The approval of immunotherapy for stage II-IV melanoma has underscored the need for improved immune-based predictive and prognostic biomarkers. For resectable stage II-III patients, adjuvant immunotherapy has proven clinical benefit, yet many patients experience significant adverse events and may not require therapy. In the metastatic setting, single agent immunotherapy cures many patients but, in some cases, more intensive combination therapies against specific molecular targets are required. Therefore, the establishment of additional biomarkers to determine a patient’s disease outcome  or response to treatment  is of utmost importance. Multiple methods ranging from gene expression profiling of bulk tissue, to spatial transcriptomics of single cells and artificial intelligence-based image analysis have been utilized to better characterize the immune microenvironment in melanoma to provide novel predictive and prognostic biomarkers. In this review, we will highlight the different techniques currently under investigation for the detection of prognostic and predictive immune biomarkers in melanoma. The yearly incidence of skin cancer in the United States is greater than all other types of cancer combined . Among sThe development of immunotherapy has served as a pivotal turning point in the treatment of many cancers and melanoma in particular . SpecifiBRAF V600-mutant metastatic melanoma , these therapies have also been associated with multiple adverse events including elevated liver enzymes, rash, pruritus and fatigue, among many others. Grade 3 or 4 adverse events have also been commonly reported, oftentimes in over 50% of melanoma patients treated in clinical trials with ICI . Given tBiomarkers are measurable biological indicators that can be subdivided into two main groups: prognostic and predictive . While pTraditionally, the major clinical and histological prognostic biomarkers of melanoma have included primary tumor  thickness, ulceration, mitotic rate, anatomic site  and sentinel lymph node (SLN) involvement . These fRegarding the N category, patients with “clinically occult” nodal metastasis  have been shown to have better survival than patients with clinically or radiographically evident disease. Thus, SLN biopsies are often performed in these patients as nodal status is an important independent predictor of prognosis . A positAlthough prognostic biomarkers have been well-established for melanoma, predictive biomarkers are not yet routinely used for ICI in melanoma due to limited clinical utility or low sensitivity and specificity . HoweverBRAF are most common, with reported frequencies of over 60% (V600E) accounts for over 90% of mutations within that locus  such as dabrafenib and trametinib have been shown to improve progression-free survival (PFS) and OS in patients with these mutations . However therapy . Additioe to ICI . Additio mutants . Nonetheelanomas .Circulating tumor DNA (ctDNA) has also emerged as a promising prognostic and predictive blood-based biomarker to monitor disease status in advanced melanoma patients . MelanomImmune surveillance has been shown to have potential value in prognostication for many solid cancers . Thus, tTo address this need, Nanostring transcriptomic technology, which analyzes the expression of multiple transcripts under varying pathological or physiological states, has been used to profile a group of 446 immune-associated candidate genes in primary melanoma. A study in 2014 found that 53 out of the 446 screened genes predicted non-progression, disease-specific survival (DSS) and prolonged recurrence-free survival (RFS) in two independent cohorts of patients with resectable stage II-III melanoma . This 53Single-cell RNA-sequencing (scRNA-seq) analysis is a valuable method for obtaining gene expression profiles of individual cells which helps to identify different cell types and pathways involved in cancer progression and resistance . HoweverA recent scRNA-seq study found that PRRT3-AS1, an important long non-coding RNA (lncRNA) that has been incorporated in prognostic models for prostate cancer, hepatocellular carcinoma and glioblastoma (GBM), may be required for tumor cell migration in melanoma, suggesting that PRRT3-AS1 is not only a potential prognostic biomarker but also a potential therapeutic target . AdditioCXCL13+ CD4+ T cells and CXCL13 expression broadly correlates with OS in a cohort of melanoma patients, independent of immunotherapy type . Studies have shown that the presence of apy type . A TEX-rapy type .+ density predicts response to ICI in the metastatic setting  cell abundance and TCR diversity—constitute promising biomarkers of ICI-induced immune-related adverse events (irAEs) in metastatic melanoma patients. Additionally, the authors identified a notable correlation between early T cell clonal expansion and the onset of severe irAEs in patients treated with combination ICI  to elaborate additional predictive information in melanoma, which has recently been shown to be a feasible technique in both fresh and frozen tissue . This pation ICI . A subsetion ICI . These sTypical approaches to immunohistochemistry (IHC) evaluation of tissue from melanoma patients have several limitations including inter-observer variability and the labeling of just a single biomarker for each tissue section. Emerging techniques, namely, multiplex IHC or immunofluorescence (mIHC/IF), have attempted to address these limitations by detecting multiple biomarkers in a single tissue section through high-throughput staining and quantitative analysis . This teTILs, a major component of the TME, have been implicated in the prevention or progression of tumor growth and invasion leading to significant interest in TILs as a potential prognostic biomarker . ConventRegarding predicting response to ICI, a multiplex chromogenic and IF study of melanoma samples showed that proximity between PD-1 and PD-L1+ cells was associated with response to anti-PD-1 therapy. Similarly, high co-localization of PD-L1 and CD8 expression was associated with increased response to targeted immunotherapy . In anotin situ on pretreatment melanoma specimens from advanced melanoma patients on ICI. In this study, higher density of early effector T cells (CD8+FoxP3+) correlated with response to anti-PD-1 therapy whereas the CD163+PD-L1- myeloid phenotype was associated with lack of response to PD-1 blockade  to melanoma cells was associated with positive response to ICI  with multiplex IF demonstrated that PD-L1 expression in macrophages but not tumor cells was a predictive marker for PFS, OS, and treatment response. Further, specific immune markers associated with PFS and OS, respectively . Cytomete to ICI . In the e to ICI .While AI, a set of sophisticated algorithms and highly advanced machine learning tools to simulate some aspects of human intelligence, has greatly expanded its reach across all of medicine, it has demonstrated new potential horizons for melanoma biomarker development.Machine learning, a subset of AI that involves computers improving performance from learned experience and pattern recognition, has been leveraged as an important tool for the identification of prognostic biomarkers. A study in 2022 utilized a machine learning classifier that accounted for multiple variables of TILs including cell type  and area of interest  to validate the prognostic value of TILs for potential pathologist-independent use in future clinical trials. In this study, machine learning found that automated TIL score is prognostic in clinically-localized primary melanoma and may assist in isolating a subgroup of stage II patients with high recurrence risk. This will ultimately enable identification of patients who would likely benefit from adjuvant therapy . In anotGSTA3 and VNN2, were important features in predicting ICI response  signatures have also stratified melanoma patients into low and high risk groups based on level of immune surveillance, which can further guide precision ICI. Subsetting these signatures based on single cell data may allow for improved accuracy.Currently, standard clinical care algorithms utilize TNM staging for prognosis. IHC has been able to elucidate many prognostic and predictive biomarkers including MART1/Ki-67, preferentially expressed antigen of melanoma (PRAME), makers of lymphovascular invasion  and mismatch repair (MMR) proteins, among many others . HoweverAdditional technologies that have elucidated cellular interactions in the TME include the use of multiplexed, quantitative IHC which has allowed for the analysis of multiple cellular phenotypes at a time, in addition to assessing proximity of individual cells to each other. In particular, multiplexing has better characterized TILs and their role in the TME.AI-based analyses have also expanded biomarker discovery in melanoma. By simulating some aspects of human intelligence in a sophisticated and automated platform, these tools have the propensity to decrease inter-observer variability and error in order to more reliably quantify biomarker presence in patient samples based on integrated clinical and histopathologic data along with image analysis.In the prognostic setting, the GEP test that classifies melanoma patients as Class 1 (low-risk) or Class 2 (high risk) for recurrence or metastasis is commercially available, which may allow clinicians to modify screening intervals and treatment regimens depending on a patient’s individual disease risk. However, studies have assessed the performance of this tool and while this GEP test generally identifies recurrence in patients with stage II disease, correctly identifying recurrence in stage I patients is poor, limiting its clinical utility . Thus, sIn the predictive setting, therapeutically targeting PD-1 and CTLA-4 correlate with clinical benefit. However, given intratumoral heterogeneity and limited ICI options, these markers are insufficient to capture the nature of all patient tumors. Additional biomarkers that have been well-explored such as the TMB and inflammatory mediators may soon be utilized in clinical settings. Newly discovered biomarkers such as antigen experienced cytotoxic T cells are likely to require additional evaluation, although preliminary data shows promise for predicting ICI treatment response.While biomarkers may serve as independent prognostic or predictive indicators, a single biomarker is usually inadequate to precisely stratify patients. Thus, multimodal investigation of biomarkers using a combination of the techniques described while also prioritizing sensitivity, specificity and cost will be important for timely assessment of future patient risk and response.


