Posttraumatic stress disorder (PTSD) is a common psychiatric disorder that can occur following exposure to traumatic events. It is characterized by re-experiencing symptoms, avoidance, and persistent hyperarousal. While 7–8% of adults in the United States will experience PTSD over the course of their lifetime (Kessler et al., 2005; Roberts et al., 2011; Kilpatrick et al., 2013), rates are much higher among military veterans. The prevalence of PTSD in veterans returning from Iraq/Afghanistan is estimated to be 23% (Fulton et al., 2015), whereas an estimated 30% of Vietnam veterans have experienced lifetime PTSD (Kulka et al., 1990). Individuals with PTSD are at increased risk for many other comorbid conditions including major depressive disorder (MDD) (Cougle et al., 2010; Rytwinski et al., 2013; Flory and Yehuda, 2015; Ben Barnes et al., 2018), substance abuse disorder (SUD) (Petrakis et al., 2011; Seal et al., 2011; Blanco et al., 2013; Stein et al., 2017; Teeters et al., 2017), sleep disorders (Pigeon et al., 2013; Lind et al., 2020), and cardiovascular disease (Pollard et al., 2016; Koenen et al., 2017). Individuals with PTSD are also at increased risk for suicidal behaviors, particularly if they experience comorbid MDD (Oquendo et al., 2005; Panagioti et al., 2012; Kimbrel et al., 2016; Livingston et al., 2020; Ursano et al., 2020). For example, Kimbrel et al. (2016) found that among Iraq/Afghanistan-era war veterans, comorbid PTSD-depression was a robust prospective predictor of future suicide attempts over a 12-month period, even after controlling for the effects of sex, age, race, sexual orientation, and lifetime history of suicide attempts. Given the rate of death by suicide among veterans is approximately 1.5 times the rate among non-veteran adults (Department of Veterans Affairs, Office of Mental Health & Suicide Prevention, 2020¹), it is clear that an improved understanding of the etiology of PTSD and depression is of utmost importance.

While most people experience at least one traumatic event in their life, only some will subsequently develop PTSD (Benjet et al., 2016; Liu et al., 2017), suggesting that a heritable component to risk for PTSD exists. Based on family and twin studies, genetic susceptibility accounts for an estimated 30–70% of the variance in PTSD risk (True et al., 1993; Yehuda et al., 2001; Stein et al., 2002; Wolf et al., 2014). There is also evidence for a shared heritable influence on PTSD and MDD (Sartor et al., 2012). Utilizing over 20,000 individuals from 11 studies, the Psychiatric Genomics Consortium-Posttraumatic Stress Disorder group (PGC-PTSD) recently reported a single nucleotide polymorphism (SNP) heritability estimate of 29% for European-American females and found evidence for overlapping genetic risk between PTSD and schizophrenia, bipolar disorder, and MDD (Duncan et al., 2018). Even with large sample sizes, it has been difficult to identify robust regions of association with PTSD, illustrating the complex and multi-genic nature of the disorder. Recent GWAS reports from the PGC-PTSD and Million Veteran Program (MVP) have had greater success in identifying PTSD-associated loci when utilizing quantitative symptom severity and symptom subdomains instead of PTSD diagnosis (Maihofer et al., submitted; Stein et al., 2021). Nonetheless, the biological mechanisms underlying these largely non-coding regions of association have not yet been elucidated.

Gene expression profiling has been widely used to identify genes and pathways that are associated with specific biological processes and to study related molecular mechanisms. A recent transcriptome-wide study in prefrontal cortex (PFC) of post-mortem human brains identified co-regulated gene networks that are altered in PTSD, including differences between men and women which could help explain the increased prevalence of PTSD among women (Girgenti et al., 2021). However, due to the extremely limited availability of human brain tissue from PTSD-affected donors, the majority of studies have focused on gene expression in human peripheral blood samples. Although this is a limitation, evidence from both human and animal studies indicate it is likely that changes in the blood transcriptome reflect at least some changes occurring in the brain. For example, inflammation in the periphery has been associated with global brain transcriptome changes in mouse (Thomson et al., 2014). In humans, Huckins et al. (2020) showed that genetically regulated transcriptomic changes in the brain correlate with measured gene expression changes in peripheral blood.

