNA extraction, PBMCs were lysed by adding 1 ml of Trizol reagent (Qiagen) and vortexing for 20 sec. The homogenized samples were incubated for 5 minutes at room temperature (RT) to permit the complete dissociation of nucleoprotein complexes. Then, 200 ul of chloroform was added to the samples, vortexed vigorously for 15 seconds, and incubated at RT for 2–3 minutes. Samples were then centrifuged at 12,000 g at 4°C for 15 min. Following centrifugation, the upper aqueous phase was carefully transferred into fresh RNase-free microtube. RNA was precipitated from the aqueous phase by mixing the aqueous phase with an equal volume of isopropanol and incubation at RT for 10 minutes followed by centrifugation at 14,000 g at 4°C for 10 min. The RNA pellet was washed by brief vortexing, mixing with 1 ml of 75% ethanol, and recentrifugated at 8,500 g at 4°C for 10 min. After removing leftover ethanol, the precipitated RNA was air-dried for 5-10 minutes and dissolved in 40 ul diethylpyrocarbonate (DEPC)-treated H2O. The quantity of extracted RNA was determined by measuring the absorbance at 260 nm and the RNA quality was assessed by determination of the ratio for absorbance at 260 nm vs. absorbance at 280 nm (A260 nm/A280 nm) using the Thermo Scientific™ NanoDrop™ One Spectrophotometer (Themo Fisher Scientific, USA). The RNA purity was also assessed by agarose gel electrophoresis. RNA samples had an A260/A280 ratio > 1.6. Contaminating DNA was removed from each RNA extract by means of DNase (Themo Fisher Scientific, USA) treatment, according to the manufacturer’s recommendations. RNA samples were stored at -80 °C.
cDNA synthesis
cDNA synthesis was performed on 1 µg of RNA in a 10 µl sample volume using High-Capacity cDNA reverse transcription kit (Applied Biosystems, USA) according to the manufacturer's instructions. Ten µl of a master mix containing 2 µl of 10X reverse transcriptase buffer, 0.8 µl of 25X dNTP mix (100 mM), 2 µl of 10 X reverse transcriptase random primers, 1 µl of MultiScribe™ reverse transcriptase, and 4.2 µl of nuclease-free H2O was added to the RNA sample. The final reaction mixture was incubated at 25°C for 10 min and 37°C for 120 min followed by 5 min at 85°C to inactivate the enzyme and then cooled to 4°C. cDNA was stored at −20°C.
Gene expression analysis by real-time reverse transcriptase polymerase chain reaction (RT-PCR)
Quantitative analysis of mRNA expression of apoptosis related genes was performed by real-time RT-PCR using a StepOnePlusTM Real-Time PCR System (Applied Biosystems, USA). Each reaction contained 7.5 µl of RealQ Plus 2x Master Mix Green High ROX™ (Ampliqon, Denmark), 1 µl of mixed forward and reverse primers (5 µM), 1 µl of cDNA, and 5.5 µl of DNase-free H2O. The sequences of primers were as follows: Forward, 5′-TGGTTCATCCAGTCGCTTTG-3′ and reverse, 5′-CATTCTGTTGCCACCTTTCG-3′ for capsae-3 gene; 5′-AGGAGCTGCTCTTCCGAATT-3′ and reverse, 5′-CCCTGCCTGGTGTCTGAAGT-3′ for caspase-8 gene; 5′-ACTAACAGGCAAGCAGCAAAGT-3 and reverse, 5′-ACATCACCAAATCCTCCAGAAC-3′ for caspase-9 gene; forward, 5′-GGTCCTGCTCAACTCTACCC-3′ and reverse, 5′-CCTGAGAGTCCAACTGCAAA-3′ for BAK gene; forward, 5′-GGGGACGAACTGGACAGTAA-3′ and reverse, 5′-CAGTTGAAGTTGCCGTCAGA-3′ for BAX gene; forward, 5′-AGGAATTCCCAGTAAGTGCG-3′ and reverse, 5′-GCCTCACTAAACCATCCAA-3′ for 18S rRNA gene. All reactions were prepared in duplicate. The PCR thermal program was 95°C for 15 min followed by 40 cycles of 95°C for 15 sec, 58°C for 30 sec, and 72°C for 30 sec. Then, a melting curve analysis was performed to confirm that a single PCR amplicon has been detected with the SYBR green. The relative expression levels of the target genes were normalized to that of the endogenous control gene, 18S rRNA. The data were analyzed using the comparative threshold cycle (2-ΔΔCT) method.
Statistical analysis
Data were expressed as mean ± SEM. Unpaired student’s t-test was used to analyze statistical differences between the means of gene expressions in ALL patients and healthy individuals (controls). P value of <0.05 was considered statistically significant. All analyses were performed using Graphpad Prism 8 software (GraphPad Software, Inc.).