Inter-relationships between pain sensitivity, drug reward, and drug misuse are of considerable interest given that many analgesics exhibit misuse potential. Here we studied rats as they underwent a series of pain- and reward-related tests: cutaneous thermal reflex pain, induction and extinction of conditioned place preference to oxycodone (0.56 mg/kg), and finally the impact of neuropathic pain on reflex pain and reinstatement of conditioned place preference. Oxycodone induced a significant conditioned place preference that was extinguished throughout repeated testing. Correlations identified of particular interest included an association between reflex pain and oxycodone-induced behavioral sensitization, and between rates of behavioral sensitization and extinction of conditioned place preference. Multidimensional scaling analysis followed by k-clustering identified three clusters: (1) reflex pain and the rate of change in reflex pain response throughout repeated testing, (2) basal locomotion, locomotor habituation, and acute oxycodone-stimulated locomotion, and (3) behavioral sensitization, strength of conditioned place preference, and rate of extinction. Nerve constriction injury markedly enhanced reflex pain but did not reinstate conditioned place preference. These results support the notion that behavioral sensitization relates to the acquisition and extinction of oxycodone seeking/reward, but suggest that generally cutaneous thermal reflex pain poorly predicts oxycodone reward-related behaviors except for behavioral sensitization. Practically, much of this interest is driven by the goal of understanding how individual differences in pain sensitivity impact vulnerability to substance use disorders 7. Evidence suggests the experience of pain is comorbid with various mood and anxiety disorders 10, and of particular interest to the current study, individuals more sensitive to pain are suggested to be more likely to develop opioid use disorders 13.There is considerable interest in the fundamental mechanisms of pain and pleasure, especially the degree to which underlying neural systems overlap and mutually impact these two psychological domains 14. Among these, the synthetic opioid oxycodone (OXY) is particularly concerning as it ranks highly among misused substances 17. Preclinical studies agree that OXY exhibits many of the characteristics of an addictive substance. For instance, animal studies show that OXY exposure induces conditioned place preference 23, is readily intravenously self-administered 28, induces behavioral sensitization 29 and in some, but not all studies, OXY solutions are preferred over water via oral consumption 32. Moreover, OXY exhibits the hallmark action of stimulating mesolimbic dopamine release 33.Despite significant concern over their abuse liability, opioids remain common for managing severe pain 35. Here we performed an ethogramic analysis of relationships between reflex cutaneous thermal pain and OXY-related drug-seeking behavior. We found that locomotor and locomotor habituation measures were generally positively related, and that rates of behavioral sensitization and extinction were associated with the strength of CPP. We found a strong positive correlation between thermal pain sensitivity and behavioral sensitization to OXY, but little other evidence of any relationship between individual differences in sensitivity to thermal pain and drug-seeking behavior. Contrary to expectation, induction of a chronic pain state through chronic nerve constriction (CCI) did not reinstate extinguished OXY-induced CPP.Generally, the relationship between individual differences in pain sensitivity and subsequent vulnerability to the misuse-related properties of opioids, especially OXY, has been little studied. Here we capitalized on the ability of the conditioned place preference (CPP) paradigm to provide multiple measures related to abuse liability to draw relationships between thermal pain sensitivity in drug-naïve animals and behavioral measures relevant to vulnerability to OXY misuse. That is, in addition to assaying the rewarding properties of drugs, the CPP paradigm delivers useful information on the psychomotor activating and behavioral sensitizing effects of drugs of abuse. Additionally, through extended testing, the CPP paradigm can model extinction of drug-seeking behavior. Finally, the CPP paradigm offers insight into some of the processes that reinstate drug-seeking behavior, a.k.a. relapse. For instance, drug re-exposure and stress readily reinstate drug-seeking behaviors in CPP The study was performed in five major phases, as depicted in Fifteen Sprague-Dawley rats  were entered the study, which was reduced to 13  following sham/CCI due to complications with surgery. To reduce the impact of differences in body weight between the sexes, all animals were delivered from the supplier  to the University of Florida vivarium weighing between 200 and 225 g. Mean (± standard deviation) animal weight on day 13 of the study was 249.4 ± 38.6 g. Animals were pair-housed by sex, provided with standard laboratory chow and water ad libitum, and maintained on a 12:12-hour light/dark cycle. Behavioral testing procedures took place approximately 2 to 8 hours into the light phase. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee. As males and female animals differed in age, data are presented pooled across the sexes. OXY hydrochloride was prepared by dissolving in phosphate buffered saline vehicle (VEH) at a concentration of 0.56 mg/ml, corrected for salt content and administered intraperitoneally (i.p.).Animals were acclimated to a thermal hindpaw reflex testing apparatus  for at least 10 minutes. A radiant heat lamp (intensity set at 45) was targeted on the left or right hind paw (random order) until the paw was withdrawn, using a cutoff time of 20 seconds to prevent tissue damage. Two or more paw withdrawal latency measures (minimum 5 s each) were taken for each paw, from which the two numerically closest values were averaged.3 outer conditioning compartments separated by a 12 × 21 × 21 cm3 center compartment. One outer compartment had black walls on which large (11 cm) white stars were superimposed. This compartment also had a floor made from 4.8 mm stainless steel rods, placed at 16 mm intervals. The other compartment had white walls on which large black circles (9 cm) were superimposed. This compartment also had a 1.25 × 1.25 cm2 stainless steel mesh floor. The center compartment had gray walls and a smooth polyvinyl chloride floor. Animal position and locomotion were recorded using photobeam breaks. Before conditioning, a 20 min initial preference test  was performed in which animals could freely explore all three compartments, and the amount of time spent in each was recorded. On days 13–16, animals underwent 30 min conditioning sessions in their assigned compartments. OXY was administered in one compartment and VEH was administered in the other. Approximately equal numbers of animals were assigned to each compartment in a pseudorandom counterbalanced fashion based on their Pre-test times, which ensured no statistical difference between the mean Pre-test time of each group between the compartments. Injections were given immediately before placement in the compartments. To minimize order effects, animals were counterbalanced such that during morning conditioning, half received OXY and the other half received VEH, with the opposite respective conditioning treatment occurring approximately 4 hours later. Ensuring a fully unbiased conditioning protocol obviated the need of an additional group of animals that received VEH in both conditioning compartments to demonstrate acquisition of OXY-induced CPP. Horizontal locomotor activity in response to VEH and OXY administration was recorded as beam break counts during conditioning sessions. The day following conditioning (day 17), and periodically thereafter , animals were tested for the expression of CPP in the same manner as the Pre-test. On day 66, which occurred after CCI or sham surgery, animals were re-tested a final time to ascertain if a chronic pain state reinstated expression of CPP.Place conditioning studies were performed in a sex-counterbalanced order in a three-compartment place conditioning apparatus . The apparatus consisted of two 28 × 21 × 21 cm2). The right hind leg was shaved and sterilized with chlorohexidine and 70% ethanol. A 5 to 7 mm incision was made in the skin below the femur and the skin separated from the muscle and connective tissue using blunt forceps. An incision was made through femoris muscles, and the sciatic nerve freed using a glass pipette with a blunt curved tip. Three ligatures were made using a double knot 1 mm apart, tightened until the loop was just snug and the ligatures were unable to slide along the nerve. Silk sutures  were used to close the muscle layer and skin, and the wound was cleaned with chlorohexidine before application of triple antibiotic ointment. Following surgery, animals were variably housed with cagemates that had undergone the same or other surgery.Chromic gut suture  was cut into 2 cm pieces and immersed in sterile saline to prevent drying. Surgery was performed under aseptic conditions on a T-pump heating pad. Animals were administered buprenorphine (SR)b (1.0 mg/kg s.c.) before being maintained under general anesthesia  for each animal. The rate of decrease in withdrawal latencies (termed “Rate of change in reflex pain”) was calculated by fitting a linear regression, with the slope being inverted such that a higher positive value indicated greater rates of decreases.The main outcome measures of interest  and their method of calculation are detailed in Basal loco”). The rate of locomotor habituation (termed “Rate of loco habituation”) during conditioning was calculated by fitting a linear regression to the locomotor activity following the four VEH administrations during conditioning on days 13 to 16, with the slope being inverted such that a higher positive value indicated faster rates of habituation. Of all the outcomes measured, only this measure presented a non-normal distribution. Consequently, values were transformed by taking the square root of the initial values to yield a dataset fitting a normal distribution, thereby allowing consistent use of parametric analysis methods across all measures.Locomotor responses following VEH administration in the first conditioning session were taken as a measure of basal locomotion (termed “Acute OXY loco”). Rates of behavioral sensitization  were calculated by first normalizing locomotor responses to OXY to same-day locomotion following VEH administration during conditioning sessions by subtracting the latter from former and then by fitting a linear regression curve to yield a slope reflecting the rate of change of locomotion over the four conditioning sessions.Locomotor responses following OXY administration in the first OXY conditioning session were normalized to locomotor responses following VEH administration during the first VEH conditioning session by subtracting locomotor counts to yield a measure of acute OXY-stimulated locomotion (termed “CPP at first test”. Rates of extinction of CPP (termed “Rate of CPP extinction”) were estimated by calculating the slope of a fitted linear regression across all post-conditioning CPP sessions  before sham/CCI surgery. The value of the slope was inverted such that a higher positive value indicated faster rates of extinction.CPP following conditioning was calculated by subtracting time spent in the OXY-paired compartment before conditioning , from times spent in the OXY-paired compartment after conditioning . The strength of OXY-induced CPP immediately following conditioning (day 17) was termed “Reflex pain – surg. paw”), relative sensitivity of the surgeried paw to the unsurgeried paw (termed “Reflex pain - surg. paw vs. non-surg. paw”) and change in sensitivity of the surgeried paw relative to the first thermal hindpaw test (day 6) taken before sham/CCI surgery (termed “Reflex pain − Δ surg. paw”).The impact of sham/CCI surgery on paw withdrawal latencies in the thermal hindpaw test was