Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes  likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/β-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit β-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to β-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of β-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of β-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect β-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development. Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (AIDS). Without intervention, HIV-1-infected individuals become increasingly susceptible to opportunistic infections due to progressive decline in their CD4+ T-cell population, which ultimately results in death. Antiretroviral therapy (ART), however, is highly effective at managing HIV-1 viral replication, preventing subsequent infection, and preserving immune function. Despite the effectiveness of ART, HIV-1-infected cells persist and can be found in the periphery and in various organ systems throughout the body . In partin vivo  in the presence or absence of Morphine sulfate pentahydrate . Alternatively, where indicated, 24 hours post-transfection, cells were treated with the β-catenin activator -6-Bromoindirubin-3′-oxime  in the presence or absence of morphine (500nM).24 hours post-transfection or post-treatment, where indicated, cells were lysed with passive lysis buffer (Promega). Cell lysates were incubated in equal volumes with the DualGlo luciferase reagent (Promega) and luciferase was measured in a luminometer. Data shown represent the average of technical triplicates.™ Protease inhibitor Cocktail (ThermoFisher) and incubated under agitation at 4°C. Lysates were briefly homogenized by syringe and debris were cleared by centrifugation. Cell lysates were incubated in equal volume Bolt™ Sample Reducing Agent (Invitrogen) and Bolt™ LDS sample buffer (Invitrogen) for 10min at 98°C. Cell lysates were separated on Bolt™ 4–12% Bis-Tris plus gels (ThermoFisher) at 200 volts. Transfer of proteins to 0.2um pore size PVDF membranes was performed using Mini Blot Module (Invitrogen) transfer cassettes and Bolt™ transfer buffer made with 10% methanol and Bolt™ antioxidant (Invitrogen). Proteins were transferred at 20 volts for 1 hour. Membranes were blocked in 5% milk in PBS with 0.1% Nonidet™ P-40 (NP-40) detergent at 25°C for 1 hour, stained with primary antibodies at 4°C overnight, washed, and stained with secondary antibodies at 25°C for 1 hour. The α-total β-catenin antibody  and the α-active β-catenin antibody  were used at a 1:1000 dilution. The antibody against total β-catenin recognizes all forms of the protein, however, the antibody against active β-catenin recognizes unphosphorylated Ser32 and Ser37. The α-GAPDH antibody  was used at a 1:3000 dilution. Membrane chemiluminescent signals were imaged with BioRad imager and Quantity One software. Images were analyzed, processed and quantitated using ImageJ software. The mean gray scale values for total and active β-catenin were normalized to GAPDH.24 hours post-treatment, cells were lysed in RIPA buffer (ThermoFisher) with HaltFor Tat Western blotting a cocktail of antibodies was used. Membranes were blocked in 5% milk + PBS-T (PBS + 0.05% Tween-20) and incubated with a cocktail of primary antibodies at 4°C overnight: α-Tat , α-Tat  from Dr. Dag E. Helland ), and α-instructions. RNA concentration was quantified by Nanodrop (ThermoFisher).48 hours post-treatment, RNA was harvested using TRIzol (Invitrogen) reagent according to manufacturer’s ® green mix, 1.5μl of primer  using 1 μg of input RNA. The cDNA samples were diluted 1:20 and 1μl of sample template was added to a reaction mix containing 2.5μl of SYBR95°C—10 min95°C—15 sec x39A—30 secT72°C—30 sect test. Where indicated, data were statistically analyzed in GraphPad PRISM using two-way ANOVA followed by Tukey’s multiple comparisons test. P-values were calculated as significant as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.Data from biological or technical triplicates in experiments were statistically analyzed in GraphPad PRISM using two-tailed unpaired Student’s 101WT) expression construct and a β-catenin responsive luciferase reporter construct in the presence of LiCl to stimulate β-catenin activity ; morphine concentrations were selected based on literature describing optimal stimulation of cells with minimal toxicity Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: No**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript by Chen and colleagues is reporting the effects of morphine on the suppression of b-catenin in the presence of HIV Tat. This is a relevant manuscript, with important findings. Yet, the manuscript presentation has several problems that need to be addressed, including focus and flow. They have been itemized below, not in order of relevance but as they appeared in the text, followed by a general summary. We believe that the findings are important, but major revision needs to be performed.1. The abstract needs to be revised for missing verbs and for improving clarity of goals, methods and results. For instance, the study is performed in astrocytes, cell lines and primary cultures. However, the relevance of studying the effects of Tat on astrocytes is not clear, in the abstract. The results should be better outlined. It is right now too brief, and short on explaining the actual findings. The importance of studying the effects on b-catenin is also not clear in the abstract. The study of miRNA or targets is not stated, or why.2) The Introduction has a lot on HAND and Tat (missing citations and important literature), but it is short on offering background about what was the actual study, which is a mechanism, not the disorder. The model, the outcomes, and their relevance all should be stated upfront.3) miRNA in the Introduction is disconnected (line 71). Terrible flow.4) Literature on the effects of Tat on RNA PolII and on dopamine pathway is outdated.5) Case for studying astrocytes, rather than other "physiologically relevant" cells, in the context of HIV, and in particular, neurological disorders, is not sufficiently strong.6) The explanations about b-catenin (line 94) may have to be focused towards astrocytes.7) Citation on numbers of opioids and HIV, and on inflammatory molecules (which by the way are not the ones investigated in the study) are needed (line 105 and 110).8) The significance of Tat mutants is not stated in Introduction.9) Results chapter start with miRNA, but I don't see results on miRNAs. Unfocused.10) Overall, It is not shown whether the transfection with low, moderate or high plasmid really corresponds to low, moderate and high protein expression, upon transduction. This is desirable, to demonstrate the efficiency of the transfection and the protein levels (which are rather more important).11) The three different models  are not well separated or justified, causing confusion and problems of flow.12) It is not clear why in primary astrocytes there are only two doses with a 2log difference.13) Tat only controls should be included in all graphs. It is very hard to appreciate the effects of interaction if not ALL controls are included in the graphs.14) Reference for serum concentration of morphine found in subjects who overdosed on heroin is not provided (line 144).15) IN line 180, all of a sudden there are donors. Human, mice, monkeys, fetal, purity of cultures, and mainly why? Only two, and with opposite results? This doesn't seem to add anything. There is zero power in this experiment, even with a paired analysis. There are two choices here. Drop the donor experiments and make a better case for astrocytes in general, introduce and discuss better the effects on b-catenin, or increase the number of donors, with data on sex, age, pre-exposure, HIV status, everything that is missing here, including statistics, to make sense. Another strong alternative is to show that the effects are reverted with interventions that restore b-catenin levels.16) Line 190, different mutants give different results. Nowhere we see why is this relevant, whether they differ in structural properties, and implications. All this should be in the discussion.17) Line 205. All of a sudden we have NLRP-1 and other things that were not introduced at all. Again 3 donors. AND, there no discussion about individual variability at all.18) IN the discussion also, comments on CCL5, iNOS, genes that were not measured.19) Paired Student's t test is not correct. Student's t test is unpaired.Very hard to follow. Rationale has to be better developed. Focus has to be largely improved. Further experiments to increase the power of readouts, with additional donors, including acknowledgment of factors that may affect individual responses in this pathway, as well as interventional studies, are recommended.Reviewer #2: The manuscript entitled "Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatoryfactors in cultured astrocytes "Since HAND increases in drug abusers, this study was directed to elucidate the role morphine on the effect of Tat HIV-1 and Tat mutants associated with neurocognitive impairment mediated by the β-catenin suppression and inflammatory processes.Although the data may be interesting, the manuscript has serious errors that must be resolved before it is acceptable for publication.2.- Some paragraphs do not have a bibliographic support and there are multiple typographical, orthographic and verb conjugation errors.3.- Throughout the writing different styles are noticed, with some secctions well written and others are not.4.- Major changes have to be made in the methods section.4.1. The subtitles in general are inadequate4.2. Given that the primary cultures of astrocytes are of fetal origin, it is important to inform about the time during the fetal development in which the cells were taken, how many donors are included in the study and if the experiments were performed in same donors?, please mention culture conditions for primary cultures and the cell line (was the same medium was used for both’), mention the passage of the culture in which the studies were made.4.3. The authors must demonstrate that the primary cultures are really astrocytes and not neural stem cells, which are also positive to GFAP. Immunohistochemistry is recommended for neural stem markers.4.3. Furthermore, depending on the fetal age in which the cells are obtained, they have different potential for differentiation. The fact that they are proliferative indicates that they are stem cells since the differentiated astrocytes do not proliferate, unless they are cancerous.4.4. The luciferase assay is not in the methodshttp://antibodyregistry.org/).4.5. Please include the data of all suppliers and the information on the antibody ID used , and others as xmRBNA/GAPDH. The authors are asked to clarify which method of expression analysis is used, and they are recommended to use 2-ΔΔCT.4.9. Statistic section is unclear, why did the authors used Paired Student’s t test. And why after ANOVA a pairwise analysis was performed and not a multiple comparison test.Results:1. The images are of very poor quality.2. In some results they refer to the donor number and in others they do not.3. The luciferase assay are confusing, perhaps because it is not described in the methods.4. Not all figure legends have the statistical test used.5. In cases where ANOVA was used, which post-test was performed.6. It is inappropriate to use the Student's t to compare more than two groups 7. The number of experiments is not clear.The discussion does not have a clear focus and repeats a large part of the results, which makes it tedious and very long.