Many previous gene expression studies of peripheral blood in PTSD patients and controls have reported that alterations in immune and inflammatory response pathways are important in PTSD pathogenesis, specifically the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid (GC) function (Segman et al., 2005). For example, FK506 binding protein 5 (FKBP5), a GC receptor inhibitor, is downregulated in PTSD cases (Yehuda et al., 2009). This finding, along with other genes in the GC receptor signaling pathway, has been widely replicated across various trauma types (Mehta et al., 2011, 2018; Sarapas et al., 2011; van Zuiden et al., 2012; Logue et al., 2015; Kuan et al., 2017). Others have reported differentially expressed genes in pathways enriched for innate immunity and inflammatory response (Zieker et al., 2007; Neylan et al., 2011; Breen et al., 2015, 2018; Guardado et al., 2016; Rusch et al., 2019). Additionally, decreased interleukin 1A (IL1A) expression was observed in the dorsolateral prefrontal cortex (dlPFC) of post-mortem PTSD brains (Morrison et al., 2019), a finding that is concordant with reports of altered cytokine expression in peripheral blood of PTSD patients (Zieker et al., 2007; Mehta et al., 2011; Breen et al., 2018).

Taken together, the findings described above suggest that altered transcripts in peripheral blood can still provide meaningful insights into the pathophysiology of PTSD. However, most of the studies to date have relied upon small sample sizes and did not include subjects from diverse ancestry groups. Accordingly, the objective of the current study was to conduct the largest meta-analysis of differential PTSD gene expression to date (n = 977), including 383 PTSD cases and 594 trauma-exposed controls from three multi-ethnic cohorts. Additional analyses aimed at uncovering genetic regulators of expression and modules of co-expression were performed in the largest cohort (n = 726). Finally, we sought to explain previously reported PTSD and MDD GWAS regions by alterations in gene expression.

A description of sample characteristics by cohort is shown in Table 1. Subjects from GMRF-QUT were significantly older, exclusively male, more likely to be non-smokers, and had a higher percentage of PTSD compared to those from MIRECC/Duke or INTRuST. The percentage of MDD in the MIRECC/Duke cohort was higher compared to INTRuST and GMRF-QUT. PTSD and MDD were significantly correlated in all three cohorts (p ≤ 0.0001) and Cramer’s V between PTSD and MDD was highest among INTRuST subjects (V_GMRF–QUT = 0.38, V_MIRECC/Duke = 0.43, V_INTRuST = 0.53). The MIRECC/Duke subset utilized in this analysis was comprised of 309 NHW and 417 NHB subjects. PCA plots of each cohort overlaid with HapMap3 samples show that the GMRF-QUT samples were primarily of European descent, whereas the INTRuST samples were multi-ethnic (Supplementary Figures 1–3).

Despite these differences in cohort characteristics, meta-analysis revealed 558 genes that were differentially expressed between PTSD cases and controls (p < 0.05, Supplementary Table 1). Inflation and bias were sufficiently controlled (λ_GC = 1.1). Meta-analysis p-values and effect sizes from the largest cohort (MIRECC/Duke) are depicted in Figure 1. For nominally significant probes (p < 0.05), the direction of effect is concordant 91% of the time for MIRECC/Duke and INTRuST, and is concordant 72% of the time for MIRECC/Duke and GMRF-QUT. Although not surpassing correction for multiple testing, the most significant genes were interleukin-1 beta (IL1B; p = 2.15 × 10^–5), and integrin-linked kinase (ILK; p = 1.10 × 10^–4). PTSD cases displayed higher expression of IL1B compared to controls, whereas ILK expression was lower in PTSD cases compared to controls. Pathway analysis of all nominally associated genes (p < 0.05) revealed an enrichment of genes involved in Toll-interleukin 1 resistance (SM00255:TIR; p = 7.01 × 10^–4), including TLR5, TLR6, TLR8, TLR10, IL1RAP, and IL18RAP, which represent seven unique expression probes that were all upregulated in PTSD cases.