averaged over three tests, which generated three measures detailed in CPP - Δ pre-test CPP”), relative to the CPP expressed immediately following conditioning , and, relative to the CPP expressed immediately before sham/CCI surgery .The impact of sham/CCI surgery on expression of extinguished OXY-induced CPP was assessed four- or five-days following sham/CCI surgery, counterbalanced across surgery treatment groups. The strength of CPP was expressed in three different ways detailed in 36). Details of individual statistical tests and the rationale for their application are detailed where appropriate. Probability (p) values represent two-tailed testing and are presented uncorrected throughout.Data are expressed as mean ± standard error of the mean unless otherwise noted and analyzed using parametric statistical methods based on normality testing of individual measures using a Shapiro-Wilk normality test  over the four days of testing before induction of CPP from an initial mean of 18.7 ± 0.3s to a final mean of 16.7 ± 0.6 s  yielded a significant difference .Repeated measures ANOVA showed paw withdrawal latencies decreased  decreased over the four days of testing from a mean of 675.3 ± 56.2 counts to 427.1 ± 39.0 counts, suggestive of locomotor habitation , which, when tested against a hypothesized mean of 0, and confirmed a statistically significant  reduction in locomotor activity following VEH administrations across the whole group of animals.Repeated measures ANOVA showed that locomotion in VEH conditioning sessions progressively and significantly  greater (65.4%) locomotion than following VEH administration on the same day , yielded a significant difference  indicating behavioral sensitization, though when tested by repeated measures ANOVA over the four administrations of OXY, normalized OXY-stimulated locomotion narrowly missed reaching statistical significance .OXY administration on the first day of conditioning induced significantly  and was 393.4 s . Groupwise, animals showed no initial bias for either of the two prospective conditioning chambers . Three days after the pre-test (days 13 to 16), animals were assigned to receive OXY in one of the two conditioning compartments  in a counterbalanced fashion such that no statistical difference in Pre-test times existed between the two assignment groups . 7,98 = 4.872, p < 0.0001) generally reflecting acquisition and extinction of OXY-induced CPP. A within subjects paired t-test showed a statistically significant  increase in time spent in the OXY-paired compartment during CPP at first test compared to Pre-test. A within-subjects paired t-test comparing time spent in the OXY-paired compartment between CPP at first test and the final test of CPP before sham/CCI surgery showed a significant  reduction in CPP, reflecting extinction of CPP. This was further reflected in a mean Rate of extinction slope of 4.4 ± 0.9 s/day , which tested against a hypothesized mean of 0  confirmed a statistically significant  extinction across the whole group of animals. Notably by the final test (day 51) before sham/CCI surgery, animals showed an apparent aversion to the OXY-paired compartment such that CPP scores were significantly lower than zero . Additional analysis showed that this apparent aversion may have been partially driven by progressive increases in the amount of time spent by animals in the center/grey compartment of the CPP apparatus (The average time in the compartment destined for pairing with OXY at pre-test (day 10) exhibited a normal distribution (Wpparatus .cor) between all major outcome measures taken prior to sham/CCI surgery. rcorr$P) arranged by angular order of the eigenvectors such that closely correlated variables are proximate in the matrix. Among all pairwise correlations, four had uncorrected p values lower than 0.05, these being Basal loco and Rate of loco habituation (positive correlation), Rate of behavioral sensitization and Rate of CPP extinction (positive correlation), Rate of change in reflex pain and Basal loco (negative correlation), and, Basal reflex pain and Rate of behavioral sensitization (negative correlation).A correlational analysis was performed by generating Pearson’s correlation coefficients  Basal loco, Rate of loco habituation and Acute OXY loco into a second cluster, (3) Rate of behavioral sensitization, CPP at first test and Rate of CPP extinction into a third cluster. These results suggest that thermal pain sensitivity, locomotion and OXY-related responses in the CPP apparatus represent distinct groups of responses, with the exception that acute locomotor responses to OXY more closely relate to locomotor behavior in general.To elucidate general relationships between measures, Pearson’s correlation coefficients were subjected to a classical multiple dimensional scaling analysis using the R function CPP − Δ post-cond. CPP measure in sham treated animals , all measures taken after sham/CCI surgery were normally distributed  when split into treatment groups. Considered across both groups, all measures were normally distributed . 11 = 1.837, p = 0.0933) in the CPP − Δ pre-test CPP measure. Notably for both treatment groups, this measure was negative, which when tested against 0 (one-sample t-test), yielded a statistically significant aversion to the OXY-paired compartment in the CCI surgeried animals  but not in sham treated animals  suggesting CCI surgery produced a stronger aversion to the OXY-paired compartment relative to pre-test than in sham surgeried animals.Except for the Reflex pain - surg. paw vs. non-surg. paw and CPP − Δ pre-test CPP showed a positive correlation that passed a p threshold of 0.05 (p = 0.018), indicating that those animals with strongest differences in withdrawal latencies between paws in the thermal hindpaw test following sham/CCI surgery showed the strongest avoidance of the OXY-paired compartment relative to pre-test. The R package NbClust (set to suggest between 2 and 4 clusters) suggested four clusters as optimal for K-clustering of the multidimensional scaling analysis, the results of which are shown in Here we found that OXY induced CPP, which extinguished during repeated testing. Cutaneous thermal reflex pain poorly predicted OXY-seeking-related behaviors, and chronic pain did not reinstate extinguished CPP. Nociceptive measures clustered together, acute locomotor measures clustered together, and CPP-related measures clustered together, suggesting that thermal pain reactivity is not strongly predictive of OXY reward.41. Notably, the dose used here was chosen for clinical translatability, corresponding to a 6.3 mg dose in a 70 kg human 42. This falls in the recommended initial prescribing range of 2.5 to 10 mg for chronic pain 43. Though expression of CPP extinguished throughout repeated testing, our study was not designed to address if this was a passive event, or due to repeated re-exposure to the CPP apparatus. Our previous studies in mice suggest the latter, however, in that, left undisturbed, morphine-induced CPP tends to incubate rather than extinguish 44.Conditioning with 0.56 mg/kg of OXY produced a significant and persistent CPP response. This is consistent with studies showing that 0.3 mg/kg of OXY is sufficient to generate CPP in rats, though most studies use higher doses 45. However, others have pointed out that animals often show biphasic dose-response relationships and as such, consideration of dose is critical to the validity of the theory 46. Relatedly, we find that acute OXY administration produces progressive increases in locomotion between 0.178 to 1 mg/kg in the same strain of rat studied here 47.We noted that the locomotor response to initial OXY administration did not correlate with CPP measured immediately following conditioning. This finding is somewhat at odds with the psychomotor stimulant theory of addiction, which theorizes that the ability of drugs to stimulate horizontal locomotion predicts their misuse potential 48. This theory suggests that repeated exposure to misused drugs produces incremental neuroadaptations that render individuals increasingly “sensitized” to drugs and associated stimuli. One of the outcomes of this is progressively increasing locomotor responses, i.e., locomotor behavioral sensitization. By extension, rates and degrees of behavioral sensitization to a particular stimulus, e.g., drugs, can be predictive of the misuse potential of that stimulus 49. We previously showed that behavioral sensitization to morphine predicts the strength of CPP in mice 50. Here, we found only a weak  albeit positive correlation between these two outcomes. Nonetheless, multidimensional scaling analysis found that the rate of behavioral sensitization clusters with the strength of OXY-induced CPP, supporting the postulate that behavioral sensitization reflects a process related to the motivating or rewarding effects of drugs.An alternative theoretical account designed to explain drug misuse is incentive-sensitization theory 51. Relatedly, there has been considerable interest in the relationship between responses to novelty and drug misuse liability 54. Based on the rationale that initial locomotor responses to VEH injection in the CPP apparatus reflect locomotion driven by novelty, one might expect that this response would predict the strength of expression CPP following OXY conditioning. However, there was in fact a weak negative correlation  between these measures.Drawing together psychomotor stimulant and incentive sensitization theory, we found little direct evidence to suggest a relationship between these two constructs, in that acute locomotor responses to OXY barely correlated with rates of behavioral sensitization. Again, however, species, strain, sex, dose and a range of other factors need to be considered in the interpretation of this negative finding. For instance, previous studies find that acute locomotor responses to cocaine across various rat strains are a poor predictor of behavioral sensitization 55. Others calculate CPP scores by subtracting time in the VEH-paired compartment from time in the drug-paired compartment 56. By this latter method of calculation, CPP at the end of repeated testing was not statistically significant different from zero . This difference draws attention to the impact of differences in calculation methods on the conclusions drawn. As far as we are aware, this issue has not been thoroughly discussed, though others have pointed out theoretical differences between two and three compartment CPP apparatuses 57.A particularly interesting finding of the study is that the rate of extinction of OXY-induced CPP correlated strongly with the rate of behavioral sensitization. Loosely interpreted, this suggests that animals undergoing the most profound adaptations driving drug-seeking behavior more readily experience reversal of those adaptations. It is noteworthy that we found a weak positive correlation  between the strength of the initially expressed CPP and rate of extinction. There is the possibility that higher rates of extinction may relate to the stronger initial CPP, providing a greater dynamic range that allows steeper extinction rates. Notably, by the end of extinction testing, animals as a group had developed a significant negative CPP, i.e., apparent avoidance. Given the opportunity to express such avoidance, one could reason that the strength of the initial CPP did not limit the rate of extinction. However, scrutiny of the data in prior to developing drug-seeking behavior and substance misuse. An unexpected finding of the current study is the lack of relationship between individual sensitivity to thermal reflex pain and behavioral measures acquired during the CPP . There was, however, one notable exception - there was a strong negative correlation  between the initial nociceptive response and development of behavioral sensitization. That is, animals most sensitive to cutaneous thermal pain exhibited the most robust behavioral sensitization. This finding agrees with the view that individuals inherently sensitive to pain may be at higher risk of undergoing drug-induced neuroadaptations underlying drug misuse. However, this