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  13 Jan 2020A fully formatted and color coded version of this response to your comments exists at the end of the combined PDF (at least in our view). This version may be easier to read than the comments copied below. We wish to thank the reviewers for their time and comments. The critiques provided were all very helpful and we have addressed them individually below. It should be noted that we wanted to do much more. In setting out to do these revisions we originally planned to expand our use of primary astrocytes so that all figures would include data from three to five different donors. This would have increased our statistical power, allowed comparison between immortalized astrocytes (U87MG) and primary cells, and have provided additional controls for each experiment. However, recent changes in the NIH guidelines on the use of fetal material has made it impossible to obtain new cultures. We had some material on hand and were able to add data points to some of the existing analyses, but not as much as we would have liked. The text of the manuscript has been updated extensively. We believe this new text provides a much clearer explanation of the rationale for the experiments and a cleaner discussion of the findings.Specific points are addressed below:Reviewer #1: The manuscript by Chen and colleagues is reporting the effects of morphine on the suppression of b-catenin in the presence of HIV Tat. This is a relevant manuscript, with important findings. Yet, the manuscript presentation has several problems that need to be addressed, including focus and flow. They have been itemized below, not in order of relevance but as they appeared in the text, followed by a general summary. We believe that the findings are important, but major revision needs to be performed.1. The abstract needs to be revised for missing verbs and for improving clarity of goals, methods and results. For instance, the study is performed in astrocytes, cell lines and primary cultures. However, the relevance of studying the effects of Tat on astrocytes is not clear, in the abstract. The results should be better outlined. It is right now too brief, and short on explaining the actual findings. The importance of studying the effects on b-catenin is also not clear in the abstract. The study of miRNA or targets is not stated, or why.We agree with the reviewer that the abstract would benefit from revision. We have rewritten the abstract as follows:“Despite antiretroviral therapy (ART), human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system (CNS) that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the CNS. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes  likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/β-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit β-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to β-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of β-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of β-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect β-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development.”2) The Introduction has a lot on HAND and Tat (missing citations and important literature), but it is short on offering background about what was the actual study, which is a mechanism, not the disorder. The model, the outcomes, and their relevance all should be stated upfront. We agree with the reviewer that the Introduction was misfocused. We have now re-written the entire Introduction to hopefully improve the clarity of the study.3) miRNA in the Introduction is disconnected (line 71). Terrible flow.We agree with the reviewer that introducing the contribution of Tat to the dysregulation of cellular miRNA pathways is out of context at this point in the introduction. We have removed this statement.4) Literature on the effects of Tat on RNA PolII and on dopamine pathway is outdated.We thank the reviewer for bringing this to our attention. We have now included two additional citations to recent reviews on Tat function. We have also removed the statement on Tat effects on the dopamine pathway as it is not relevant to the current study.5) Case for studying astrocytes, rather than other "physiologically relevant" cells, in the context of HIV, and in particular, neurological disorders, is not sufficiently strong.We thank the reviewer for bringing this to our attention. We have largely rewritten the Introduction to provide a stronger case for studying HIV-1 and neurological disorders in astrocytes as opposed to other cells of the CNS. Some newly added statements include:“CNS-resident, long-lived cell types such as monocytes, macrophages, and astrocytes have all been implicated in HIV-1 infection and neuroinflammation, though the establishment of latent and prolonged infection in these cell types within the CNS remains controversial [15]. Astrocytes, specifically, can be permissive to HIV-1 infection, but these cells are restrictive to productive replication, and there is limited data demonstrating infection in vivo [16-18]. Within the CNS, astrocytes maintain the homeostatic balance and play key roles in mediating cerebral blood flow by maintaining the BBB, providing neuronal support through neurotransmitter processing, regulating nutrients and growth factors, and modulating inflammation [19-22] . HIV-1 does not infect neurons, implying that development of HAND might be mediated indirectly, for example through astrocyte dysregulation and inflammation. A measurable percentage of astrocytes in the CNS can harbor HIV-1 integrated DNA, making astrocytes a likely contributor in the development of HAND [23-26].”6) The explanations about b-catenin (line 94) may have to be focused towards astrocytes. We agree with the reviewer and have now provided information regarding the role of B-catenin in astrocytes:“The Wnt/ β-catenin pathway has been implicated in suppressing replication of HIV-1 in multiple cell types, including astrocytes, and conversely, downregulation of Wnt/β-catenin signaling by inflammatory cytokines promotes HIV-1 replication [59-63]. Interestingly, HIV-1 Tat has also been shown to inhibit Wnt/β-catenin signaling in astrocytes, therefore alleviating, in part, the negative regulation of Tat-mediated transactivation of the LTR by β-catenin .” … “Compared to other cell types and despite robust levels of signaling, less is known about the role of Wnt/ β-catenin in astrocyte function . Approximately 150 β-catenin-regulated genes are estimated in primary astrocytes are comprising at least five broad categories including inflammation, transport, exocytosis, apoptosis, and trafficking [61]. Of these, β-catenin responsive genes that are neuroinflammatory in nature include BDNF (Brain-Derived Neurotrophic Factor), a member of the BDNF/TrkB (Tropomyosin receptor kinase B) signaling axis [66-68].”7) Citation on numbers of opioids and HIV, and on inflammatory molecules (which by the way are not the ones investigated in the study) are needed (line 105 and 110).We agree with the reviewer that a citation on the numbers of opioids and HIV is needed. We have revised the statement for accuracy as follows:“In addition to neurotoxicity associated with HIV-1 infection, mounting evidence suggests exacerbation of these effects in the presence of opiates [69-72]. Illicit use and drug abuse contribute significantly to HIV-1 infections and transmission [73], therefore elucidating the interaction between the cells of the CNS, opiates, and HIV-1 Tat is necessary to understanding HAND disease progression.” We have also removed the information about inflammatory molecules as they are not related to the study.8) The significance of Tat mutants is not stated in Introduction.We agree with the reviewer that stating the significance of the Tat mutants in the Introduction will add to the clarity of the manuscript. We have now added the following statements to the Introduction: “HIV-1 is subject to genetic variation, which may alter the functionality of the encoded viral proteins. In vitro, Tat expressing cells have been demonstrated to readily secrete Tat extracellularly, a function mediated by a tryptophan at amino acid position 11 (W11), as well as residues 49-51 within the basic domain . Tat variants containing mutations at this position, such as W11F or W11Y, are deficient in both secretion from Tat-producing cells and cytoplasmic translocation in recipient cells . In the CNS, Tat can also be secreted from HIV-1 infected cells, such as macrophages and astrocytes, which results in direct neurotoxicity; therefore, Tat secretion-deficient mutants may alleviate this phenotype within patients [49-55]. Finally, mutations at a conserved cysteine at amino acid position 31, C31, which is within the cysteine-rich domain, have been shown to abrogate synaptodendritic injury in neurons and normalize levels of amyloid beta production, therefore conferring protection from Tat-mediated neurotoxicity .”9) Results chapter start with miRNA, but I don't see results on miRNAs. Unfocused.We agree with the reviewer that the Results section required additional clarification as to our interest in pursuing this study. We have modified the beginning of the Results section as follows: “We have recently determined that HIV-1 Tat-mediated suppression of specific miRNAs affects downstream signaling from the Wnt/�-catenin pathway [78]. The modulation of �-catenin activity by Tat could alleviate repression of basal HIV-1 transcription, resulting in enhanced neuroinflammation in the CNS . To confirm our previous results and to address the ability of Tat to modulate β-catenin activity in physiologically relevant cells, we utilized the U87MG astrocytic cell line and primary fetal astrocyte (PFA) donors in our study.”10) Overall, It is not shown whether the transfection with low, moderate or high plasmid really corresponds to low, moderate and high protein expression, upon transduction. This is desirable, to demonstrate the efficiency of the transfection and the protein levels (which are rather more important).We agree and have added a new supplemental figure demonstrating detection of Tat expression in the three conditions. Tat is clearly visible in the moderate and high doses, but not in the low. However, an LTR-Luciferase