Quantitative trait loci analysis identified many SNPs associated with expression levels: 2,216 in the NHW MIRECC/Duke subset and 1018 in the NHB subset (FDR q < 0.05). Subsequently, we performed an interaction analysis and identified several SNPs that were associated with gene expression at levels surpassing correction for multiple testing, but only in those with either PTSD or MDD. In the NHB subset, rs28842268 was associated with ILMN_1764090 in AK3L1 (AK4), but only among those with PTSD (p = 7.9 × 10^–6, q = 0.0473, Supplementary Figure 4A). Likewise, in the NHB subset, rs11823726 was associated with ILMN_1759789 in KAT5, but only among those with PTSD (p = 8.24 × 10^–6, q = 0.0473, Supplementary Figure 4B). In the NHW subset, we observed two eQTLs that were significant only in those with MDD. rs28536123 was associated with ILMN_2134888 in TUBE1 (p = 3.43 × 10^–9, q = 4.25 × 10^–5, Supplementary Figure 4C) and rs687562 was associated with ILMN_1872122 in LCOR (p = 1.63 × 10^–6, q = 0.0101, Supplementary Figure 4D), but only among those with MDD. These results should be viewed as preliminary due to small sample size and require replication in independent cohorts.

Next, Bayesian colocalization analysis was used to detect regions with high probability of a shared causal variant for PTSD or MDD and gene expression (Table 2). One eQTL in Ubiquitin Like Modifier Activating Enzyme 7 (UBA7; PP_H4 = 0.9604) colocalized with PTSD associated variants, whereas two eQTLs were found to colocalize with MDD associated variants: one in HLA-F (PP_H4 = 0.9844) and one in HSPA1B (PP_H4 = 0.9509). Several other regions displayed suggestive evidence for colocalization, including two separate eQTLs in RERE (PP_H4 = 0.8757 and 0.8488) with MDD associated variants. Interestingly, the eQTL in UBA7 deemed to colocalize with PTSD associated variants also showed suggestive evidence for colocalization with MDD associated variants (PP_H4 = 0.8178).

Weighted gene co-expression analysis in all MIRECC/Duke subjects identified 17 modules, none of which were associated with PTSD. When stratifying by ancestry, 16 independent co-expression modules were identified in the NHB subset and 18 were identified in the NHW subset. While none of the module eigengenes were associated with PTSD in the NHB subset, four modules significantly associated with PTSD status in the NHW subset (p ≤ 0.05, Figure 2). Two modules (MEtan and MEred) were upregulated in PTSD cases and two modules (MEpink and MEpurple) were downregulated in PTSD cases. MEpurple was also associated with PTSD severity as measured by total DTS score (p = 0.0356), and with current MDD status (p = 0.0007), an association that surpassed adjustment for multiple testing (Bonferroni adjustment; 18 tests; p = 0.0028). Additionally, MEred was also associated with MDD status (p = 0.0118) and MEtan was also associated with current smoking status (p = 0.041). These associations were in the same direction as were those for PTSD: MEpurple genes were downregulated in MDD and associated with decreasing total DTS score, MEred genes were upregulated in MDD, and MEtan genes were upregulated in smokers. Pathways significantly enriched in the PTSD-associated co-expression modules are listed in Table 3. The genes in MEtan showed significant enrichment for gene ontology (GO) terms involving plasma membrane/transmembrane, glycoprotein, and signal peptide (FDR q < 0.05), whereas genes in MEred were enriched for protein transport, alternative splicing, and phosphoprotein GO terms (FDR q < 0.05). MEpink genes were enriched for KEGG pathway term “metabolic pathways” (FDR q < 0.05) and MEpurple showed enrichment for actin binding, alternative splicing, and adaptive immunity GO terms (FDR q < 0.05).