interpretation should be viewed cautiously, as multidimensional scaling analysis did not suggest a strong association between reflex pain measures and measures derived in the CPP apparatus. It must also be borne in mind that pain is a multi-dimensional phenomenon, presenting across many modalities, such as nociceptive , inflammatory, neuroplastic and neuropathic pain 58. Here, we studied only the relationship between one of these – cutaneous thermal pain – and measures related to drug misuse.Though there are many human studies on vulnerability to substance use in pain sufferers, little is known about the relationship between individual differences in pain sensitivity 60. Regardless, various stimuli can reinstate extinguished CPP, notable among these being stress 61. A particularly common method of demonstrating stress-induced reinstatement is use of foot shock, which is an inherently acute painful stimulus. As far as we are aware, few studies have investigated the effect of chronic pain on reinstatement of CPP, though operant self-administration studies indicate that analgesics are more reinforcing in animals in chronic pain 64. Here we used an established model of chronic neuropathic pain, CCI, to study the impact of chronic pain on reinstatement of drug-seeking behavior. The impact of CCI on nociceptive responses was clear from its ability to significantly reduce paw withdrawal latencies; however, CCI did not appear to affect reinstatement of extinguished CPP. In fact, CCI operated animals tended to show greater avoidance of the OXY-paired compartment, a finding that concords somewhat with studies showing chronic pain can reduce the reinforcing and rewarding impact of misused drugs (reviewed in 66) and does not reinstate opioid seeking behavior 67. Relatedly, nerve ligation suppresses development of morphine CPP in mice 68. On face value, this finding contradicts a prediction that animals in most pain would seek out OXY to a greater extent. However, it is important to note that animals were not conditioned to OXY in a pain state, and as such are unlikely to associate the OXY-paired compartment with pain relief. Previous studies show that CPP to morphine is enhanced in animals experiencing a chronic pain state during conditioning 69. However, studies pairing noxious stimuli with opioid self-administration show that exposure to the noxious stimuli does not reinstate opioid seeking 67.Re-instatement of CPP has been suggested to model some of aspects of relapse, though technically reinstatement of CPP does not model reinstatement of drug-taking in humans as animals do not actually resume drug intake21. Third, outcome measures rarely reflect pure processes. For instance, locomotor responses following VEH and OXY administration could reflect animal handling and injection procedures to some degree. Changes in paw withdrawal latencies during repeated reflex pain testing could partially reflect sensitization to nociceptive pain or locomotor habituation. Finally, even though animals were derived from different litters as far as possible, genetic variation may not be sufficient to uncover some correlations between behaviors, especially when group sizes are small.There are several caveats requiring consideration in the interpretation of findings in the current study. First, given that the sexes differed also in age, data were pooled. However, four of the outcome measures showed statistically different means between the sexes (data not shown). As such, this could cause some correlations to stem from general sex- or age-related differences. Arguing against this, we noted that when split into separate sexes, all correlations with p values less than 0.05 maintained their directionality, suggesting that even though sex/age differences in means exist, the direction of correlations remain. Second, various methods have been used to define acute horizontal locomotor responses, behavioral sensitization, and establishment of CPP. Here we used time in the OXY-paired compartment as our measure of CPP. As introduced above, some studies express CPP as the difference in time spent in the OXY and VEH paired compartment. Our choice of using absolute measures in the OXY-paired compartment to define the existence of CPP was based on the conservative view that ultimately time spent in this compartment alone most simply defines “preference” for the OXY-paired compartment. This method has been applied elsewhere in studies of OXY CPP in mice In summary, we studied inter-relationships between behavioral phenotypes relevant to pain sensitivity, drug reward, and drug misuse. We found the strongest evidence of inter-relationships between (1) cutaneous thermal reflex nociception, (2) acute locomotion-related measures, and (3) behavioral sensitization and induction/extinction of CPP. Placing animals in a chronic pain state did not re-instate extinguished OXY-seeking behavior. These findings help elucidate relationships between commonly acquired preclinical measures of pain and reward, provide some supportive evidence for current theories of drug misuse, and suggest that chronic pain does not reinstate drug seeking behavior.


Bronchopleural fistulas (BPFs) are a dreaded complication following pulmonary surgery. Endobronchial valves (EVs), with endobronchial sealant (ES), instilled with robotic bronchoscopy (RB), allow occlusion of BPF, avoiding surgery. The patient was a 71-year-old woman with a history of chronic obstructive pulmonary disease and bronchiectasis who underwent bilateral lung transplantation and wedge resection of the right middle lobe and left lingula. A BPF was discovered on postoperative day (POD) 21. Conservative measures with chest tubes failed, and robotic-assisted bronchoscopy aided in reaching the bronchial segment and instilling ES, and EV was deployed with the conventional bronchoscope. The pneumothorax was cleared 12 days later, and on POD 56, she was discharged. The RB procedure was successful, with no pneumothorax or BPF symptoms after a median follow-up of POD 284. Robotic endobronchial closure of BPF with EV and ES is an effective treatment option avoiding invasive surgeries. Bronchopleural fistula (BPF) after lung transplant is infrequent . CurrentA 71-year-old female with end-stage lung failure underwent a bilateral lung transplant with wedge resection of the right middle lobe and left lingular of donor lungs using Endo-GIA staplers due to oversized lungs. On postoperative day (POD) 21, a BPF was noticed from the right middle lobe at the staple line  with perRAB has the potential to increase accessibility to peripheral lung lesions. Due to the smaller size of bronchoscopes, electromagnetic or shape sensing technology and the stable instrumentation platform provided by robots, RAB allows the direct visualization of up to the ninth airway segment lesions, including BPF . We chosAs the late BPF is challenging to treat, we preferred Histoacryl and lipiodol instead of Progel  to provide more permanent closure. The EBV  allows air expiration from a treated bronchus but prevents re-inspiration, thus limiting the reopening of the BPF and facilitating closure , 5.Bronchoscopic intervention for small BPFs using the RAB system is a valuable therapeutic alternative that improves the precision of localization and intervention in the areas difficult to access surgically or with conventional bronchoscopy.


We compared the transcriptomic responses induced by febuxostat and other xanthine oxidase inhibitors to a database of 111 different compounds profiled in the human vascular model. Of the 111 compounds in the database, 107 are clinical-stage and 33 are FDA-labelled for increased cardiovascular risk. Febuxostat induces pathway-level regulation that has high similarity to the set of drugs FDA-labelled for increased cardiovascular risk. These results were replicated with a febuxostat analog, but not another structurally distinct xanthine oxidase inhibitor that does not confer cardiovascular risk. Together, these data suggest that the FDA warning for febuxostat stems from the chemical structure of the medication itself, rather than the target, xanthine oxidase. Importantly, these data indicate that cardiovascular risk can be evaluated in this in vitro human vascular model, which may facilitate understanding the drug candidate safety profile earlier in discovery and development.Some health concerns are often not identified until late into clinical development of drugs, which can place participants and patients at significant risk. For example, the United States Food and Drug Administration (FDA) labeled the xanthine oxidase inhibitor febuxostat with a”boxed” warning regarding an increased risk of cardiovascular death, and this safety risk was only identified during Phase 3b clinical trials after its approval. Thus, better preclinical assessment of drug efficacy and safety are needed to accurately evaluate candidate drug risk earlier in discovery and development. This study explored whether an  A 2016 meta-analysis revealed that 90% of compounds failed in phase 2/3 clinical trials, 30% due to safety concerns, reflecting high failure rate in the drug discovery process [In vitro models have advantages over more traditional in vivo safety and efficacy studies, such as decreased ethical considerations, increased experimental control and throughput. However, in vitro models often lack physiological relevance bringing into question the translatability of data obtained. A strategy we have taken is to design advanced human in vitro systems that restore the physiological microenvironment and cellular responsiveness of multicellular tissues across several disease states, such as propionic acidemia [Numerous strategies are commonly deployed in drug development for de-risking pre-clinical molecules. acidemia –6, canceacidemia , liver dacidemia ,9 includacidemia –13, and acidemia –17 incluacidemia –21. ThesIn this study, we sought to test the utility of a human drug-response database for understanding cardiovascular safety of front-line gout medications. These medications have a well-developed clinical profile, yet recent clinical trial data appears to contradict United States Food and Drug Administration (FDA) warnings of cardiovascular risk.Fig 1).Gout is the most common inflammatory arthritis, and the frequent presence of specific comorbidities, including cardiovascular disease and chronic kidney disease increase the mortality rate. In addition to anti-inflammatory therapies for flares, gout is primarily managed with the xanthine oxidase inhibitors febuxostat or allopurinol to lower serum urate levels. Febuxostat is widely prescribed as a second-line therapy where allopurinol is ineffective, not tolerated, or contraindicated –24 and cPrimary human endothelial cells (ECs) and smooth muscles cells (SMCs) of aortic origin were purchased from Lonza (USA). ECs and SMCs were maintained in EGM-2 BulletKit (Lonza CC-3162) and SmGM-2 BulletKit (Lonza CC-3182) media, respectively.4 for three days. Next, the oxidized LDL (oxLDL) was dialyzed in PBS containing 50 μM EDTA to remove excess copper. The oxidative state of LDL was then confirmed via electrophoretic migration of oxLDL versus nLDL [Human native low-density lipoprotein LDL (nLDL) was purchased from Kalen Biomedical (770200) and oxidized via the following procedure: nLDL was dialyzed overnight in PBS to remove EDTA, then dialyzed in PBS containing 13.8 μM CuSOsus nLDL .Fig 2B. Briefly, the top and bottom surface of a porous polycarbonate transwell membrane  was coated with 0.1% gelatin. First, human SMCs were plated on the bottom transwell surface, and then human ECs were plated on the top transwell surface two days later. Transwells with cells were then placed in fresh media  until shear stresses were applied the next day.The hemodynamic device with the transwell co-culture plating was previously detailed  and illuFig 2A and 2C). Wall shear stress values were calculated from the blood velocity profiles and applied in the EC layer of the co-culture transwell using a direct drive cone-and-plate device  [Human hemodynamic shear stress profiles were derived from magnetic resonance imaging of the human common carotid artery and internal carotid sinus, areas of healthy, atheroprotective regions, or areas prone to atherosclerosis (atheroprone), respectively ,28  ,18. DextFig 2C).Cells were primed with atheroprotective or atheroprone hemodynamics for 18 hours, and then exposed to physiologically relevant concentrations of human oxidized low-density lipoprotein  and human recombinant tumor necrosis factor-α  in the atheroprone condition for an additional 6 hours prior to test compound addition . All experiments were terminated after 48 hours and cells harvested for RNA-seq studies and data used to construct that human response database.111 test compounds were initially selected to span a diverse range of mechanisms, structures, and known clinical outcomes. Following priming, cells were exposed to a vehicle control  or compounds at concentrations approximating Cmax (based on human PK at therapeutic doses), or 10x Cmax, for 24 hours , the allopurinol active metabolite oxypurinol , topiroxostat , and a structural analog of febuxostat . For xanthine oxidase inhibitors, minimal to no effects were observed for compounds dosed at lower concentrations, and thus 10x concentration data is presented.After 48 hours of test compound exposure under atheroprone conditions, RNA was separately harvested from ECs and SMCs using the Invitrogen Purelink RNA Mini kit (12183018A) and RNA concentration determined with the Nanodrop according to manufacturer’s instructions. 