assay reveals strong activation (5-fold) at the lowest dose, supporting the presence of Tat protein.11) The three different models  are not well separated or justified, causing confusion and problems of flow.We agree with the reviewer that clarification of which cells were used for given experiments was needed. We have now indicated use of either the U87MG astrocytic cell line or any of three primary fetal astrocyte (PFA) donors  on each figure. We have also clarified which cells were used in each relevant section of the Results and in the Figure Legends. Finally, the source of the cells has been clarified in the Materials and Methods.As noted above, recent changes in the NIH guidelines on the use of fetal material has made it impossible to obtain the new cultures needed to expand on the data as originally presented. Using material on hand we were able to include new data in some of the figures.12) It is not clear why in primary astrocytes there are only two doses with a 2log difference.We agree with the reviewer that some clarification is needed for the details of the LiCl treatment, Morphine treatment, and Tat transfection between U87MG cells and PFAs. The LiCl concentrated used (50mM) is consistent across all experiments. In U87MG cells, we include additional concentrations of Morphine and Tat due to the ease and adaptability of using an immortalized cell line. Based on the results from the U87MG-based experiments, we optimized conditions for the PFA-based experiments, which are more challenging and of limited supply. For example, we see little to no difference in the effect of 0.35 Tat on B-catenin activity , therefore, this concentration was not included in the experiment in Figure 1B or Figure 2B. Additionally, we expected to see the largest effect on B-catenin activity in PFAs at the highest concentration of Morphine tested (500nM).We have indicated the above information in revised versions of the Figures and Figure Legends which we hope will clarify how the experiments were performed.13) Tat only controls should be included in all graphs. It is very hard to appreciate the effects of interaction if not ALL controls are included in the graphs. The figures now include Tat only controls. When using a B-catenin activator (LiCl or Bio) we have not included analyses of Tat effect on the cells in the absence of activator. In these systems we have needed to activate B-catenin in order to detect activity and see changes. Figure 4C does not include no Tat control as the figure exists to compare within the different mutants tested – in this figure Tat WT serves as the control. Additionally, we now include a Western blot demonstrating the varying amounts of Tat detectable at the amounts transfected.14) Reference for serum concentration of morphine found in subjects who overdosed on heroin is not provided (line 144).The statement describing the rationale for the amount of morphine use has been adjusted and no includes proper references.15) IN line 180, all of a sudden there are donors. Human, mice, monkeys, fetal, purity of cultures, and mainly why? Only two, and with opposite results? This doesn't seem to add anything. There is zero power in this experiment, even with a paired analysis. There are two choices here. Drop the donor experiments and make a better case for astrocytes in general, introduce and discuss better the effects on b-catenin, or increase the number of donors, with data on sex, age, pre-exposure, HIV status, everything that is missing here, including statistics, to make sense. Another strong alternative is to show that the effects are reverted with interventions that restore b-catenin levels.We agree with the reviewer that additional clarification was needed at this point in the manuscript. We believe that steps taken to clarify the difference between the U87MG cell line used and PFA donors (discussed in point #11) will help alleviate the confusion. We agree with the reviewer that two donors may be insufficient to draw significant conclusions. As noted above, the resources needed to perform these experiments  are not available due to changes in NIH funding guidelines. The error bars present in the figures are the result of biological replicates performed with the same donor astrocytes. As such, we that descriptions of Tat and morphine changes in each donor are valid. The text has been updated to provide clear explanation of where changes are common between the donors used.All donor material is HIV negative and derives from gestational age of 16-18 weeks as noted in materials and methods.16) Line 190, different mutants give different results. Nowhere we see why is this relevant, whether they differ in structural properties, and implications. All this should be in the discussion.We agree with the reviewer that additional clarification is needed for both the two different forms of Tat, Tat86 and Tat101 as well as the Tat mutants C31R, W11Y, and W11F. We have added the following clarifying statements to the appropriate section of the Results: “Tat is encoded by HIV-1 on two exons, the first of which is sufficient for the primary function of Tat to act as the viral transactivator and drive the transcriptional elongation of viral genes [83-85]. The functions of Tat encoded by the second exon contribute to viral replication and the modulation of interferon-stimulated genes in antigen presenting cells . Widely used laboratory-adapted strains of HIV-1 produce a truncated form of Tat, encoded by the first exon plus 14 amino acids of exon 2, Tat86. The full-length transcript, however, encodes for the full-length form of Tat, Tat101. To determine if the two different Tat variants have an effect on suppression of β-catenin activity, we co-transfected two PFA donors (Donor #1 and Donor #2) with increasing concentrations of a Flag-tagged truncated Tat (Tat86) and or a Flag-tagged full length (Tat101) Tat expression construct and a β-catenin responsive luciferase reporter construct. In this experiment we utilized the compound BIO , a potent GSK3� inhibitor, to stimulate �-catenin activity [88]. Although PFAs from two different donors differentially activated β-catenin signaling in the presence of BIO, both Tat86 and Tat101 effectively suppressed β-catenin activity in a dose-dependent manner . These data suggest that the effect of Tat on β-catenin activity is not due to Tat function attributed to the second exon.” We have also added the following clarifying statements to the appropriate section of the Discussion:“To further explore the mechanism of Tat suppression of β-catenin activity, we utilized Tat expression constructs that encoded Tat proteins of various lengths or containing functional mutations. In the absence of Tat, a transcriptional inhibitor complex including β-catenin can bind upstream of the viral promoter, preventing the binding of RNA pol II, therefore effectively stalling transcription . In the presence of Tat, Tat antagonizes β-catenin, displaces the complex, and promotes HIV-1 transcription through recruitment of cellular factors. Tat also enhances the degradation of β-catenin. The ability of Tat to suppress β-catenin signaling has been associated with the first 72 amino acids of the Tat protein [63]. In accordance with these findings, we show that both truncated (Tat86) and full-length Tat (Tat101) can suppress β-catenin signaling . Tat also contains several functional domains with variable and conserved residues. In particular, a highly conserved tryptophan at residue 11 in the proline-rich domain is required for efficient secretion of Tat. We observed that mutations at this position, W11F and W11Y, which abrogate Tat secretion and uptake, are unable to suppress β-catenin activity compared to WT Tat in the presence or absence of morphine . Tat produced during the viral life cycle can be efficiently exported from the infected cell; therefore, this mechanism likely contributes to antigenicity and neurotoxicity in the CNS environment. Loss of suppression by the W11Y mutant suggests that efficient Tat secretion may be important in suppression of β-catenin signaling, perhaps via bystander effect. Therefore, we expect in an environment where few astrocytes are infected that Tat will not induce efficient β-catenin suppression in bystander cells if its secretion function is lost. Transfection of Tat101 W11Y at high levels suppressed β-catenin similar to WT (data not shown). We speculate that at low concentrations of Tat, the protein can freely move to a neighboring cell and cause β-catenin inhibition in bystander cells allowing our assay to observe a greater overall reduction in β-catenin activity. Whereas Tat W11Y loses the ability to exit the cell and is limited to downregulating β-catenin in the local astrocyte. Further experiments will be required to test this hypothesis. HIV-1 Tat also contains a cysteine-rich domain containing highly conserved cysteine residues that are critical for disulfide-bond formation (amino acids 22-37). We also observed a loss of suppression of β-catenin activity by a C31R Tat mutant in the presence or absence of morphine . Interestingly, HIV-1 subtype C virus contains a polymorphism at position 31 (C31S) which does not suppress β-catenin activity. This data supports the importance of Tat directed β-catenin suppression in subtype B induced neuroinflammation.”17) Line 205. All of a sudden we have NLRP-1 and other things that were not introduced at all. Again 3 donors. AND, there no discussion about individual variability at all.We agree with the reviewer that additional introduction of the genes of interest in this study is needed. We have now added the following statements to the Introduction:“Approximately 150 β-catenin-regulated genes are estimated in primary astrocytes are comprising at least five broad categories including inflammation, transport, exocytosis, apoptosis, and trafficking [61]. Of these, β-catenin responsive genes that are neuroinflammatory in nature include BDNF (Brain-Derived Neurotrophic Factor), a member of the BDNF/TrkB (Tropomyosin receptor kinase B) signaling axis [66-68].”We have also added the following statements to the Results:“Our previous analysis of cellular miRNAs modulated by Tat expression revealed multiple protein targets and pathways that may contribute to neuroinflammation, such as glucocorticoid signaling, axonal guidance, neurotrophin/Trk signaling, and cytokine/chemokine signaling [78]. We were particularly interested in Trk signaling because TrkB is a receptor for BDNF and the dysregulation of this pathway has been implicated in degenerative changes in the CNS [90]. Additionally, BDNF gene expression has been shown to be directly regulated by the Wnt/β-catenin pathway . Furthermore, NLRP-1 was of interest because it is a gene responsive to glucocorticoid signaling; glucocorticoids being inhibitors of the Wnt/β-catenin signaling cascade [91]. Based on these predictions, and the correlates to Wnt/β-catenin signaling, we measured gene expression changes of TrkB, BDNF and NLRP-1 in U87MG cells and PFAs using RT-qPCR.”18) IN the discussion also, comments on CCL5, iNOS, genes that were not measured.We apologize for the confusion. Mention of genes that were not measured in this study have been removed.19) Paired Student's t test is not correct. Student's t test is unpaired.We agree with the reviewer and have revised the statistical tests used throughout the manuscript. The tests used have now been indicated in the Materials and Methods and in the Figure Legends. Where indicated that Student’s t test was used, we now used unpaired. Reviewer #2: The manuscript entitled "Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes "Since HAND increases in drug abusers, this study was directed to elucidate the role morphine on the effect of Tat HIV-1 and Tat mutants associated with neurocognitive impairment mediated by the β-catenin suppression and inflammatory processes.Although the data may be interesting, the manuscript has serious errors that must be resolved before it is acceptable for publication.2.