This is the largest meta-analysis of differential PTSD gene expression to date. Results not only identified genes in pathways relevant to PTSD pathogenesis, but also showed PTSD and MDD GWAS signals may be driven by differences in gene expression. The most significant differentially expressed gene was interleukin-1ß (IL1B), a pro-inflammatory cytokine that is associated with innate immunity and has been extensively studied for its role in autoinflammatory diseases (Dinarello, 2011). Indeed, those with PTSD are at increased risk for autoimmune disorders including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, and multiple sclerosis (Ali et al., 2014; O’Donovan et al., 2015; Song et al., 2018; Bookwalter et al., 2020), and display elevated levels of pro-inflammatory cytokines, including interleukin-1ß (Spivak et al., 1997; Tursich et al., 2014; Passos et al., 2015). Moreover, inflammation and dysregulation of the immune system has been associated with other psychiatric disorders that frequently co-occur with PTSD such as MDD (Howren et al., 2009; Dowlati et al., 2010), schizophrenia (Miller et al., 2011), and alcohol use disorder (Crews et al., 2017; Patel et al., 2019). Animal studies have shown that mice subjected to a model of depression display increased levels of interleukin-1ß in the hippocampus (Goshen et al., 2008), a brain region integral to the neuropathology of PTSD due to its role in fear learning and contextual processing (Maren et al., 2013), as well as memory formation and retrieval (Bremner et al., 1997); in fact, a recent study in post-mortem human brain observed increased IL1B expression in dlPFC of PTSD cases compared to controls (Girgenti et al., 2021). The increase in IL1B expression observed among PTSD cases in the present research adds additional evidence to the growing body of work demonstrating widespread inflammatory response and dysregulation of the immune system in PTSD pathology.

We also detected reduced expression of ILK, the gene encoding ILK, a key scaffold protein that localizes to focal adhesions and is involved in the regulation of many cellular processes including cell growth, survival, adhesion, invasion, and migration. ILK is highly expressed in several brain regions including hippocampus, cerebellum, and frontal cortex (Mills et al., 2003; Xu et al., 2015) and is essential for neurite outgrowth (Mills et al., 2003), dendritogenesis (Naska et al., 2006), and survival signaling in hippocampal neurons (Gary et al., 2003). Although this gene has not previously been implicated in PTSD, indirect evidence points to ILK as a plausible candidate for further interrogation. In mouse, knockdown of brain-derived neurotrophic factor (BDNF), a gene associated not only with PTSD, but also MDD, bipolar disorder, and schizophrenia, results in dysregulation of hippocampal neurogenesis and memory deficits (BDNF+/-; Ernfors et al., 1994). Overexpression of ILK in the hippocampus of these mice rescues the hippocampal neurogenesis and memory deficits (Xu et al., 2015). Additionally, in a rat model of fetal alcohol spectrum disorder, alcohol-exposed pups displayed impaired contextual fear conditioning and memory performance, along with reduced hippocampal ILK activity (Bhattacharya et al., 2015). This pattern has also been observed in a mouse model for Alzheimer’s disease (AD), where ILK protein levels are significantly decreased in the hippocampus of APP/PS1 mice, which display perturbed neurogenesis and memory deficits, and can be rescued by overexpressing ILK (Xu et al., 2018). The direction of the effects in these animal studies is consistent with our observation that ILK expression is reduced in PTSD cases. Relatedly, several studies have reported a bi-directional association between PTSD and dementia (reviewed in Desmarais et al., 2020); those with PTSD are at increased risk for dementia and vice versa. More research is necessary to investigate the potential therapeutic value of ILK signaling pathway targets in PTSD, as have been suggested for AD (Li et al., 2012; Xu et al., 2018).

In addition to IL1B and ILK, pathway analysis of all gene expression probes nominally associated with PTSD revealed an enrichment of toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) genes that share the conserved toll/IL-1R homologous region (TIR) and are involved in innate antibacterial and antifungal immunity, consistent with previous reports from gene expression (Breen et al., 2015) and methylation studies (Uddin et al., 2010; Smith et al., 2011) in PTSD. TLRs play an important role in pathogen recognition and activate a signaling cascade which leads to upregulation of pro-inflammatory cytokines, including interleukin-1ß. Identification of not only IL1B, but also several TLR genes, demonstrates an increased burden of dysregulated innate immunity and inflammatory response genes among PTSD cases.

Correlation network analysis identified many modules of co-expression in the MIRECC/Duke cohort. Four modules identified in the NHW samples were associated with PTSD, two of which were also associated with MDD. Identification of coordinated expression associated with both PTSD and MDD confirms not only a strong correlation between the two phenotypes, but also shared biological pathways underlying the two conditions. We also identified eQTLs that were only significant in those with either PTSD or MDD, demonstrating genotype × environment interactions affecting gene expression. For instance, rs28842268 was associated with adenylate kinase 4 (AK4) expression, but only among PTSD cases; there was no effect among the control samples. AK4 localizes to the mitochondrial matrix and is involved in the cell survival response to oxidative stress (Liu et al., 2009; Kong et al., 2013), a molecular state that is activated by the persistent hyperarousal and fear experienced by those with PTSD (reviewed in Miller et al., 2018). Using a convergent functional genomics approach, Le-Niculescu et al. (2009) implicated AK4 in bipolar disorder, another neuropsychiatric condition which frequently co-occurs with PTSD (Cerimele et al., 2017). These data suggest there are genetic regulators of expression that are specific to the elevated stress and inflammatory state induced by PTSD.