250 ng RNA per sample was submitted to Expression Analysis, Inc. for Illumina-based RNA deep sequencing and approximately 20 million, 50 base pair, paired-end reads were generated per sample. For each treatment, 4 EC and 4 SMC samples were run per experiment.Following RNA sequencing, reads were aligned to the human transcriptome build GRCh38, version 91 using the Salmon RNA-seq quantification tool . Gene-leThe human response RNA-seq data for 111 compounds in the human hemodynamic co-culture model underlying this study were created as a proprietary dataset by HemoShear Therapeutics. Together, these compound responses constitute a human drug-response database (HRDB) of transcriptomic data generated in the same experimental system across 111 compounds tested in the system. Of those 111 compounds, 107 are clinical stage compounds with 33 carrying FDA boxed warnings for cardiovascular risk. The full list of compounds and any information regarding their boxed warning for cardiovascular risk can be found in 10.5281/zenodo.8144264.We made functional comparisons to test compound treatments in the database by comparing the log fold changes of genes across Reactome pathways. For a given Reactome pathway, the Spearman correlation of log fold changes was calculated between the treatments in this study and the treatments in the HRDB. Only genes that had treatment p-value < 0.05, in either of the two contrasts being compared, were considered for analysis. Correlation values were calculated across all elements of the Cartesian product of treatments in the HRDB, the xanthine oxidase inhibitor treatments in this study, and all Reactome pathways. The resulting set of correlation values allowed us to identify pathways where the xanthine oxidase inhibitor treatments had an exceptionally high degree of similarity with HRDB drugs that are associated with cardiovascular risk. This assessment was made by identifying pathway-drug combinations where cardiovascular-risk drugs tended to have a higher correlation than drugs without known cardiovascular risk. This identification was performed with the Mann-Whitney U-test. In addition, Fisher’s exact test was used to assess the enrichment of cardiovascular-risk drugs among drugs with positive pathway-level correlation. The ultimate result of this analysis was a list of pathways for each xanthine oxidase inhibitor, where each pathway is similarly regulated by the xanthine oxidase inhibitor and the set of cardiovascular-risk drugs in the HRDB. All data and code are available at DOI: in vitro vascular system aimed to capture key pathological drivers of atherosclerosis. Atherosclerosis is a focal inflammatory disease found in bifurcating or bulbous vessel geometries, such as in the internal carotid sinus, which are characterized by low mean shear stresses and a high oscillatory shear index [Fig 2A and 2B) towards a disease phenotype. Eighteen hours later, tumor necrosis factor-α and oxidized low-density lipoprotein were added to heighten the atheroprone state [Fig 2C). Twenty-four hours later, vehicle, or compounds were added to the atheroprone model. Test compounds were incubated for an additional twenty-four hours . EC and SMC transcriptomic responses were captured separately within the atheroprone model and compared to a non-disease control that utilized human atheroprotective hemodynamics derived from the common carotid artery, a vessel region that does not develop vascular disease. This experimental paradigm was completed with 111 compounds that span a diverse range of structures, targets and disease indications with the goal of developing a predictive vascular pharmacology model . Of these compounds, 107 are clinical stage and 33 carry FDA cardiovascular risk warnings (S1 Table). Many of the compounds are well studied with known side effect profiles and were selected to aid predictions around potential safety concerns, such as cardiovascular risk. Cardiovascular risk associated with each of the test compounds are varied in target mechanism and no common chemical motif can explain the observed cardiovascular risk. Machine learning classification models were created to determine if the transcriptomic profiles encode information predictive of adverse events. Indeed, the system demonstrated good predictive performance across a diverse set of side effects described in SIDER DB [Fig 3B), including arteriosclerosis . Together, these results show that the HRDB contains transcriptomic patterns that correlate with many adverse events, which include cardiovascular risk.The advanced ar index ,28. By car index ,28,35. Tne state  SIDER DB  Fig 4A). These differences were evident in the DEG cluster heatmaps . Subsequent cluster pathway analysis showed febuxostat downregulated nitric oxide (NO) signaling pathway expression concomitant with increased reactive oxygen species (ROS) and inflammatory signaling pathway expression in ECs . Related to the reduced NO signaling expression in ECs, febuxostat-regulated SMC pathways were associated with contractility  . Expression of pathways with strong inflammatory effects on vascular cells [Fig 4D and 4E). Collectively, febuxostat, but not oxypurinol, modulation of these pathways may be indictive of deleterious effects of febuxostat under atheroprone conditions [Differential expression and pathway analysis was performed for the transcriptomic response of XOis using the same protocol as the 111 compounds used to construct the HRDB. Vehicle treated ECs and SMCs exhibited a large number of differentially expressed genes (DEGs)  in the atheroprone model compared to the atheroprotective control, as expected. Furthermore, amongst the XOis, febuxostat elicited the strongest DEG response in both cell types from the atheroprone model  relative to the vehicle control. The allopurinol active metabolite, oxypurinol, elicited only a modest response. Principle component analysis revealed febuxostat clearly separated from oxypurinol and the vehicle control in atheroprone ECs and SMCs, demonstrating these XOis elicit transcriptionally distinct biologic responses . The dataset underwent statistical analysis for quantification of shared pathways regulated by compounds with known cardiovascular risk. In SMCs, the febuxostat transcriptomic response strongly correlated with 29 pathways regulated in common by drugs FDA-labelled for increased cardiovascular risk . This correlation was not driven solely by higher febuxostat-mediated DEG response, as atorvastatin (a cholesterol-lowering drug with no known cardiovascular risk) exhibited a strong DEG response , but did not correlate with cardiovascular risk-associated pathways , in keeping with its clinical findings. Interestingly, no correlation was observed following oxypurinol treatment in either cell type , and heightened cardiovascular risk is not associated with this compound. As additional controls, no correlation was observed following randomization of the febuxostat-regulated transcript response  and observed correlations in the SMCs are greater than expected by chance. Lastly, minimal overlap in pathways modulated by drugs labelled for increased cardiovascular risk was observed in the ECs for each of the XOis evaluated .Next, utility of the HRDB to detect cardiovascular risk was tested for several xanthine oxidase inhibitors . Similar to febuxostat, febuxostat-1 strongly correlated with cardiovascular risk-associated pathways in SMCs while topiroxostat showed no correlation . Pairwise comparison of febuxostat and febuxostat-1 indicated strong DEG overlap, which was not observed with any other compound. As above, neither febuxostat-1 nor topiroxostat showed association with cardiovascular risk in ECs . Together, transcriptional profiling in this advanced in vitro model suggests that differences in cardiovascular risk can be detected pre-clinically and that these data support the FDA cardiovascular warning in individuals with cardiovascular risk for febuxostat, while echoing the safety profile clinically observed with allopurinol and topiroxostat.Because febuxostat is structurally unrelated to purine-based allopurinol or oxypurinol, we hypothesized that the observed cardiovascular risk stems from chemical structure. To test this hypothesis, two additional non-purine compounds were tested under atheroprone conditions: a febuxostat structural analog , labelled febuxostat-1, and topiroxostat, a structurally distinct compound  . In contThis study also extends earlier work examining vascular xanthine oxidase . Here, fOur study has several limitations. Many mechanisms likely contribute to cardiovascular risk, and the role of chemical motifs or causal pathways for the 33 test articles carrying FDA-labeled cardiovascular risk remain unknown. Thus, we took an unbiased approach to determine if cardiovascular signaling pathways regulated by febuxostat may overlap with pathways modulated by drugs with known cardiovascular risk. Regardless, it is critical that future work interrogate pathways we identified to evaluate their correlation to cardiovascular risk. Additionally, compounds were only evaluated in the context of an atheroprone environment, which is more similar to the CARES rather than the FAST trial; however, these data suggest this platform should be used towards that end in future work. Despite these limitations, we suggest these data both support CARES while providing an excellent platform to better understand both FAST trial outcomes and novel medications under development in future work.In summary, comparing the transcriptomic response of novel drug entities within advanced human disease model systems could provide insight into potential liabilities that may not otherwise be uncovered until large and late-stage clinical trials are conducted. This potential utility is demonstrated here using the FAST-CARES trial inspired controversy over FDA cardiovascular risk labelling of the gout medication febuxostat. Due to febuxostat-mediated signaling within human SMCs in a pattern similar to other drugs FDA-labeled for heightened cardiovascular risk, this model supports the FDA warning and further suggests that this liability may be related to chemical structure rather than on-target activity. Results of this study also support the promise of developing and utilizing new drug discovery tools to uncover potential risk earlier in the drug development process and could help enable best-in-class and first-in-class future therapeutics.S1 Table111 compounds alone or in combination were utilized to create the HRDB, of which many of these have known cardiovascular risk. Abbreviations: MI: Myocardial infarction; GI: Gastrointestinal; OTC: Over-the-counter.(XLSX)Click here for additional data file.