- Some paragraphs do not have a bibliographic support and there are multiple typographical, orthographic and verb conjugation errors.We agree with the reviewer and hope that the largely revised version of the manuscript has addressed the grammatic, syntax, and bibliographic errors.3.- Throughout the writing different styles are noticed, with some sections well written and others are not.We agree with the reviewer and to improve clarity and read-ability, the majority of the text has been re-written to maintain “one voice” throughout the manuscript.4.- Major changes have to be made in the methods section.We agree with the reviewer and have rewritten the Materials and Methods section to include all pertinent information including the information outlined in the subsections below.4.1. The subtitles in general are inadequateWe agree with the reviewer and have re-written the subtitles to be more inclusive and descriptive.4.2. Given that the primary cultures of astrocytes are of fetal origin, it is important to inform about the time during the fetal development in which the cells were taken, how many donors are included in the study and if the experiments were performed in same donors?, please mention culture conditions for primary cultures and the cell line (was the same medium was used for both’), mention the passage of the culture in which the studies were made.We agree with the reviewer that additional information is needed. We have now included a statement about the origin of the primary cells as follows:“Fetal brain tissue  was obtained from elective abortion procedures performed in full compliance with National Institutes of Health and Temple University ethical guidelines. The tissue was washed with cold Hanks balanced salt solution (HBSS), meninges and blood vessels were removed. For glial cultures, tissue in HBSS was digested with 0.25% trypsin for 30 min at 37C. Trypsin was neutralized with FBS, and the tissue was further dissociated to obtain single-cell suspensions. For glial cultures, cells were plated in mixed glial growth media . The mixed culture was maintained under 10% Co2 for 5 days, and the medium was fully replaced to remove any cell debris. To enrich for microglia and astrocytes, flasks were placed on an orbital shaker for 14-18 hours at 200 rpm in growth media. Detached cells constitute the microglial component of the culture and were collected and plated into a new flask containing microglial media. Monolayers that remain after shaking constitute the astrocytes and are fed with astrocyte media.”We have now included culture conditions for the U87MG cell line and the primary cells (PFAs) as follows:“U87MG cells were maintained in Eagles Minimal Essential media (EMEM) supplemented with 10% Fetal Bovine Serum (FBS), 4.5g/L of L-glutamine, and 1% penicillin/streptomycin. Primary fetal astrocyte (PFAs) were maintained in Astrocyte Growth Media composed of Dulbecco’s Modified Eagle Media (DMEM) supplemented with 15% FBS (exosome free), 4.5 g/L of L-glutamine, 100 ug/mL gentamicin, 10 ug/mL Amphotericin B, and 20ug/mL insulin.”4.3. The authors must demonstrate that the primary cultures are really astrocytes and not neural stem cells, which are also positive to GFAP. Immunohistochemistry is recommended for neural stem markers.We agree with the reviewer and have now included the following statement regarding the purity of the primary cells: “Purity of cell type specific cultures was assessed by immunolabeling with anti-GFAP and -GLAST1 for astrocytes, -lba-1 and -CD11b for microglia and -MAP2 or -neurofilament for neurons.”4.3. Furthermore, depending on the fetal age in which the cells are obtained, they have different potential for differentiation. The fact that they are proliferative indicates that they are stem cells since the differentiated astrocytes do not proliferate, unless they are cancerous.We agree that the age of the cells, both in terms of when during development they are derived AND when in the lifetime of an individual the cells are examined. In this case, the cells are derived from the fetal tissue by the cell core at Temple University Comprehensive NeuroAIDS Center. They are expanded during this process as they are differentiated appropriately and will expand a limited number of times in culture and therefor have a certain amount of ‘stemness’ The manuscript includes mention of the difference in age of the cultures used.4.4. The luciferase assay is not in the methodsWe thank the reviewer for bringing this to our attention. We have now included the B-catenin luciferase assay in the Materials and Methods section as follows: “24 hours post-transfection or post-treatment, where indicated, cells were lysed with passive lysis buffer (Promega). Cell lysates were incubated in equal volumes with the DualGlo luciferase reagent (Promega) and luciferase was measured in a luminometer. Data shown represent the average of technical triplicates.”http://antibodyregistry.org/).4.5. Please include the data of all suppliers and the information on the antibody ID used  and the �-active β-catenin antibody  were used at a 1:1000 dilution. The antibody against total β-catenin recognizes all forms of the protein, however, the antibody against active β-catenin recognizes unphosphorylated Ser32 and Ser37. The �-GAPDH antibody  was used at a 1:3000 dilution.”4.6. It is not clear how long after the transfections and treatments the tests were performed.We thank the reviewer for bringing this to our attention. We have now included additional clarifying information on the experimental design both in the Materials and Methods and in the Figure Legends. In general, cells were transfected the day after plating, when cells were approximately 90% confluent. 24 hours post-transfection, at approximately 90% transfection efficiency, cells were treated with the indicated compounds. 24 hours post-treatment, cells were collected and lysed either for luciferase assay or Western blot. 48-hours post-treatment, cells were collected for qPCR analysis.4.7. In the section of reverse transcription authors mention that it was done as for the microRNAs, but in the manuscript, there is no analysis of these molecules. Please describe how the reverse transcriptions were made.4.8. The use of 1μl of cDNA without quantifying the amount of cDNA used in the PCR reactions is inadequate, please mention the amount of cDNA used.We have removed the erroneous text and include proper methods on RT for the qPCR4.9. Graphs on PCRs are inaccurate some are as relative expression , and others as xmRBNA/GAPDH. The authors are asked to clarify which method of expression analysis is used, and they are recommended to use 2-ΔΔCT.We thank the reviewer for bringing this to our attention. We have now re-plotted the data in Figure 5 as “Gene Fold change 2^ ΔΔCT.” Values less than one are indicative of a decrease in gene expression and values greater than one are indicative of an increase in gene expression. The Figure Legend has been updated with the following statement: “Fold change values were calculated by normalizing relative mRNA gene expression (Ct) to GAPDH (�Ct) relative to the control (��Ct). Data is plotted as the fold change of the gene over the conditions (2��Ct).”4.9. Statistic section is unclear, why did the authors used Paired Student’s t test. And why after ANOVA a pairwise analysis was performed and not a multiple comparison test.We agree with the reviewer and have revised the statistical tests used throughout the manuscript. The tests used have now been indicated in the Materials and Methods and in the Figure Legends. Where indicated that Student’s t test was used, we now used unpaired. Where indicated that ANOVA was used, we now indicate use of multiple comparison test with Tukey’s analysis.Results:1. The images are of very poor quality.We thank the reviewer for the comment. We have attempted to adjust the quality and clarity of the figures submitted in the revised version of the manuscript including re-graphing the data to and clearly stating the cells used.2. In some results they refer to the donor number and in others they do not.We agree with the reviewer that clarification of which cells were used for given experiments was needed. We have now indicated use of either the U87MG astrocytic cell line or any of three primary fetal astrocyte (PFA) donors  on each figure. We have also clarified which cells were used in each relevant section of the Results and in the Figure Legends. Finally, the source of the cells has been clarified in the Materials and Methods.3. The luciferase assay are confusing, perhaps because it is not described in the methods.We agree with the reviewer that additional clarification of the luciferase assay is needed. An example of the type of clarifying statements we have included in the manuscript is as follows: To address the ability of Tat to modulate B-catenin activity in physiologically relevant cells, we utilized the U87MG astrocytic cell line and primary fetal astrocyte (PFA) donors in our study. We co-transfected these cells with increasing concentrations of full-length WT Tat and a B-catenin responsive luciferase reporter construct in the presence of LiCl to stimulate B-catenin activity. 