Results
Two important characteristics (age, and sex distribution) of ALL patients were nearly similar to those of healthy subjects (Table 1). Of 20 patients, 16 (80%) patients had B-cell precursor ALL, and 4 (20%) patients had T-cell ALL.
Melting curve experiments were carried out after real-time RT-PCRs to exclude non-specific reactions (false-positives). The amplified products were treated with the following thermal program: heating at 95°C for 15 s, cooling to 60°C, holding at 60°C for 1 min, and then slowly heating again to 95°C in increments of 0.3°C. Melt curves were generated by continuous fluorescence acquisition in the temperature elevating period from 60 to 95°C. Melt curve analysis showed a single sharp park indicating formation of a single product.
For gene expression analysis, threshold cycle (CT) values were measured during the exponential phase of the PCR reaction. ΔCT was then calculated by the difference between the CT values of the Caspase-3, 8, and -9, BAX, BAK and the CT value of the reference gene, 18S rRNA. Finally, relative expressions were calculated by 2-ΔΔCT equation.
Analysis the mRNA levels of apoptosis related genes (Caspase-3, 8, 9, BAX, and BAK) in PBMCs showed that expression of some apoptosis related genes were significantly altered in PBMCs of ALL patients (Figure 1). The expression of the initiator caspase-8 and -9 genes were significantly upregulated while the expression of the proapoptotic BAX and the executioner caspase-3 genes were significantly downregulated in PBMCs of ALL patients as compared with PBMCs of healthy individuals (p< 0.0001 for each). The expression of the proapoptotic BAK gene was also upregulated in PBMCs of ALL patients as compared with PBMCs of healthy individuals (individuals (p< 0.01).
The mRNA levels of caspase-8, -9 and BAK in the PBMCs of leukemia patients were 2.43, 5.56, and 1.52 folds higher than that in PBMCs of healthy subjects, respectively. In contrast, the mRNA levels of BAX and caspase-3 in the PBMCs of leukemia patients were 2.3 and 1.72 folds lower than that in the PBMCs of healthy subjects.
Caspase-8 is the initiator caspase in the extrinsic pathway of apoptosis. Caspase-8 is essential for induction of the extrinsic (the death receptor) apoptotic pathway. Activated caspase-8 initiates the proteolytic cleavage of caspase-3, -7 or BID. In certain cell types, known as Fas type I cells, the activation of caspase-8 is sufficient to induce the activation of downstream caspases such as caspase-3 [16]. In Fas type II cells, caspase-8 cleaves BID, leading to activation of the downstream components of the mitochondrial apoptotic pathway [17]. In some lymphoid cell lines, caspase-8 is vigorously activated and activates the downstream procaspases such as proscaspase-3 [18]. Caspase-9 is the initiator caspase in the intrinsic pathway of apoptosis and directly activates the effector caspase-3 and -7. Caspase-3 is at the crossroad of both the extrinsic and intrinsic pathways of apoptosis. Activated caspase-3 cleaves multiple cellular substrates involved in cellular integrity and metabolism which contribute to the cell death. Upregulation in caspase-3 gene expression ensures the presences of sufficient amounts of the proenzyme to eliminate apoptotic cells.
Low levels of caspases can be contributable to the decrease in apoptosis in cancer cells. Teitz and colleagues reported that the caspase-8 gene is frequently inactivated in childhood neuroblastomas [19]. Winter and colleagues found no significant differences in the mRNA levels of caspase-1, -3, and -9 in prostate tumors relative to the normal gland. But, protein expression analysis revealed that more than 80% of prostate tumors exhibit total lack of caspase-1 and reduced caspase-3 protein expression [20]. Devarajan and colleagues showed that about 75% of tumor and normal peritumoral tissue samples obtained from breast cancer patients lack caspase-3 mRNA and protein expression. Caspase-3 mRNA levels were also undetectable in commercially available total RNA samples isolated from breast and cervical tumors and were substantially decreased in RNA samples isolated from ovarian tumors. Protein expression levels of caspase-8 and -9 were normal in all of the caspase-3-negative breast tumor samples [21]. Fong and colleagues observed lower expression of caspase-8 and -10 mRNA and/or protein in choriocarcinoma and hydatidiform mole when compared with normal placentas [22]. Shen and colleagues found that the expression of caspase-9 mRNA is downregulated in stage II colorectal cancer tissues and correlates with poor clinical outcome [23]. The expression of caspases-2, 3, and 6 genes was heterogeneous in bone marrow samples obtained from patients with de novo acute myeloid leukemia (AML) (n=15). mRNA levels of caspase-3 were higher in most AML bone marrow samples compared to bone marrow samples obtained from healthy donors (n=2). Conversely, mRNA levels of the short isoform of caspase-2, that encodes an anti-apoptotic protein, were lower in most AML bone marrow samples than that in normal bone marrow cells [24]. In our study, mRNA levels of caspase-8, -9 were higher in the PBMCs of adult ALL patients compared to PBMCs of healthy individuals. In contrast, the mRNA levels of caspase-3 in the PBMCs of leukemia patients were lower than that in the PBMCs of healthy subjects. It is possible that cancer cells undergo apoptosis, recover, and proliferate if caspase-3 activation is inhibited.