The construction and understanding of the abiotic stress regulation network will provide valuable genomic resources for molecular breeding of agriculture. In this special issue, Li et al. focused on the studies of regulatory mechanisms of non-coding RNAs (ncRNAs) in response to drought stress in Triticum aestivum (wheat). In this study, the long non-coding RNAs (lncRNAs), micro RNAs (miRNAs), and mRNAs related to drought stress were identified by transcriptome sequencing. On the combined analyses of these differentially expressed lncRNAs (DELs), differentially expressed miRNAs (DEMs), and differentially expressed genes (DEGs), 10 lncRNA-miRNA-mRNA regulatory modules related to wheat drought stress response were screened and two important candidate modules of them were verified by qRT-PCR. The results showed that ncRNAs play an important role in drought stress response by regulating gene expression. The prediction of regulatory patterns of these candidate lncRNA-miRNA-mRNA modules related to drought tolerance provided useful information in molecular breeding of wheat. Yang et al. also studied the differential expression of ncRNAs in response to drought stress in Phyllostachys aureosulcata f. spectabilis (bamboo). In this research, ncRNAs including circular RNAs (circRNAs) in addition to conventional lncRNAs and miRNAs were identified and analyzed. A total of 2,419 differentially expressed ncRNAs were detected, and the performed Gene Onthology (GO) and KEGG enrichment analysis showed that the host genes were enriched mainly in biochemical reactions involved in some metabolites, as well as in organelle activities. Thus, ncRNAs may regulate the metabolism of multiple metabolites to cope with drought stress. Understanding the regulatory mechanisms of ncRNAs in response to drought will provide a molecular basis for plant resistance studies. Fan et al. focused in a review paper on the research of links between circadian clock and alternative splicing (AS) of plant life activities responsive to abiotic stresses. They reviewed prior studies demonstrating that splicing factors could affect circadian rhythm by itself and the AS of core circadian genes under abiotic stresses. In turn, some genes could undergo AS according to circadian rhythm changes in abiotic stress responses. The crosstalk of circadian clock and AS under abiotic challenges in plants suggests a novel regulatory network that offers new ways to deal with abiotic stress. Leaf senescence is also a response to abiotic stress such as nitrogen deficiency. Wen et al. present the regulatory network of N deficiency-induced leaf senescence in Malus domestica (apple), and shows that overexpressing MdNAC4 enhanced the senescence phenotype of leaves after N deficiency. Instead, overexpression of MdAPRR2 stimulates the expression of chlorophyll synthesis-related genes, which can delay leaf senescence. It is suggested that there may be a balancing mechanism between MdNAC4 and MdAPRR2 in response to N deficiency through the interaction between MdNAC4 and MdAPRR2. It is necessary to summarize the existing research on the molecular mechanisms of plant stress response, especially to sort out the crosstalk among the different regulatory networks, and construct a systematic regulation network for plant abiotic stress responses. It will ultimately be applied to the breeding of highly resistant crops.In recent years, extreme weather conditions occur more frequently as the global climate changes. As a consequence, abiotic stresses such as drought, high/low temperatures, salinity, and flooding are becoming more prominent, which adversely affecting plant growth, development and agricultural productivity. To cope with the challenge of abiotic stress, plants transmit the stress signals within cells and across cells and tissues JH drafted the manuscript. HA and DJ revised the manuscript. All authors approved the final version of manuscript.


Age-associated cardiovascular disease is becoming progressively prevalent due to the increased lifespan of the population. However, the fundamental mechanisms underlying the aging process and the corresponding decline in tissue functions are still poorly understood. The heart has a very high energy demand and the cellular energy needed to sustain contraction is primarily generated by mitochondrial oxidative phosphorylation. Mitochondria are also involved in supporting various metabolic processes, as well as activation of the innate immune response and cell death pathways. Given the central role of mitochondria in energy metabolism and cell survival, the heart is highly susceptible to the effects of mitochondrial dysfunction. These key organelles have been implicated as underlying drivers of cardiac aging. Here, we review the evidence demonstrating the mitochondrial contribution to the cardiac aging process and disease susceptibility. We also discuss the potential mechanisms responsible for the age-related decline in mitochondrial function. Unfortunately, this has also led to an increase in many age-associated diseases such as dementia, cancer, and cardiovascular disease (CVD). In fact, aging alone represents one of the biggest risk factors for developing CVD. A better understanding of the mechanisms underlying the aging process is urgently needed so that improved therapies can be developed that prevent or treat age-related diseases.Improved health care, hygiene, diet, healthier lifestyles, and medical care have all contributed to a substantial increase in lifespan, with an average life expectancy of 76.1 years in the US [Life Expectancy in the USA. Dropped for the Second Year in a Row in 2021  and oxidative stress. Interestingly, various interventions or treatments that directly or indirectly target mitochondria to reduce ROS generation, promote oxidative metabolism or enhance quality control have all been demonstrated to delay cardiac aging and alleviate disease development–7.The beating heart is a highly energy-consuming organ and the cellular energy needed to sustain contraction is primarily generated by mitochondrial oxidative phosphorylation (OXPHOS). Mitochondria are also involved in supporting various metabolic processes, as well as activation of the innate immune response and cell death pathwaysAging is a complex biological process that is associated with a gradual decline in the function of various tissues, leading to increased susceptibility to stress and disease development. However, the fundamental mechanisms contributing to the aging process and development of age-related pathologies are still poorly understood. Given the central role of mitochondria in energy metabolism and cell survival, unraveling the mechanisms underlying age-related mitochondrial impairments is currently of great interest. In this review, we describe existing evidence implicating mitochondrial impairment in the cardiac aging process and disease susceptibility. We also discuss the potential mechanisms responsible for the age-related decline in mitochondrial function and how alterations in the signaling pathways regulating mitochondrial quality control contribute to the accumulation of dysfunctional mitochondria..Mitochondria are the main source of ROS in the aging heart. They are generated as a by-product of electron transfer during oxidative phosphorylation (OXPHOS) when electrons that leak out of the electron transfer chain react with oxygen to produce superoxide. The superoxide can be converted to hydrogen peroxide by superoxide dismutase 2 in the matrix. The hydrogen peroxide, in turn, can be converted into a highly reactive hydroxyl radical. Complex I and III of the respiratory chain are the main sites of ROS production in mitochondria . NAD(P)H. Many genes involved in ROS homeostasis are downregulated in cardiac tissues of elderly humans and rats, which correlate with increased superoxide production. A key role for mitochondrial ROS in cardiac aging and lifespan has been demonstrated in studies utilizing mice with overexpression of a mitochondria-targeted catalase (mCAT). Catalase mitigates the toxic effects of hydrogen peroxide by catalyzing its conversion to water and oxygen and targeting this enzyme to mitochondria attenuates cardiac aging and prolongs median lifespan in mice,12. Hearts of aged mCAT mice also have diminished characteristics of aging, including reduced mitochondrial protein oxidation and mtDNA damage, as well as decreased fibrosis and fiber size. In addition, reducing oxidative stress pharmacologically in aged mice through infusion of a mitochondria-targeted antioxidant, MitoTEMPO, improves cardiac function. Similarly, delivery of SS-31 , a synthetic tetrapeptide that associates with the mitochondrial phospholipid cardiolipin, for 8 weeks improves diastolic function and reduces oxidative stress in the hearts of aged mice. Overall, these studies suggest that ROS generated by mitochondria contribute to the age-related decline in cardiac function and their targeting represents a potential approach to preserve the health of the aging heart.Because of their high reactivity with lipids, proteins, and DNA, ROS pose a constant threat to mitochondrial function. To ensure a reliable supply of energy, cardiac myocytes are equipped with a strong antioxidant system that quickly neutralizes the various ROS species and minimizes oxidative damage. However, excessive ROS generation by dysfunctional mitochondria can lead to an imbalance between production and removal, resulting in oxidative stress [et al. reported a correlation between the accumulation of damaged mtDNA in elderly individuals and decreased strength, cognition, metabolism, and cardiovascular health. They further noted that individuals with a higher burden of mtDNA mutations are at an increased risk of dementia and stroke. In addition, a specific 4977 bp mtDNA deletion that results in the loss of several genes encoding important OXPHOS subunits is commonly observed in cells from aged tissue. This deletion is associated with various age-related diseases, including Alzheimer’s disease and CVD–17.Although mitochondria have transferred most of their genome to the nucleus, they still contain their own DNA which encodes tRNAs, rRNAs, and thirteen critical OXPHOS subunits necessary for mitochondrial function. Due to the location near the OXPHOS complexes and the lack of protective histones, mtDNA is vulnerable to oxidative damage . MtDNA m,19. The POLG mutant mice have an increased burden of somatic mtDNA mutations and develop premature aging with thymic involution, osteoporosis, anemia, alopecia, kyphosis, and reduced lifespan. The accumulation of mtDNA also leads to increased nuclear DNA strand breakage and oxidative damage. These mice also develop cardiac hypertrophy at a younger age than age-matched WT mice. Overall, studies in these mice clearly demonstrate a relationship between mtDNA mutations and aging. However, it is important to bear in mind that although the POLG mutant mice are suitable for examining the consequences of mtDNA mutations, the mutational load in these mice is significantly higher than is found in normal aging and findings might not reflect what occurs in normal physiological aging.Studies in the “mtDNA mutator” mice have provided direct evidence that mtDNA mutations can contribute to the aging process. These mice have a knock-in mutation at D257A in the mitochondrial DNA Polymerase gamma (POLG) that causes inactivation of the proofreading exonuclease activity. ROS produced inside mitochondria cause mtDNA damage which affects the synthesis of functional respiratory chain subunits. This, in