24-hours post-transfection, cells were lysed with passive lysis buffer and were incubated with equal volumes of the DualGlo luciferase reagent. Luciferase was measured in a luminometer. Data are presented as B-catenin activity over the experimental conditions.4. Not all figure legends have the statistical test used.We agree with the reviewer and have added the appropriate statistical tests to the figure legends.5. In cases where ANOVA was used, which post-test was performed.When two-way ANOVA was used it was followed by Tukey’s multiple comparisons test. This is now included in the methods and figure legends.6. It is inappropriate to use the Student's t to compare more than two groups We agree with the reviewer and have revised the statistical analyses used as indicated in the figure legends. Student’s unpaired t-test is now used to compared two groups only. 7. The number of experiments is not clear. We agree with the reviewer and have added the number of experiments to each figure legend.The discussion does not have a clear focus and repeats a large part of the results, which makes it tedious and very long.We agree with the reviewer and have largely revised and rewritten the Discussion for clarity.AttachmentPLoS ONE reviewers comments FINAL.docxSubmitted filename: Click here for additional data file. 4 Mar 2020Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytesPONE-D-19-16014R1Dear Dr. Klase,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Figure 6 indicates that the results are 2 ^ ΔΔC and should say 2 ^ -ΔΔCT. Please confirm that the data is calculated correctly. And although it is not mandatory, I suggest that the way in which the analysis was performed must be in the methods section rader than in the figure legend.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? 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The knowledge about the molecular alterations which are found in head and neck squamous cell carcinomas (HNSCC) has much increased in recent years. However, we are still awaiting the translation of this knowledge to new diagnostic and therapeutic options. Among the many molecular changes that are detected in head and neck cancer, the abnormalities in several signaling pathways, which regulate cell proliferation, cell death and stemness, seem to be especially promising with regard to the development of targeted therapies. Canonical Wnt signaling is a pathway engaged in the formation of head and neck tissues, however it is not active in adult somatic mucosal cells. The aim of this review paper is to bring together significant data related to the current knowledge on the mechanisms and functional significance of the dysregulation of the Wnt/β-catenin pathway in head and neck tumors. Research evidence related to the role of Wnt signaling activation in the stimulation of cell proliferation, migration and inhibition of apoptosis in HNSCC is presented. Moreover, its role in promoting stemness traits in head and neck cancer stem-like cells is described. Evidence corroborating the hypothesis that the Wnt signaling pathway is a very promising target of novel therapeutic interventions in HNSCC is also discussed. Head and neck squamous cell carcinoma (HNSCC), which is most frequently located in the oral cavity, lip, pharynx or larynx, is one of the most common types of cancer, comprising around 4% of new cancer cases each year. HNSCC incidence is higher in men than in women ,2. The dCTNNB1 mutations and APC mutations were present infrequently [FAT1 tumor suppressor, which encodes a protocadherin protein that binds and inactivates β-catenin, were detected in some cases of HNSCC [TP53 (p53R(175)H missesnse mutation) led to a significant induction of a gene expression profile matching Wnt/β-catenin pathway activation [Recent genomic analyses have shown the prevalence of genetic driver alterations in HNSCC, which most frequently affect genes associated with receptor tyrosine kinase , PI3K, p53, Notch or Hippo signaling pathways. On the other hand, genetic alterations in genes related to canonical Wnt signaling were rarely detected in HNSCC ,9. In coequently ,14,15,16of HNSCC . Howeverof HNSCC . In anotof HNSCC . The staof HNSCC . Other sof HNSCC . The treof HNSCC . Additiotivation .The central regulatory protein in the canonical Wnt pathway is β-catenin. Briefly, in unstimulated cells, cytoplasmic β-catenin undergoes proteasomal degradation, which is stimulated by its phosphorylation by the components of the destruction complex, which comprises casein kinase 1 (CK1), GSK-3β, APC and AXIN . On the Importantly, apart from playing a key role in Wnt signaling, β-catenin has crucial functions related to the regulation of cell-cell adhesion by the formation of adherens junctions (AJ). It forms complexes with E-cadherin, and, together with α–catenin, takes part in associating E-cadherin to actin cytoskeleton in epithelial cells. Thus, although debatably, it is believed that there are two, relatively separate, pools of intracellular β-catenin—membranous and cytoplasmic, with distinct functions, which are regulated by partly independent mechanisms . The canCDH1 gene [DPAGT1, which encodes N-acetylglucosamine 1-phospho-transferase [N-glycosylation. Glycosylated E-cadherin shows reduced ability to form stable AJ [The reduction in membranous β-catenin level is frequently associated with the reduction in E-cadherin level ,55,57, wDH1 gene ,58. The DH1 gene . On the nsferase . This entable AJ . Indeed,DPAGT1 was associated with a reduction in β-catenin-dependent reporter gene expression in vitro [The frequently observed cytoplasmic delocalization of β-catenin cannot be considered by itself as a direct evidence of canonical Wnt pathway activation in HNSCC. However, there is also other evidence corroborating this hypothesis. The loss of membranous FAT1 is observed in poorly differentiated OSCC, and this is associated with the cytoplasmic and nuclear accumulation of β-catenin, upregulation of cell growth and loss of cell adhesion . Also, tin vitro .In oropharyngeal tumors, delocalized expression of β-catenin was associated with increased expression of LEF1. Importantly, LEF1 expression was absent from normal epithelium. The majority of tumors were LEF1-positive, including all advanced stage carcinomas, most recurrent cases and two thirds of tumors which were associated with death during follow-up. Interestingly, some LEF1-positive tumors were negative for β-catenin, but these tumors were positive for γ-catenin, which is redundant with β-catenin, when it comes to nuclear transcription regulation . AnotherThe overexpression of CIP2A, which is frequently observed in oral dysplasia and carcinoma ,68, may SFRP1, SFRP2 and SFRP5 genes were frequently hypermethylated in OSCC cell lines and in primary tumor samples, while their ectopic expression suppressed the growth of OSCC cells in vitro [SFRP2 was found methylated in the majority of OSCC samples, in contrast to corresponding normal mucosa, and its overexpression reduced tumor growth in vivo [SFRP1 and SFRP5 expression, which was induced by gene promoter methylation, was associated with cytoplasmic/nuclear delocalization of β-catenin in oral lesions [SFRP1 and SFRP2, also WIF1, DKK1 and PPP2R2B were methylated in LSCC cell lines and primary tumors [WIF1 promoter [WIF1 in OSCC was found to be cancer-specific when compared to normal mucosa [SFRP2 and WIF1 was shown to be already present in dysplastic lesions and the rate of methylation increased with progression to OSCC [WIF1 predicted shorter survival in OSCC patients [CBY promoter methylation [Epigenetic alterations play an important role in Wnt pathway activation in HNSCC. The activity of the pathway is regulated physiologically by a series of antagonistic proteins which downregulate Wnt signaling by sequestrating extracellular Wnt ligands (SFRP and WIF proteins), blocking the receptor complex proteins (DKKs), antagonizing intracellular signal transduction  or inhibiting the formation of active β-catenin transcriptional complex . The expression of these genes is frequently reduced by promoter methylation in cancer. Indeed, in vitro ,71. Addi in vivo . It was  lesions . Apart fy tumors . The expy tumors , and thipromoter . The metl mucosa ,75. The  to OSCC . Anotherpatients , howeverpatients . Also, thylation .CCND1 and c-MYC. It was found that arecoline leads to epigenetic silencing of these miRNAs, and the induction of their re-expression by a combined treatment with DNA methyltransferase and histone deacetylase inhibitors reduced cell proliferation and migration [DKK2, which acts as the antagonist of LRP6 co-receptor. Oral carcinomas were characterized by increased expression of miR-21 and loss of DKK2 expression [MINCR, PLAC2 and UCA1 was frequently observed in head and neck cancers, and functionally, it promoted malignant progression by the activation of Wnt/β-catenin pathway [MINCR expression correlated with lymph node metastasis and shorter survival of OSCC patients. The knockdown of MINCR reduced β-catenin-dependent reporter gene expression, and also reduced the expression of Wnt target genes—CCND1 and c-MYC—but elevated the level of E-cadherin. This resulted in reduced cell viability, reduced cell migration and invasion, and cell cycle arrest and increased apoptosis in cancer cell lines [PLAC2 in tongue carcinoma cells resulted from the enrichment of its promoter region in H3K27 acetylation, which was dependent on CBP acetyltransferase activity. PLAC2 knockdown also diminished cell proliferation and migration [UCA1 in laryngeal cancer cells was associated with reduced GSK-3β phosphorylation and elevated β-catenin level. The inhibition of Wnt signaling using IWP-2, a Porcupine inhibitor, reduced the stimulatory effects of UCA1 overexpression on cell proliferation and migration [Recent studies have shown the important involvement of regulatory RNA molecules in the regulation of Wnt/β-catenin signaling, and abnormalities in the expression of miRNA were implicated in the activation of the Wnt pathway. The epigenetic silencing of miR-329 and miR-410, which may be induced by arecoline, the carcinogenic ingredient of betel quid, was associated with the upregulation of WNT-7B expression and the induction of expression of igration . Also, migration . In contpression . Moreove pathway ,83,84. Tll lines . The eleHNSCC is largely induced by environmental carcinogens, and it has been shown that both HPV infection and chemical carcinogens can stimulate canonical Wnt signaling in epithelial cells. Based on the analysis of the level of expression of β-catenin target genes, it has been suggested that HPV-negative tumors show stronger activation of Wnt/β-catenin signaling, at least in patients using tobacco . Also, tCcnd1, Mmp-2 and Mmp-9, was also elevated [4-Nitroquinoline-1-oxide (4-NQO) is a synthetic compound mimicking the action of tobacco carcinogens, and is used for the experimental induction of tongue carcinomas in rats. It leads to the progressive development of oral lesions, from hyperplasia through dysplasia to carcinoma, which show similar characteristics to human oral cancers . The dyselevated . Finallyelevated .WNT-1 expression in oral cancer cells inhibited Wnt/β-catenin signaling and reduced the expression of Vimentin, a marker of epithelial-to-mesenchymal transition [MMP-7, which resulted in enhanced cell migration and invasion [It is believed that, within a tumor, cancer cells can be divided into two general groups: most cells form the bulk of tumor, and these cells are usually targeted by classical chemotherapy, while the group of tumor propagating cells, or the so called cancer stem-like cells (CSC), remain resistant to standard