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The statistical analyses have now been revised properly.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
There is a bit of confusion related to statistical analyses as evidenced by a reviewer. Please, specify clearly these aspects.
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No comment.
No comment.
1) In response to the reviewer's question "Based on the authors' explanation that the data is expressed as proportions / percentages, the control cultures will always = 100% and therefore will not accurately reflect standard deviation", the authors responded "The proportional data of IP-HPLC need to do multiple repeats to compare different assay series. In data sheet, the standard deviations of control assays were not listed to get proper graph plot, but calculated simultaneously".
-The reviewer recommends that the authors include the standard deviations of control assays. All underlying data should be provided.
2) In response to the reviewer's question "The chi-square test is not an appropriate statistical test. Chi-square test is used when evaluating the relationship between two categorical variables (i.e., race, sex, age group, educational level). The study does not evaluate categorical variables", the authors responded "In this study, every protein assay is independent with each other, and more, the repeated rate of each protein assay is relatively low, only 39.9% of protein assays is adequate for n-value. However, the protein assays showed standard error, √(σ^2/n). Therefore, it is agreed that the great variability of protein expression data is inadequate for statistical analysis of Chi-square test and probability (p-value) t-test, but still can be simply evaluated by standard error of the mean in this study. The data of protein expression levels were reanalyzed by standard error concept."
-The authors revised the manuscript text from "Results were analyzed using the Chi-squared test" to "Results were analyzed by measuring standard error (s=±√(σ^2/n))", but it does not appear that the data were reanalyzed since none of the study outcomes changed. Furthermore, it is unclear to the reviewer how results can be analyzed by standard error.
Authors have improved the text and the overall quality of the article.
Authors have included in situ proliferation data, explain rational to select 48 h of treatment for most of the studies.
Authors have explained in the text regarding RAW264.7 response to pamidronate for proliferation and apoptotic instigation is based on expression of p53 and Fas associated genes.
This revised manuscript is very much improved, and authors have added some contents according to reviewers' comments. I have no further comment.
The authors need to perform revisions as suggested by reviewers.
The manuscript entitled “Global protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC” by Lee et al analyzed protein expression changes with IP-HPLC technology in RAW 264.7 cells treated with pamidronate. The authors showed that pamidronate treatment induced RARP-1 and FAS-mediated apoptosis signaling, while suppressed inflammation, cellular differentiation, survival, angiogenesis, and osteoclast genesis signaling. The manuscript was properly written. Figure quality is ok. But the manuscript still needs a minor revision before it can be accepted for publication.
Though the authors have utilized the technology for several times, how can the authors make sure that the 218 antibodies are all specific? The authors should at least explain in the manuscript.
Second, the authors only used 218 antigens to do IP, so this is not a true global protein expression change, the authors should change the manuscript title to a much proper way.
Experimental design is ok.
I noticed that the authors had published another paper on Scientific Reports in 2018, investigating protein expression changes of RAW264.7 treated with dialyzed coffee extract. So, what is the specificity of RAW264.7 cells treated with pamidronate compared with dialyzed coffee extract? It is better the authors compare these two treatments in this manuscript.
The Introduction section does not provide accurate background information. Moreover, the authors' statements are not supported by adequate citations / references. The reviewer provides examples of statements made by the authors which need to be revised:
1) The following statement is inaccurate: “Bisphosphonates inhibit the digestion of bone by causing osteoclasts to undergo apoptosis and thereby inhibit bone loss”
-BPs antiresorptive actions are not limited to induction of osteoclast apoptosis. BPs impair osteoclasts’ ability to form a ruffled border, to adhere to the bone surface, and to synthesize protons necessary for bone resorption. Furthermore, BPs suppress osteoclast activity by decreasing osteoclast progenitor development and recruitment.
2) The following statement is inaccurate: “In osteoporosis and Paget's disease, the most popular first-line bisphosphonates are alendronate and risedronate, but when they are ineffective or digestive tract problems develop, intravenous pamidronate may be used”
-IV zoledronic acid is the first-choice treatment for Paget disease because of its efficacy and ease of administration. The choice of zoledronic acid as the initial agent for most patients with active Paget disease is consistent with both the 2014 clinical practice guidelines of the Endocrine Society and the 2019 Paget's Association guidelines.
3) The following statement is inaccurate: “They also change bone ultrastructures, e.g., they obliterate haversian canaliculi and deposit irregular and thick reversal lines”
-BPs have been shown to obliterate haversian canals, not haversian canaliculi.