Utilizing summary statistics from PTSD and MDD GWASs (Wray et al., 2018; Maihofer et al., submitted), we identified four genes (UBA7, HLA-F, HSPA1B, and RERE) with high probability of a shared causal eQTL variant with PTSD and/or MDD GWAS variants, thereby providing a potential mechanism by which the GWAS variant contributes to disease risk. UBA7 encodes an E1 ubiquitin-activating enzyme shown to be involved in STAT1-mediated interferon-α (INF-α) regulation of hippocampal neurogenesis and apoptosis (Borsini et al., 2018), processes triggered by a pro-inflammatory state. The UBA7 eQTL shares genetic effects with both PTSD and MDD GWAS variants, suggesting a potential pleiotropic mechanism at this locus. Recently, UBA7 was implicated in a transcriptome-wide association study (TWAS) of PTSD using brain tissue (Girgenti et al., 2021), confirming the association we observe in whole blood. The summary statistics utilized in Girgenti et al. were obtained from MVP (Stein et al., 2021) as opposed to the current study, which used summary statistics from PGC-PTSD (Maihofer et al., submitted), demonstrating an independent replication of the UBA7 finding. We also identified two eQTLs involving major histocompatibility complex (MHC) genes, HLA-F and HSPA1B, that displayed evidence for a shared genetic effect with MDD GWAS variants. The MHC group of genes encode proteins integral to immune response and variants in MHC genes have been associated with increased risk for many autoimmune diseases (reviewed in Matzaraki et al., 2017). As mentioned above, those with PTSD are at increased risk for autoimmune disorders and display increased levels of pro-inflammatory cytokines, including interleukin-1ß. The identification of multiple shared genetic effects of MHC gene expression and MDD risk increases the body of evidence implicating inflammatory and immune response pathways in both PTSD and MDD. Finally, two separate eQTLs for Arginine-Glutamic Acid Dipeptide Repeats (RERE) colocalize with MDD GWAS variants. Of note, a chromatin accessibility QTL (cQTL) in RERE identified in dlPFC of post-mortem human brains was shown to colocalize with schizophrenia GWAS variants (Bryois et al., 2018), another psychiatric disorder that co-occurs with PTSD.

The majority of these findings represent eQTLs from a PTSD-enriched dataset that colocalize with MDD GWAS variants. This further demonstrates not only the phenotypic comorbidity between PTSD and MDD, but identifies genomic regions of possible pleiotropy. Functional studies are necessary to further investigate these associations. Still, these results suggest biological mechanisms by which non-coding and common genetic variants may influence risk for PTSD and/or MDD.

While we have identified plausible novel candidates for PTSD, and confirmed many previously reported genes and pathways, this study is not without limitations. We acknowledge that the associations we observed in the differential expression analysis failed to achieve correction for multiple testing. While we have assembled the largest study of gene expression in PTSD to date, still larger sample sizes are required to reach an adequate power threshold to detect more modest differences. Despite this, IL1B has been repeatedly implicated in PTSD, therefore our replication of increased IL1B expression in PTSD cases is important. Because ILK has not previously been associated with PTSD, replication in independent datasets is necessary. Secondly, gene expression was measured in peripheral blood as opposed to brain tissue. We did attempt to impute genetically-regulated dlPFC expression in the NHW MIRECC/Duke subjects using a large eQTL reference panel (Huckins et al., 2019), but were only able to impute 6,928 genes with high confidence. Among the genes that were differentially expressed in both blood and imputed brain, nearly 70% showed a concordant direction of association with PTSD. The high percentage of concordance, as well as replication of pathways from the few brain-based studies available, lends credence to the hypothesis of at least partial overlap between blood and brain transcriptomes. Third, despite having compiled a large veteran cohort with respect to gene expression, it is difficult to identify robust interactions, particularly when SNPs involved have low minor allele frequency (MAF). As such, the eQTL interactions we have identified, particularly those involving SNPs with lower MAF, should be viewed as preliminary and need to be replicated in larger studies. Also, we did not investigate sex-specific associations due to the relatively small number of female samples available in the current study. Ascertainment of female veterans should be prioritized to facilitate research of possible sex-specific genetic factors influencing PTSD risk. Finally, gene expression in this study was determined using microarrays and not RNA-sequencing. Future studies that interrogate gene expression using sequencing approaches would be beneficial to obtain a truly global depiction of the transcriptome.