turn, can impact mitochondrial respiration and further increase ROS production and mtDNA damage. As discussed in the next section, oxidized mtDNA is also a potent activator of the innate immune system, leading to chronic inflammation. Thus, the continuous ROS-mtDNA damage cycle likely underlies the aging process and onset of age-associated diseases. Several studies have linked oxidative stress to tissue dysfunction and the premature aging phenotype in POLG mutator mice–24. In addition, overexpression of mCAT in POLG mutant mice partially rescues the age-dependent cardiomyopathy that these mice develop at 13-14 months of age. Suppression of mitochondrial hydrogen peroxide levels in these mice leads to reduced cardiac hypertrophy and dilatation, improved systolic and diastolic function, and decreased cardiac fibrosis. Also, a recent study reported that increased oxidative stress and chronic inflammation are responsible for the age-dependent decline in cardiac function in POLG mutant mice. Overall, these findings clearly demonstrate that mtDNA mutations and oxidative stress can be driving forces of aging.Increased oxidative stress within the mitochondria is thought to play a significant role in the aging process. For instance, oxidized fragmented mtDNA in the cytosol activates the NLR family pyrin domain containing 3 protein (NLRP3) inflammasome [. NLRP3 inflammasome activation is also an underlying factor in low-grade age-related inflammation. NLRP3-deficient mice have improved health spans with attenuated age-related changes, including reduced bone loss, improved cognitive function, and motor performance compared to aged WT mice. Furthermore, NLRP3−/− mice have increased muscle strength and endurance compared to age-matched WT mice, suggesting that these mice are protected from the loss of muscle mass that occurs with age. In contrast to WT mice, NLRP3-deficient mice do not develop cardiac hypertrophy and fibrosis with age and have extended lifespans. Overall, these findings suggest that cardiac inflammation due to mitochondrial dysfunction and NLRP3 activation contributes to age-related inflammation and that limiting activation of the inflammasome downstream of mitochondria can limit cardiac aging.Inflammation is a response aimed at limiting and repairing damage caused by acute traumatic injury or invading pathogens. However, chronic low-grade inflammation is another major contributor to various age-related pathologies and biological aging. Dysfunctional mitochondria are thought to underlie the chronic inflammation in tissues through activation of the innate immune response. Because of their bacterial origin, mitochondrial DNA (mtDNA) is a potent damage-associated molecular pattern (DAMP) and induces an inflammatory response by activating pathways that are normally involved in pathogen-associated responsesammasome . The NLR,32. The cGAS protein functions as a cytoplasmic mtDNA sensor and activates STING, which in turn phosphorylates the transcription factor IRF3, leading to the expression of type I IFN genes [ and cellular senescence. Accumulation of mtDNA mutation POLG mutator mice also leads to aberrant activation of the cGAS-STING signaling pathway in macrophages which contributes to the cardiac dilatation and increased mortality in these mice. These mice have persistent activation of the cGAS-STING–IFN-I axis in macrophages and tissues, which leads to repression of nuclear factor erythroid 2-related factor 2 (NRF2) activity. NRF2 is a transcription factor that is involved in the cell’s antioxidant defense by inducing transcription of antioxidant and cytoprotective genes. Disrupting the IFN-I signaling in POLG mutator mice delays the age-dependent decline in cardiac function and extends lifespan, confirming the contribution of persistent inflammation to the accelerated aging phenotype in these mice. Thus, these studies clearly demonstrate a link between mitochondria and the innate immune response in aging.Moreover, cytoplasmic mtDNA can also activate the innate immune system via the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway, a major driver of type I interferon-mediated inflammatory responsesFN genes . ActivatmtUPR). Because mtDNA does not encode any stress response genes, mitochondria must communicate the stress to the nucleus to launch a repair response. The mtUPR is a conserved specific transcriptional stress response program aimed at reducing misfolded proteins inside mitochondria. Studies in worms have provided important insights into this stress response and ATFS-1 has been identified as a key regulator of the mtUPR in C.elegans [. However, upon mitochondrial stress, the import of ATFS-1 into the matrix is abrogated, allowing it to re-localize to the nucleus and activate transcription of mitochondrial chaperones and other genes required for repair–40. ATF5 has been reported to function as a mammalian orthologue of ATFS-1. Like ATFS-1, ATF5 induces transcription of mitochondrial chaperones and proteases to reduce proteotoxic stress. ATF5 overexpression also restores the mtUPR response in ATFS-1-deficient worms, further confirming its function in communicating mitochondrial stress to the nucleus.Mitochondrial protein homeostasis is ensured by an intricate protein quality-control network composed of resident chaperones and proteases that are regulated by the mitochondrial unfolded protein response , an enzyme that catalyzes the formation of hydrogen sulfide (H2S). Conversely, loss of Atg4 in hematopoietic stem cells leads to defects associated with an aging-like phenotype.Mitochondrial stress also activates the integrated stress response (ISR) via phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), which leads to decreased global protein synthesis and induction of specific genes such as ATF4 (activating transcription factor 4),51 [,51. The cleaved Dele1 fragment activates the eIF2a kinase heme-regulated inhibitory (HRI) in the cytosol. HRI, in turn, phosphorylates eIF2α to turn on the ISR,51. The importance of Dele1 in communicating mitochondrial stress and activating the ISR in the heart was recently demonstrated by two different groups. Ahola et al. reported that ISR signaling via Oma1-Dele1-Atf4 protects hearts against the development of cardiomyopathy in mice with OXPHOS deficiency. This group also demonstrated that cardiac-specific deletion of Dele1 leads to accelerated disease development in the OXPHOS mutant mice. Similarly, Huynh et al. demonstrated that deletion of Dele1 in myocytes is detrimental in mouse models of fetal and adult mitochondrial cardiomyopathy. Interestingly, this study monitored mice with cardiac-specific Dele1-deficiency for up to one year and noted that the lack of Dele1 in myocytes has no effect on cardiac structure or function at baseline. The lack of an accelerated aging cardiac phenotype suggests that Dele1 is not critical for baseline homeostasis and suppression of normal aging. Instead, it is likely that the primary function of this pathway is in the adaptation to severe stress. Since aged individuals are known to be more susceptible to stress, it will be interesting to investigate if activation of this pathway is decreased with age.A novel pathway of ISR activation involving mitochondria was recently identified where dysfunctional mitochondria activate the ISR via DAP3 Binding Cell Death Enhancer 1 (Dele1),51 . Upon mi. Thus, a decline in this process is associated with the accumulation of autophagic cargo, including dysfunctional mitochondria. Mitophagy is a highly regulated process and requires coordinating induction of autophagy with labeling of mitochondria for selective autophagic degradation. Autophagosome formation is initiated upon activation of the serine/threonine kinase Ulk1. Ulk1 activates the Beclin1-Vps34-Vps15 complex, which, in turn, recruits various autophagy-related genes (Atg) proteins responsible for autophagosome nucleation and elongation,56. In addition, loss-of-function mutations in Atg1 (Unc-51), Atg7, Atg18, and Beclin 1 (Bec-1) decrease the lifespan of C. elegans. Autophagic activity is also reduced in aged mouse hearts,59 and the negative impact of impaired autophagic activity on cardiac aging has been confirmed in mouse models with autophagic deficiency. For instance, cardiac-specific deletion of the core autophagy protein Atg5 abrogates autophagosome formation in mouse hearts and leads development of cardiac dysfunction and accelerated cardiac aging,60. In contrast, mice with ubiquitous overexpression of Atg5 in tissues have an anti-aging phenotype, including extended lifespans. In addition, interventions that enhance autophagy extend the lifespans of organisms and improve cardiovascular health. Many studies have demonstrated that longevity is linked to the activation of autophagy. In fact, interventions, including caloric restriction and spermidine, that are known to extend lifespan in various organisms depend on autophagy to promote longevity–66. Also, rapamycin is a potent inducer of autophagy and long-term treatment ameliorates many age-related effects, improves cardiac function and extends lifespan,67. Although Rapamycin affects many pathways, its effect on longevity is lost when autophagy is impaired, confirming that some of its anti-aging effects depend on autophagic activity,69. Moreover, Beclin1 is sequestered by Bcl-2 under baseline conditions, which prevents it from forming the complex with Vps34 to initiate autophagosome formation. Mice with a knock-in mutation in Becn1 that disrupts its interaction with Bcl-2 have increased basal autophagic activity in tissues as well as extended lifespan and health span. At 20 months of age, these mice have reduced cardiac hypertrophy and fibrosis compared to age-matched WT mice. Whether mitochondrial fitness is also improved in these mice remains to be investigated. Beclin1 plays a specific role in mitophagy and future studies should investigate if targeting Beclin1 can selectively enhance mitochondrial turnover in aging myocytes.When damage to a mitochondrion exceeds the repair capacity, the entire organelle must be eliminated before it causes harm to the cell. The primary mechanism by which mitochondria are removed occurs through mitochondrial autophagy or mitophagy. In mitophagy, mitochondria are selectively engulfed by autophagosomes for delivery to lysosomes, where they are degraded which leads to recruitment and activation of the E3 ubiquitin ligase Parkin,74. Parkin ubiquitinates various OMM proteins which are recognized by adaptor proteins such as p62, optineurin (OPTN), NDP52, TAX1BP1, and NBR1,76. These adaptor proteins are responsible for linking the ubiquitinated mitochondria to LC3 on the autophagic membranes via their LC3 Interacting Region (LIR) motifs. Several studies suggest that a decline in the removal of damaged mitochondria is associated with aging. A study utilizing fluorescent mitophagy reporter mice demonstrated a decrease in mitophagy levels in the brains of aged mice compared to young mice. Also, PINK1 deficient mice exhibit accelerated decline in mitochondrial function and accumulation of megamitochondria in the cortex. Cardiac mitochondria in PINK1−/− mice are dysfunctional by 2 months of age with concurrent development of left ventricular dysfunction and cardiac hypertrophy. Although Parkin and PINK1 function in the same pathway, Parkin-deficient mice have a less severe cardiac phenotype, and despite the accumulation of abnormal mitochondria at ~ 6 months of age, cardiac function is still normal,82. This suggests that additional E3 ubiquitin ligases