chemotherapeutics. Thus, it is currently believed that targeting CSC is the key to successful chemotherapy. Recent findings have linked abnormalities in canonical Wnt signaling to HNSCC tumor propagating cells. Indeed, higher activity of the canonical Wnt pathway correlated with stem cell traits, e.g., enhanced expression of CD44 and LGR5 . LGR5 isansition . The intinvasion . Additioinvasion . All thiCTNNB1 significantly reduced tumor growth in xenograft mice [Many studies have analyzed the significance of therapeutic targeting of the Wnt/β-catenin pathway in HNSCC. Due to the central role of β-catenin in mediating Wnt signal transduction and transcription activation, this protein attracted much attention when it comes to assessing the anti-cancer effects of its downregulation. The induction of β-catenin transcriptional activity in HNSCC cell lines using lithium chloride, an inhibitor of GSK-3β, led to an increased cell proliferation rate, enhanced cell migration and invasion and a reduced rate of apoptosis . Accordiaft mice  and led aft mice . Becauseaft mice .c-MYC and MMP-2, which resulted in increased cell invasion. TWIST1-induced Wnt activation was mediated by PI3K, which phosphorylated and inactivated GSK-3β, leading to β-catenin stabilization. Thus, TWIST1-dependent activation of β-catenin was inhibited by a PI3K inhibitor [Arecoline-induced β-catenin overexpression in oral epithelial cells was reduced by ERK and PI3K small molecule inhibitors . It has nhibitor . Emodin nhibitor . These fThe silencing of galectin-3 in tongue cancer cells led to the induction of β-catenin degradation, which resulted in decreased level of MMP-9 expression and diminished cell migration and invasion capacity . InteresGAD1 suppressed the nuclear translocation of β-catenin and reduced the level of MMP-7, which led to reduced cell migration and invasion [The activation of canonical Wnt signaling in HNSCC has been shown to result from the overexpression of several other proteins as well. CUL4B, which takes part in protein ubiquitination, may activate the Wnt/β-catenin pathway by reducing the level of DKK1 and PPP2R2B antagonists. CUL4B was overexpressed in around 50% of HNSCC cases. It has been shown that the knockdown of CUL4B reduced the level of active β-catenin, decreased β-catenin-dependent reporter gene transcription and led to the reduction in the level of expression of cyclicn D1, c-MYC, MMP-7 and vimentin, while it increased the level of expression of E-cadherin. It resulted in reduced cell growth and diminished cell migration and invasion. Importantly, the effects exerted by CUL4B overexpression were abolished by treatment with a tankyrase inhibitor—XAV-939, which blocks the canonical Wnt pathway by preventing AXIN degradation and subsequent destabilization of β-catenin destruction complex, which are stimulated by active tankyrase . Also, ginvasion . On the invasion .Several natural agents have been shown to exert anti-cancer effects by attenuating Wnt signaling in HNSCC. Ellagic acid prevented the formation of DMBA-induced tumors in the buccal pouch hamster model by downregulating the expression of Frizzled and Dishevelled-2 and reducing the activation of the Wnt/β-catenin pathway . AstaxanThe inhibition of canonical Wnt signaling was also exerted by several pleiotropic therapeutics. Pyrithione zinc inhibited PI3K/Akt signaling and the downstream activation of the Wnt/β-catenin pathway. It reduced the level of expression of c-MYC and cyclin D1. It reduced cell proliferation, migration and invasion and led to the induction of apoptosis and reduced xenograft tumor growth . Nano-foCCND1, BIRC5) expression [Several potential molecular targets have been proposed for the effective attenuation of the Wnt/β-catenin pathway in head and neck cancer cells . Since Hpression . Moreovepression .RAP1 diminished the level of β-catenin activation by lithium chloride or WNT-3A. It decreased MMP-7 expression and blocked cell invasion in HNSCC cell lines [RAP1 is another protein which enhances the nuclear translocation of β-catenin and facilitates its transcriptional activity. The silencing of ll lines . This sull lines ,129. Thell lines . Moreovell lines ,131. Canll lines  or LF3, ll lines , which rHNSCC tumors are frequently resistant to standard chemotherapy, and the mechanisms of chemoresistance are poorly understood. Different observations have shown that the activation of the Wnt pathway may be responsible for chemo- and radio-resistance in HNSCC, which may be contradicted with the use of Wnt pathway inhibitors. The knockdown of β-catenin sensitized HNSCC stem-like cells to cisplatin . 14-3-3σMuch evidence has accumulated pointing to the significance of activated Wnt/β-catenin signaling in driving carcinogenesis in the head and neck region. The most important molecular alterations related to Wnt pathway activation in HNSCC and promising therapeutic targets are listed in


Background: Gemcitabine is widely used in the treatment of breast cancer (BC). However, the resistance to drugs remains a tough concern. The study explored the potential mechanism concerning gemcitabine resistance in triple-negative BC (TNBC) in vitro. Methods: TNBC cells (TNBCC) and gemcitabine-resistance cell lines (GRC) were used. We investigated the sensitivity to gemcitabine responsive to regulation of Nod-like receptor protein 3 (NLRP3) expression in TNBCC in different gemcitabine concentrations. RT-PCR checked NLRP3 mRNA expression and MTT assessed the cell cytotoxicity. Gemcitabine resistance was studied in GRC exposed to 0, 1, 3, 5 nm gemcitabine after GRC were treated with NLRP3 agonist Nigericin sodium salt (NSS) or antagonist CY-09. Epithelial-to-mesenchymal transition (EMT) biomarkers were evaluated via RT-PCR and inflammasome IL-1β, β-catenin content and GSK-3β activity were measured by ELISA methods. Last, we inactivated the signaling and examined the NLRP3, EMT mRNA expression by RT-PCR, IL-1β, β-catenin content and GSK-3β activity by ELISA and cell cytotoxicity through MTT. Results: NLRP3 up-regulation improved cell survival and reduced sensitivity to gemcitabine (P<0.05). NLRP3 had higher expression in GRC than TNBCC. GRC cell viability dropped as the gemcitabine concentration increased. NLRP3 up-regulation added to resistance to gemcitabine in GRC (P<0.05). NLRP3 agonist might induce EMT process, activate wnt/β-catenin signaling and IL-1β, while inactivation of wnt/β-catenin signaling could result in the inhibition of NLRP3, IL-1β and EMT as well as cell viability in GRC (P<0.05). Conclusion: NLRP3 could enhance resistance to gemcitabine via IL-1β/EMT/Wnt/β-catenin signaling, which suggested that NLRP3 antagonist CY-09 might be incorporated into gemcitabine treatment for TNBC. Gemcitabine has been widely introduced to the treatments of cancers and has been proved as a curative drug alone or combined with other drugs, including cisplatin . HoweverPreviously, it was reported that mRNAs were involved with chemoresistance in BCs. PTEN could obstruct the resistance against gemcitabine and inhibited epithelial-to-mesenchymal transition (EMT) via AKT signaling . Howeverin vitro nor in vivo.NLRP3, Nod-like receptor protein 3, belongs to the canonical inflammasome family of leucine-rich repeat containing proteins (NLRs) and mediates cancer pathogenesis via mediation of apoptosis proteins and immune responses ,10. Alsoin vitro it presents high EMT process, which could be manifested through changes in expression of EMT-gain biomarkers N-cadherin, Vimentin and EMT-loss biomarker E-cadherin. Previous research concluded that Wnt/β-catenin signaling pathway was significant for regulation of metastasis, cell proliferation and EMT in BC even though the localization of β-catenin in cancer cells differed depending on the specific types of BC [BC is highly invasive and es of BC . In the es of BC . Researces of BC . Based oin vitro.Therefore, in the present study, we investigated the regulatory role of NLRP3 in resistance to gemcitabine in TNBCs and its underlying interaction with Wnt/β-catenin 2, 95% O2 condition at 37°C .TNBC cell lines (TNBCC), MDA-MB-231 and MDA-MB-468 were purchased . The gemcitabine-resistance cell lines (GRC), MDA-MB-231-R and MDA-MB-468-R were produced by treating TNBCC with increasing gemcitabine concentrations (0.1–15 nm) for 12 months (GI50 < 7.0) . GemcitaEach cell line was also incubated with NLRP3 agonist Nigericin sodium salt (NSS) and antagonist CY-09 separately  to differentiate NLRP3 expression in both TNBCC and GRC. Wnt inhibitor Wnt-C59 was added into subgroups of GRC so as to inactivate signaling pathway .All the cell lines were exposed to the 0, 1, 3, 5 nM gemcitabine, respectively, for 72 h for the following assays.Ct−ΔΔ method.The total RNA was extracted from cells with different drug treatments by TRIzol based on the guidebook of the producer . IScript Advanced cDNA synthesis kit was applied to synthesize cDNA . Cells were selected and planted into 96-well plates for RT-PCR assays. SYBR Green qPCR Master Mix  was adopted for PCR analysis. The sequences for the primers were listed as below. NLRP3: F, 5′-ACTCTGTGAGGGACTCTTGC-3′; R, 5′-GGTCGCCCAGGTCATTGTT-3′ . E-cadheThe cells from MDA-MB-231, MDA-MB-468, MDA-MB-231-R, MDA-MB-468-R, MDA-MB-231-R CY-09, MDA-MB-468-R CY-09, MDA-MB-231-R Wnt-C9, MDA-MB-468-R Wnt-C9 groups were collected and cultured till they reached ∼80% confluence. Then lysis buffer in the ELISA Kits mentioned below were added to lyse the mixture. BCA Assay Kit was utilized to quantify the protein in each group. A total of 10 μg protein extracts of all groups were used for each of the following ELISA methods based on the manufacturer’s instructions. IL-1β ELISA Kit was used according to the provided instructions from the producer to measure the IL-1β cytokine activity in each group mentioned above . GSK3β activity was evaluated using GSK-3β (pS9) + Total GSK-3β Simple Step ELISA Kit strictly guided by the instructions on the guidebook . β-catenin accumulation was detected using β-catenin ELISA Kit .2 at 37°C for a day. Then cells were exposed to different gemcitabine concentrations . After 48-h exposure, we added 10 μl MTT (5 mg/ml) solution into each well and left the plates to incubate inside the incubator for another 4 h. Thereafter, 100 μl Formazan solution was put in each well and the plates were in incubation for another 4 h. Absorbance values were evaluated at 570 nm.Cells were seeded into 96-well plates with 2800 cells each well and the plates were incubated in the incubator with 5% COP-values, double-tailed Student’s t test. All the data were shown in figures as mean and standard deviation values. GraphPad Prism 8  contributed to the formation of figures in tiff format based on the raw data.All the assays were performed for three times in independence. SPSS Statistical 19  was applied for P<0.05, P<0.05, In both the TNBC cells, NLRP3 was either inhibited by its antagonist CY-09 or agonized through its agonist NSS. RT-PCR measured