This is the largest study of differential gene expression between PTSD cases and controls to date, and these analyses have identified many logical candidate genes and pathways for further research. Notably, while we did not replicate the FKBP5 finding (p > 0.05), the present findings provide additional support for the role of IL1B and other inflammatory response and innate immunity pathway genes in PTSD, and have identified ILK as a novel candidate. Further, we identified modules of co-expressed genes that were associated with both PTSD and MDD, suggesting common biological pathways underlie the two comorbid disorders. Finally, we identified changes in gene expression that may help explain several PTSD and MDD GWAS signals. These data provide further insight into genetic factors and pathways associated with PTSD, as well as potential regions of pleiotropy between PTSD and MDD.

Mira Brancu, Ph.D., Patrick S. Calhoun, Ph.D., Eric Dedert, Ph.D., Eric B. Elbogen, Ph.D., John A. Fairbank, Ph.D., Robin A. Hurley, M.D., Jason D. Kilts, Ph.D., Angela Kirby, M.S., Sara Martindale, Ph.D., Scott D. McDonald, Ph.D., Scott D. Moore, M.D., Ph.D., Rajendra A. Morey, M.D., M.S., Jennifer C. Naylor, Ph.D., Jared Rowland, Ph.D., Robert Shura, Ph.D., Cindy Swinkels, Ph.D., Steven T. Szabo, M.D., Ph.D., Katherine H. Taber, Ph.D., Larry A. Tupler, Ph.D., and Ruth E. Yoash-Gantz, Psy.D.

Sarah McLeay, BSc(Hons), Ph.D., Wendy Harvey, BSc(Hons), MBBS, MPH, Madeline Romaniuk, BA, GradDipPsych, BBehSc(Hons), DPsych(Clinical), Darrell Crawford, MBBS, FRACP, M.D., David Colquhoun, MBBS, FRACP, Ross McD Young, Ph.D., Miriam Dwyer, BSc, HDipEd, John Gibson, MBBS, FRANZCP, Robyn O’Sullivan, MBBS, FRACP, Graham Cooksley, MBBS, M.D., FRACP, Christopher Strakosch, M.D., FRACP, Rachel Thomson, MBBS, GradDipClinEpi, Ph.D., FRACP, Joanne Voisey, BSc(Hons), Ph.D., Bruce Lawford, MBBS, FRANZCP, FAChAM (RACP).

The datasets presented in this article are not readily available because the institutional review board (IRB) protocols governing these datasets do not allow sharing of individual level data. Requests to access the datasets should be directed to the senior author, AA-K at allison.ashleykoch@duke.edu.

The studies involving human participants were reviewed and approved by the Durham (NC) VA Medical Center IRB, McGuire (Richmond, VA) VA Medical Center IRB, Hampton (VA) VA Medical Center IRB, W.G. (Bill) Hefner (Salisbury, NC) VA Medical Center IRB, Greenslopes Research and Ethics Committee, Queensland University of Technology Human Research Committee, San Diego (CA) VA Medical Center IRB, Togus (ME) VA Medical Center IRB, Cincinnati (OH) VA Medical Center IRB, Puget Sound (WA) VA Medical Center IRB, Dartmouth College (NH) IRB, University of California San Diego (CA) IRB, and Human Research Protections Office of the Department of Defense. The patients/participants provided their written informed consent to participate in this study.

AA-K, MH, and MG contributed to the design of the study. DM, MD, CM, GG, MS, NK, JB, MH, and AA-K assisted in the data collection, generation, and preparation. MG, XQ, and DM performed the analyses. MG prepared the first draft of the manuscript. AA-K, MH, NK, and JB contributed to the comments and edits during manuscript drafting. All authors reviewed the manuscript, provided feedback, and approved the manuscript for submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.