4) The authors make the statement, “In addition, bisphosphonate-related osteonecrosis of the jaw (BRONJ) may develop in patients who have used bisphosphonates long term (Marx et al. 2005; Ruggiero et al. 2004)”
-The authors need to address the incidence of BRONJ in patients being administered oral bisphosphonates versus IV bisphosphonates. BP route of administration critically influences the incidence of BRONJ.
-The authors should cite the specific research studies which have shown that long-term use of bisphosphonates is a risk factor for BRONJ.
5) The following statement is inaccurate: “BRONJ is a microbial infection that occurs after dental extraction or surgery and constitutes a severe complication of suppurative and necrotizing osteomyelitis in jaws (Chirappapha et al. 2017; Choi et al. 2017; Park et al. 2009)”
-The pathophysiology of BRONJ is currently unclear. BRONJ has been attributed to infection, suppressed bone turnover, vascularity, genetic predisposition, etc.
-BRONJ is not always caused by dentoalveolar surgery. BRONJ can occur spontaneously.
-The current evidence does not support the view that BRONJ is a complication of suppurative and necrotizing osteomyelitis in jaws.
6) The authors make the statement, “Pamidronate (pamidronate disodium or pamidronate disodium pentahydrate) is a nitrogen-containing bisphosphonate and used to prevent bone loss due to steroid use or to treat certain cancers with propensities for bone, such as multiple myeloma”
-The reviewer is unaware that pamidronate is used to treat steroid-induced bone loss. Please cite / reference the society guidelines demonstrating this clinical use.
7) The following statement is inaccurate: “Due to its ability to sequester calcium in bone, pamidronate is frequently used to treat high calcium levels”
-BPs do not sequester calcium in bone. BPs inhibit calcium release from bone by impairing osteoclast-mediated bone resorption.
8) The authors make the statement, “Furthermore, multiple trials have shown that IP-HPLC can be used to rapidly determine multiple protein levels accurately (< ±5% standard deviation) and reproducibly.”
-The authors need to provide citations / references supporting this statement.
9) The authors make the statement, “When pamidronate is injected into blood vessels, it is immediately neutralized by cationic molecules like albumin and calcium and engulfed by macrophages, which suggests its various pharmacologic effects may be associated with the cellular functions of pamidronate-laden macrophages.”
-The reviewer is not aware that when pamidronate is injected into blood vessels, it is neutralized by albumin and calcium and engulfed by macrophages. Please provide citations / references supporting this statement.
The reviewer does not feel that the statistical analyses are acceptable, which is explained below.
1) Based on the authors' explanation that the data is expressed as proportions / percentages, the control cultures will always = 100% and therefore will not accurately reflect standard deviation.
2) The authors explain that analyses were repeated two to six times until standard deviations were ≤±5%. Experiments should have been repeated at least three times in order to have n-values = 3 per group.
3) The chi-square test is not an appropriate statistical test. Chi-square test is used when evaluating the relationship between two categorical variables (i.e., race, sex, age group, educational level). The study does not evaluate categorical variables.
The data should be re-analyzed applying appropriate statistical analyses methods.
The reviewer cannot evaluate the validity of the findings since the data were not analyzed appropriately.
The balance between the process of osteoblasts and osteoclasts is critical for maintaining the integrity of bone mass. Earlier studies demonstrate bisphosphonates potential to inhibit osteoclasts and suppressing proinflammatory cytokines. In this manuscript, author have investigated the effect of Pamidronate on the expression of multiple genes involved in range of cellular pathways. This manuscript is well conceptualized and written, data extracted through IP-HPLC method further signifies the quantitative assessment of biological fluids to obtain multiple readouts.
This is very compressive study, still authors need to conduct some additional experiments to improve the outcomes of this study. Some examples where the authors could provide more insight includes-
1. Figure 1- It is better to provide DIC images without colour background to observe the cell morphology, also include scale bar.
2. Figure 1- Authors should provide the complete dose-time response analysis to validate 6.5 μM concentration was best dose for this study. Given that most of the protein induction was observed at 48 h of treatment, a dose-response analysis needs to be included.
3. For cell proliferation assay authors should provide some additional biochemical based studies.
Figure 2 & 5- results shown here an increase in expression of tumor suppressor p53 and pro-apoptotic protein suggesting more cell death, however till 24 h more proliferation observed, please explain these opposite observation.
This study will highlight the potential of Pamidronate in regulating the expression of multiple pathways through tuning the expression of respective proteins. Still authors need to add some more data that will further enhance the outcomes of this manuscript.
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