the mRNA expression and observed an increase in agonist group and a decrease in CY-09 group, using a non-treatment group as a control P<0.05, A,C. MTT P<0.05, P<0.05, P<0.05, First, differential mRNA expression of NLRP3 was measured in TNBCC, GRC, GRC treated with NSS and GRC treated with CY-09 groups, and it was discovered that NLRP3 had higher expression in GRC than in normal BC cells (TNBCC) P<0.05, A,C. It aP<0.05, P<0.05, P<0.05, P<0.05, RT-PCR observed higher Vimentin and lower E-cadherin in GRC than TNBCC, suggesting that EMT process was promoted in gemcitabine-resistance cells P<0.05, A. HoweveP<0.05, P<0.05, P<0.05, P<0.05, ELISA confirmed that GSK3β activity was up-regulated and β-catenin content was inhibited in the GRC incubated with Wnt-C59 P<0.05, E. BesideBC is mainly constituted of three subtypes, HR positive, human epidermal growth factor (HER2)-enriched and TNBC according to immunohistochemical expression of the hormone receptors (HR): estrogen receptor (ER) and progesterone receptor (PR) as well as HER2 . TNBC prin vitro and in vivo in oral squamous cell carcinoma [Researchers unveiled that NLRP3 stimulation could mediate the resistance to 5-Fluorouracil both arcinoma . Besidesarcinoma . Howeverarcinoma .in vitro, increased IL-1β induced higher invasiveness of TNBC cells [Recently NLRP3 was exposed to play a role in regulation of BC metastasis by activation of IL-1β . BesidesBC cells . Thus, wBC cells . Further9 and Tyr216. Phospho-GSK3β-Ser9 suppresses GSK3β activity, thereby inducing the β-catenin content while Phospho-GSK3β-Tyr216 promotes the activity and inhibits the β-catenin [9, total GSK3β and β-catenin levels by ELISA and calculated the GSK3β activity accordingly in TNBC, GRC, inhibited NLRP3 and signaling blocked groups (Supplementary Tables S1 and S2). Meanwhile, we also evaluated the β-catenin content by ELISA method. Our results present that the lower p-GSK3β-Ser9 induced by NLRP3 inhibitor CY-09 or signaling suppressor Wnt-C59 indicates higher GSK3β activity and suppresses β-catenin. Taken together, we assessed the potential influence of NLRP3 expression on IL-1β/EMT and Wnt/β-catenin signaling and and discovered that higher NLRP3 expression resulted in more active IL-1β, EMT process and Wnt/β-catenin signaling.EMT and Wnt/β-catenin signaling regulate the sensitivity to chemodrugs in TNBC ,34. GSK3-catenin . In our in vitro.In TNBC, wnt3A inhibitor was revealed to slow down EMT process with a hint that Wnt/β-catenin signaling could modulate EMT . Hence, in-vitro findings that NLRP3 down-regulation could increase the gemcitabine sensitivity in TNBC cells and that in gemcitabine-resistant cells, NLRP3 up-regulation could enhance the drug resistance via activation of IL-1β, EMT and Wnt/β-catenin signaling, which posed a potential possibility that NLRP3 antagonist could be a therapeutic strategy that can be incorporated into gemcitabine treatment to promote the efficacy by reducing the drug resistance in TNBC in the future.The study presented the The current research features the contribution of NLRP3 to gemcitabine resistance in TNBCC.in vitro.It was disclosed that up-regulated NLRP3 reduced sensitivity to gemcitabine in TNBC cells and inhibition of NLRP3 alleviated the gemcitabine resistance in TNBC in vitro.NLRP3 antagonist inhibited NLRP3 expression, resulting in the suppression of cell viability, EMT process and IL-1β through inactivation of Wnt/β-catenin signaling pathway in gemcitabine-resistant TNBC Inactivation of Wnt/β-catenin signaling inhibited NLRP3 expression, deterring IL-1β, EMT and cell viability of GRC in gemcitabine-resistant TNBC.Click here for additional data file.


ANGPTL7 is a member of the angiogenin-like protein family. Compared to other members, ANGPTL7 is the least known. Recent studies have explored the relationship between ANGPTL7 and multiple pathological processes and diseases. However, there is no research about ANGPTL7 in neonates. This study was designed to investigate the concentration of ANGPTL7 in cord blood of preterm infants.  Singleton infants born in November 2017 to June 2019 in the study hospital were enrolled in the study. Maternal and neonatal clinical data were collected. ANGPTL7 levels in cord blood and serum on the third day after birth were measured by an enzyme-linked immunosorbent assay. t = 15.4, P < 0.001). In multiple line regression analysis, ANGPTL7 levels independently correlated with gestational age . There is also no correlation between preterm outcomes and ANGPTL7 levels. Cord blood levels of ANGPTL7 were significantly higher than those in serum on the third day after birth .  A total of 182 infants were enrolled in this study. Patients were categorized into two groups by gestational age . ANGPTL7 levels in preterm infants were significantly higher than that in full-term babies ( Cord blood ANGPTL7 levels are higher in preterm infants than full-term babies. The levels are independently influenced by gestational ages and attenuated significantly after birth. The underlying mechanism needs to be further studied. Preterm birth is a worldwide epidemic, and the global incidence of preterm birth is approximately 15 million every year . Among pAngiopoietin-like proteins (ANGPTLs) are a family of secretory proteins that have a similar structure to angiopoietin proteins, consisting of eight members (ANGPTL1-8) . All proFrom November 2017 to June 2019, 182 infants born in Guangdong Women and Children's Hospital were included in this study. Inclusion criteria: (a) born in the study hospital; (b) singleton birth; (c) without congenital abnormalities; (d) the mother was negative for hepatitis B (HBsAg and/or HBeAg) and C virus (anti-HCV), syphilis, HIV (anti-HIV-1 and anti-HIV-2) and IgM against cytomegalovirus, rubella, toxoplasma, and herpes simplex virus; and (e) with written consent form.A cord blood sample and a serum sample on the third day after birth were collected from every included infant if a signed informed consent form was obtained from the parent or guardian, centrifuged, and then, stored at –80°C. ANGPTL7 levels were measured using a human ANGPTL7 enzyme-linked immunosorbent assay (ELISA) Kit  following the manufacturer's instructions.T test was used to compare the quantitative variables between two groups. Chi-square was used to compare the qualitative variables between the two groups. Pearson/Spearman's correlation coefficients were estimated to determine associations between ANGPTL7 levels and variables. Multiple linear regression analysis was used to estimate the unique independent predictive contribution of neonatal/maternal on ANGPTL7. The variables' distribution characteristics were estimated with single sample Kolmogrov-Smirnov test. Statistics were considered to be significant when P value < 0.05.Statistical analysis of the data was performed using SPSS software version 20.0 . Baseline continuous variables were expressed as mean ± standard deviation or median (interquartile range). Categorical variables were represented by numbers (percentages). Independent samples t = 15.4, P < 0.001).A total of 182 infants  met the inclusion criteria and were included in the study population. 103 (56.6%) were boys, 33.5% preterm, and 23.1% full-term. Gestational age is 25-41 weeks, and birth weight is 750-4180 g; there were significant difference in gestational age, birth weight, delivery mode, asphyxia, maternal age, antenatal steroids use, and ANGPTL7 levels between preterm and full-term groups . ANGPTL7r = −0.223, P = 0.024, r = −0.743, P < 0.001) and birth weight . Maternal age , asphyxia , maternal hypertension , and antenatal steroid use  were positively associated with cord blood ANGPTL7 concentrations. We found no significant correlation between the cord blood levels of ANGPTL7 in infants and sex, delivery mode, and maternal diabetes. We used multiple regression analysis to control for potential confounding factors. The variables “birth weight” and “maternal age” were not introduced in the same model, as well as the variables “asphyxia,” “maternal hypertension,” and “antenatal steroids use,” due to collinearity. In multiple regression analysis, gestational age was the independent predictors of cord blood levels of ANGPTL7 . The levels of ANGPTL7 were higher in infants who later developed BPD, NEC, IVH, ROP, and sepsis than those who did not, but the results all did not reach statistically significant differences .t = 13.88, P < 0.001, We also measured the ANGPTL7 levels in serum on the third day after birth. Since the consents for the collection of serum samples on day 3 were not obtained from all the parents or guardians, only 57 cord blood samples in preterm infants were finally collected. We found no maternal or neonatal characteristics have correlations with ANGPTL7 levels on day 3. However, serum levels of ANGPTL7 were significantly lower when compared with their levels in cord blood (In this study, we show for the first time the levels of ANGPT7 in preterm infants. Cord blood levels of ANGPTL7 are higher in preterm infants than that in full-term infants and have a negative correlation with gestational age. Serum levels of ANGPTL7 on day 3 of life were lower than those in cord blood. Collectively, our data shed the light on the expression pattern of ANGPTL7 in preterm infants.ANGPTL7 is a newly discovered member of the angiogenin-like protein family. Previous studies have shown the biological function of ANGPTL7 in hematopoietic stem and progenitor cells , 17, traHypertensive disorders of pregnancy were reported to be significant risk factors for preterm birth . PreviouIn this study, no significant differences were observed when ANGPTL7 levels were compared in each preterm outcome, even though the ANGPTL7 levels were higher in infants diagnosed with BPD, ROP, NEC, IVH, and sepsis than who was not. ANGPTL7 was originally discovered from corneal cells and named cornea-derived transcript 6 . It was Our study has several limitations. First, sample size was limited. Only 182 infants were included in this study. The results could be caused by random error. Second, only 9 variables were included in the multiple regression analysis; we may have ignored other factors that related to ANGPTL7. Third, as there are few studies on ANGPTL7 by now, we cannot give a reasonable reason for its negative correlation with gestational age at present. Therefore, further studies with larger sample sizes are required to validate these results.In conclusion, preterm infants have higher levels of ANGPTL7 than term babies. ANGPTL7 levels decreased during gestation, and attenuated significantly after birth. To the best of our knowledge, this is the first study that investigates the distribution of ANGPTL7 in preterm infant. However, there also remain some limitations. Thus, researches with larger sample size and explorations about the specific mechanism of the negative association between ANGPTL 7 and gestational age will be important areas of future research.