can function downstream of PINK1 in the absence of Parkin. Moreover, enhancing the mitophagy pathway can delay cardiac aging, where cardiac-specific overexpression of Parkin improves mitochondrial quality in aging hearts. However, Parkin overexpression in POLG mutant mice does not rescue the accelerated cardiac aging phenotype but results in cardiac fibrosis, likely due to an imbalance between ubiquitination and autophagic degradation. Interestingly, Parkin deficiency in POLG mutator mice does not accelerate cardiac aging, suggesting that alternative mitophagy pathways might be activated in these hearts.In mitophagy, the PINK1-Parkin pathway is involved in labeling dysfunctional mitochondria for autophagic degradation . PINK1 . There are several proteins in the OMM that can function as mitophagy receptors and the best well-characterized proteins include FUNDC1g to LC3 . In addi, suggesting that these proteins are important in the normal turnover of mitochondria to maintain homeostasis. Bnip3 protein levels are also increased in aged mouse hearts, which might represent an attempt by the heart to increase turnover of less efficient old mitochondria. However, because mitophagy relies on autophagosome formation which is reduced in the aged heart,59, increasing Bnip3 levels without restoring autophagosome formation will be ineffective.Currently, studies investigating the impact of aging on mitophagy receptors in the heart are lacking. However, simultaneous loss of Bnip3 and Nix in the mouse heart leads to accelerated accumulation of dysfunctional mitochondria and cardiac dysfunction in mice, exercise,96, and nicotinamide riboside, spermidine or rapamycin,99 treatments, are mediated at least in part through the preservation of mitochondria. Pre-clinical studies clearly suggest that directly targeting mitochondrial ROS production or enhancing repair and turnover may have promising therapeutic benefits in the aging heart. For example, administration of the mitochondria-targeted antioxidant MitoTEMPO to aged mice reduces oxidative stress and improves systolic and diastolic function, while MitoQ administration ameliorates vascular endothelial dysfunction in aged mice. Treatment of aged mice with the mitochondrially-targeted tetrapeptide SS-31 (elamipretide) for 8 weeks leads to reduced oxidative stress in hearts with improvements in cardiac function and reversal of cardiac hypertrophy. There is also great interest in developing small molecules that can directly activate mitophagy in cells. VL-004 is a small molecule that increases mitophagy and longevity in C.elegans via dct-1, the worm homolog of mammalian mitophagy receptors BNIP3 and BNIP3L/NIX. VL-004 failed to extend lifespan in dct-1 mutant worms, confirming that the effect on lifespan extension is dependent on mitophagy. Although these studies targeting mitophagy are promising, whether the beneficial effects are preserved in larger organisms remains to be investigated.Currently, effective treatments to prevent age-related cardiovascular dysfunction are lacking, but there is a strong interest in developing therapeutics that are aimed at preserving or improving mitochondrial health in cells. Many interventions that protect against cardiac aging, including caloric restriction. Similarly, too much activation of mitophagy can lead to excessive clearance of mitochondria. If the clearance exceeds the cells’ capacity to generate new mitochondria, it can lead to a catastrophic energy deficiency. Thus, the therapeutic window of these interventions needs to be clearly defined. There are also alternative pathways to traditional autophagy in place that can deliver mitochondria to lysosomes–105, but how these are affected in the aging heart has not been investigated.Due to the longer lifespan in the population, treatments that prevent late-life morbidities and increase health spans are urgently needed. Dysfunctional mitochondrial are clearly major contributors to cardiac aging . AlthougIn sum, mitochondria fitness is key to maintaining a healthy heart during aging, and a better understanding of the molecular basis for mitochondrial dysfunction during aging will provide increased knowledge that can be used to develop new therapies to combat age-related morbidities.


Prostate cancer is the second leading cause of cancer-related deaths in men in the United States. Imaging techniques such as CT, MRI, and bone scans have traditionally been used for diagnosis and staging. Molecular imaging modalities targeting the prostate-specific membrane antigen (PSMA) have recently gained attention due to their high affinity and accuracy. PSMA PET has been combined with other modalities such as multiparametric MRI for better diagnostic and prognostic performance. PSMA imaging has been studied at different clinical settings with a wide range of disease aggressiveness. In this review we will explore the role of PSMA PET in high-risk prostate cancer staging, biochemical recurrence, and castration-resistant prostate cancer. The primary focus of this review article is to examine the latest developments in the use of PSMA imaging and emphasize the clinical situations where its effectiveness has been demonstrated to significantly impact the treatment of prostate cancer. In addition, we will touch upon the potential future advancements of PSMA PET imaging and its evolving significance in the management of prostate cancer. Prostate cancer is the most common cancer in men in the United States and second leading cause of cancer-related death in men . CT, MRIThe role of PSMA has been studied at different clinical settings with a wide range of disease aggressiveness . RecentlThis review article focuses on recent advances in the applications of PSMA imaging and highlights the clinical scenarios where its effectiveness has been proven to significantly alter the management of prostate cancer.Since its discovery in 1987, multiple iterations of PSMA-targeting molecules for the purpose of diagnosis and treatment of prostate cancer have been developed . Among nThe European Association of Nuclear Medicine (EANM) and the Society of Nuclear Medicine and Molecular Imaging’s (SNMMI) joint procedure standard for 68Ga-PSMA-11  recommenThe estimated coefficient for effective dose from PSMA PET ligands ranges between 0.0116 and 0.022 mSv/MBq, resulting in calculated effective doses ranging between 3.4 and 6.6 mSv depending on radioligand type and injection dose . These nThe increased turnover and production of phosphatidyl choline in cellular membranes is the basis of this radiotracer . CholineFluciclovine PET targets amino acid transporters for L-lerucine. FluciclThe initial staging of prostate cancer is a crucial step in determining the most appropriate treatment plan, including options such as prostatectomy, radiation therapy with or without local lymph node treatment, or systemic therapy  Fig 2)..2). RiskThe majority of studies examining the role of PSMA in prostate cancer staging have reported favorable specificity and predictive values. Hope et al.  in theirSome studies have compared the performance of PSMA PET versus conventional imaging. In the proPSMA study, a multicentric randomized clinical trial of 302 patients aimed at investigating the accuracy of Ga68-PSMA-11 in staging prostate cancer in high-risk patients at initial presentation compared to conventional imaging, PSMA PET showed superior accuracy , sensitivity (85% versus 38%) . Nearly Biochemical recurrence occurs when the serum PSA level increases to a specific threshold following definitive therapy. In radiation therapy, this threshold is more than 2.0 ng/mL above nadir, while in radical prostatectomy, it is 0.2 ng/mL on two consecutive reads , 36. PSAEarly detection of biochemical recurrence (BCR) may provide a window of opportunity for effective treatment with directed radiation therapy and/or surgery before the onset of clinical recurrence. For example, salvage radiation therapy which has been shown to be the most effective treatment plan after radical prostatectomy and BCR, is usually done without conventional imaging, potentially missing distant lesions . TherefoThere is a large body of evidence supporting use of PSMA PET in BCR , 41–50. Castration resistance in prostate cancer occurs when the disease continues to progress, even in the presence of low levels of prostate-specific antigen (PSA) and despite treatment with androgen deprivation therapy. Distinguishing between metastatic and nonmetastatic castration-resistant prostate cancer (CRPC) is crucial for determining the appropriate treatment plan . PSMA PEThe life prolonging treatments for CRPC are diverse and include chemotherapy, hormonal therapy, immunotherapy and radioligand therapy Fig . With reTumor localization within the prostate can be helpful in biopsy targeting, targeted therapy and potentially eliminating the need for biopsy in patients with clinically insignificant prostate cancer. For the latter aim, PRIMARY trial  has beenDespite the name, PSMA is not specifically expressed on prostate and a wide range of other cancers have PSMA uptake including renal cell carcinoma, transitional cell carcinoma, primary brain tumors, thyroid carcinoma, breast cancer, hepatocellular carcinoma, and lung Fig.  65]. Pe. Pe65]. Although PSMA-targeted imaging has been very effective in detection of PSMA expressing tumors, prostate cancers with neuroendocrine differentiation do not express PSMA due to FOLH1 gene suppression and thus, making this imaging technique ineffective . AlternaAndrogen blockade is an integral part of the several treatment options for prostate cancer due to its significant role in improving survival rate and treatment effectiveness . PSMA exDetermination of malignancy or benignity of solitary bone lesions can be challenging in PSMA PET as this could change patient management and false positive interpretations would cause unnecessary harm to the patient . SolitarThe utilization of sensitive prostate-specific antigen (PSA) detection methods and improved PET radiotracers has resulted in an increased identification of “oligometastatic disease,” thereby raising interest in metastasis-directed therapy (MDT) , 76 Fig.  EmergiIn addition to its predictive role in determining the degree of response to 177Lu-PSMA therapy and aiding in patient selection, PSMA PET may also be utilized for evaluating treatment response . The LuPPROMISE is a molecular imaging TNM staging system which is proposed to facilitate reporting and study design of prostate cancer molecular imaging , 80. UndProstate-specific Membrane Antigen-Reporting and Data System (PSMA-RADS) is a proposed reporting and data system with ultimate goal of establishing a uniform and structured reporting system for PSMA-targeted PET studies . This frPSMA PET is considered a valuable tool for various aspects of patient care in the clinical management of prostate cancer and has shown promising results in initial staging of prostate cancer, localization of recurrent or persistent disease, and staging before PSMA-directed radioligand therapy. It also has potential applications in guiding prostate biopsy, guiding metastatic-directed therapy, and monitoring systemic and radioligand treatment response. While the impact on patient outcomes and management is still being assessed, PSMA PET has been included in clinical guidelines and consensus documents, highlighting its superior accuracy and additional value in prostate cancer staging. However, further research and evaluation are needed to fully establish its role in treatment monitoring and patient outcomes.