BAX and BAK have crucial roles in the mitochondrial pathway of apoptosis. Dysregulation of BAX has been proposed to be involved in the survival of B cell chronic lymphocytic leukemia [25, 26]. Numerous other studies showed the role of BAX in leukemia progression as well as cancer cell resistance to apoptosis induced by chemotherapy or radiotherapy [27, 28]. Genomic loss of BAX has been observed in human colon cancers [29, 30]. Loss of BAX expression was also found in some other cancer types. Cartron and colleagues examined the expression of BAX and BAK in 50 human glioblastoma multiforme tumors and observed that all the tumors expressed BAK, while three did not express BAX. The levels of antiapoptotic proteins BCL-2 and BCL-XL or the BH3-only proapoptotic BID were approximately similar between BAX-expressing and BAX deficient tumor cells, while the levels of BAK were upregulated in BAX-deficient tumor cells [31]. Protein levels of BCL-2, MCL-1. BCL-XL, and BAX in pretreatment AML (n=123) and ALL (n=36) samples varied widely between different samples [32]. Low BCL-2 and high BAX protein levels were found in AML cells undergoing spontaneous apoptosis in vitro, whereas nonapoptotic AML cells showed high BCL2 and low BAX levels[33]. In another study, no clear correlation between both BCL-2 and BAX protein levels and the extent of spontaneous apoptosis were observed in adult AML cells in vitro [34]. Assessing the protein levels of BCL-2, BAX, MCL-1, and BCL-X in lymphoblasts from 47 childhood ALL showed that BCL-2, BAX, and MCL-1 were expressed in all samples. BCL-2 levels and BCL-2/BAX and MCL-1/BAX ratios were significantly higher in B lineage as compared with T-lineage disease. High BAX protein levels were significantly correlated with increased risk of relapse in childhood ALL [35]. Higher expression of BAX gene was found in 55 childhood primary B cell ALL patients when compared with their normal counterparts, B cell precursors [36]. BAX, BCL-2, and BCL-xL mRNA levels were significantly higher in 46 childhood ALL patients at their initial diagnosis than in 50 ALL patients in remission, while BAD mRNA levels were found to be lower in ALL patients than in remission patients [37]. In our study, mRNA levels of BAX in the PBMCs of adult ALL patients were lower than that in the normal PBMCs, while mRNA levels of BAK in the PBMCs of adult ALL patients were higher than that in PBMCs of healthy subjects. These results indicate downregulation of BAX gene expression and compensatory gene expression of the alternative mitochondrial pore-forming protein, BAK, in the PBMCs of ALL patients.
Although some differences in the levels of expression of apoptosis related genes in leukemia patients reported in various studies might be due to the fact that nonquantitative methods were used in some of that studies, overall results suggest that the expression of some apoptosis related genes is heterogeneous among leukemia patients. Nevertheless, it is now obvious that the gene expression and function of certain molecules that regulate or induce apoptosis are important factors in cancer cell survival and resistance to chemotherapeutic drugs. Induction of apoptosis is a complicated process and is tightly regulated by the activity of several genes. Furthermore, synthesis of new apoptosis related proteins is not required for induction of apoptosis. However, accumulation of zymogen forms of multiple caspases and BCL-2 family proteins through direct transcriptional mechanism occurs in cancer cells. Our results indicate that induction of apoptosis initiate in adult ALL cells, but cancer cells can block it. These findings suggest that choosing appropriate class of chemotherapy drugs based on alterations in apoptosis related genes such as BAX and caspase-3 genes may be beneficial to improve therapeutic efficacy of chemotherapy in leukemia patients.
Defects along apoptotic pathways can play a crucial role in carcinogenesis. Defects in apoptosis also play a key role in cancer cell resistance to some anticancer cytotoxic drugs. Regulation of apoptosis related genes transcription as well as compensatory expression of alternative apoptosis related genes affect the survival of cancer cells as well as the sensitivity of cancer cells to multiple apoptotic stimuli exploited by anticancer therapy. Reduced expression of apoptosis related genes such as the executioner caspase-3 and the proapoptotic effector BAX can be contributed to evasion of apoptosis. Further studies on the effects of these altered gene expressions in apoptosis-mediated cancer cell death may allow the identification of novel therapeutic targets or appropriate combinational therapy for ALL patients.
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.