Graphene has excellent mechanical, thermal, optical and electrical properties and this has made it a prime target for use as a filler material in the development of multifunctional polymeric composites. However, several challenges need to be overcome to take full advantage of the aforementioned properties of graphene. These include achieving good dispersion and interfacial properties between the graphene filler and the polymeric matrix. In the present work, we report the thermal and mechanical properties of reduced graphene oxide/epoxy composites prepared via a facile, scalable and commercially viable method. Electron micrographs of the composites demonstrate that the reduced graphene oxide (rGO) is well dispersed throughout the composite. Although no improvements in glass transition temperature, tensile strength and thermal stability in air of the composites were observed, good improvements in thermal conductivity (about 36 %), tensile and storage moduli (more than 13 %) were recorded with the addition of 2 wt% of rGO. Due to its remarkable mechanical, electrical and thermal properties, graphene has evoked a lot of interest amongst researchers working on the development of multi-functional composites for a variety of applications /epoxy composites are produced in a facile, scalable and commercially viable way. This involves dispersing freeze-dried GO in a diglycidyl ether of bisphenol-A (DGEBA) epoxy resin by shear mixing. The GO is then reduced in situ (to rGO) using a relatively high-temperature cure schedule for the epoxy resin system, after adding the hardener. The thermal and mechanical properties of the composites produced are characterized and the viability of the processing technique is demonstrated. The advantages of this processing route when compared to those adopted by others are: (1) it does not involve the use of solvents or hazardous GO reducing agents such as hydrazine hydrate; and (2) in situ reduction of the GO during the curing of the epoxy eliminates the extra step in the production process of having to reduce the GO prior to incorporation in the matrix.A standard DGEBA resin , having an epoxide equivalent weight (EEW) of 185 g/eq, was used. The curing agent was an accelerated methylhexahydrophthalic acid anhydride , having an anhydride equivalent weight (AEW) of 170 g/eq.A suspension of graphene oxide was prepared via a modified Tour et al.  as described elsewhere  was carried out on the composites and GO samples using a TGA/DSC 1 apparatus . A sample of the freeze-dried GO was thermally reduced according to the cure schedule employed for processing the composites . To investigate the thermally induced dissociation of the GO and rGO, samples were analysed in a nitrogen atmosphere using a heating rate of 5 °C/min. Thermal oxidation of the composites, on the other hand, was analysed in air using a heating rate of 10 °C/min. All samples were analysed over the temperature range 30–800 °C.3 were subjected to temperature sweeps from 30 to 200 °C at a heating rate of 2 °C/min in a dual-cantilever mode using a frequency of 1 Hz and an oscillation strain of 0.05 %. The number average molecular weight, Mnc, between cross-links was calculated from Eq. , R is the universal gas constant (8.314 cm3 MPa/K mol) and ρ is the density of the epoxy determined at room temperature [1.2 g/cm3  from TA Instruments, UK. Rectangular samples with dimensions 60 × 10 × 3 mm−9 mbar. XPS spectra were acquired using an MXR1 monochromated Al Kα X-ray source (hυ = 1486.6 eV). An X-ray spot of ~400 μm radius was employed. High-resolution, core level C1s spectra were acquired using a pass energy of 20 eV. The GO sample spectra were charge referenced against the C1s peak at 284.4 eV to correct for charging effects during acquisition.X-ray photoelectron spectroscopy (XPS) analyses were performed using a Theta Probe spectrometer . The instrument was operated at a base pressure of 1 × 10Uniaxial tensile tests were conducted on the rGO/epoxy composites at room temperature using a universal tensile testing machine . Dumbbell specimens having a gauge length of 25 mm were used. A displacement rate of 1 mm/min was adopted. The tensile properties were averaged from the results obtained from a minimum of five specimens. The strain was measured using a clip-on extensometer.2 samples which were 2 mm thick. A temperature range of 30–60 °C was used, at 10 °C intervals, with a laser voltage of 450 V and a pulse width of 0.8 ms. The samples were coated with a thin layer of graphite prior to testing to increase absorption and transmission of the infrared radiation energy.The thermal conductivities of the rGO/epoxy composites were determined from their thermal diffusivities measured by the laser-flash technique using an LFA-427 Nanoflash . Three shots each were made on 10 × 10 mmThe morphology of the fracture surfaces of the composites was examined using a Leo 1525  scanning electron microscope equipped with a field emission gun (FEG-SEM). An accelerating voltage of 5 kV was used, with the samples being sputter-coated with a thin layer of chromium prior to examination.Figure Figure The morphologies of the fracture surfaces of the composites are shown in Fig. It can also be seen in Fig. The thermo-mechanical properties of the in situ thermally reduced rGO/epoxy composites are shown in Fig. Eg, of the composites, measured at 35 °C, can be seen to increase with increasing rGO content; rising from 2640 MPa for the neat epoxy polymer to 2990 MPa in the epoxy composite filled with 2 wt% rGO , from which the Mnc were calculated according to Eq. (Mnc increases from 285 g/mol in the neat epoxy to 728 g/mol in the composite filled with 2 wt% rGO. Chen et al. (Mnc for the neat epoxy. The reaction between pendant OCFGs on the GO surface and the epoxy matrix in the liquid phase is well known. Yang et al. (Mnc increases and the glass transition temperature decreases, as the rGO concentration increases. Addition of GO has also been reported to change the molecular weight of polyurethane (Kim et al. From Fig. rve Fig. , the incrve Fig.  as the region at 80 °C, fregion at 80 °C, frn et al.  obtainedg et al.  has showg et al.  preparedFigure Figure Figure A facile, scalable and commercially viable method has been developed to prepare polymeric composites of a DGEBA epoxy resin with varying rGO content, having improved thermal conductivities. It consists of dispersing freeze-dried GO in a DGEBA epoxy resin using a three-roll mill and subsequently curing at a relatively high temperature with an anhydride curing agent, thereby both partially reducing the GO to rGO in situ and curing the epoxy matrix. The rGO/epoxy composites so produced exhibit a good dispersion of rGO, as observed by electron microscopy, which ensures significant improvements in the thermal conductivity, storage modulus and tensile modulus. Other properties are however slightly reduced, such as the thermal stability, glass transition temperature and tensile strength, probably due to oxygen groups remaining on the embedded rGO and an increase in the molecular weight between cross-links for the epoxy matrices. These results show that it is possible to tune the properties of an epoxy polymer with a simple and viable method of GO addition.