Genome-wide association studies have implicated thousands of noncoding variants across common human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate that signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in cortical layer V excitatory neurons. We also show that differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in cortical layers II-III, IV, and V, as well as the dentate gyrus. Finally, we leverage these data to fine-map variants in 177 schizophrenia loci nominating variants in 104/177. We integrate these data with transcription factor binding site, chromatin interaction, and validated enhancer data, placing variants in the cellular context where they may modulate risk.

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A, and RAD21, cohesin in somatic cells contains one of two orthologous STAG subunits, STAG1 or STAG2. How these variable subunits affect the function of the cohesin complex is still unclear. STAG1- and STAG2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of STAG1 and STAG2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either STAG1 or STAG2. Following rapid depletion of either subunit, we perform high-resolution Hi-C, gene expression, and sequential ChIP studies to show that STAG1 and STAG2 do not co-occupy individual binding sites and have distinct ways by which they affect looping and gene expression. These findings are further supported by single-molecule localizations via direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging. Since somatic and congenital mutations of the STAG subunits are associated with cancer (STAG2) and intellectual disability syndromes with congenital abnormalities (STAG1 and STAG2), we verified STAG1-/STAG2-dependencies using human neural stem cells, hence highlighting their importance in particular disease contexts.

Previous studies indicated that circular RNAs (circRNA) played vital roles in the development of non–small cell lung cancer (NSCLC). Although hsa_circ_0014130 might be a potential NSCLC biomarker, its function in NSCLC remains unknown. Thus, this study aimed to investigate the role of hsa_circ_0014130 in the progression of NSCLC. The levels of hsa_circ_0014130 in NSCLC tissues and adjacent normal tissues were determined by qRT-PCR. In addition, the expressions of Bcl-2 and cleaved caspase-3 in A549 cells were detected with Western blot analysis. Meanwhile, the dual luciferase reporter system assay was used to determine the interaction of hsa_circ_0014130 and miR-136-5p or Bcl-2 and miR-136-5p in NSCLC, respectively. The level of hsa_circ_0014130 was significantly upregulated in NSCLC tissues. Downregulation of hsa_circ_0014130 markedly inhibited the proliferation and invasion of A549 cells via inducing apoptosis. In addition, downregulation of hsa_circ_0014130 inhibited the tumorigenesis of subcutaneous A549 xenograft in mice in vivo. Meanwhile, mechanistic analysis indicated that downregulation of hsa_circ_0014130 decreased the expression of miR-136-5p–targeted gene Bcl-2 via acting as a competitive "sponge" of miR-136-5p. In this study, we found that hsa_circ_0014130 was upregulated in NSCLC tissues. In addition, hsa_circ_0014130 functions as a tumor promoter in NSCLC to promote tumor growth through upregulating Bcl-2 partially via "sponging" miR-136-5p.

Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttranslational modification enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hypercitrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps (NET). NETs were first described as antimicrobial fibers that bind and kill bacteria. However, it is not known whether PAD4 can mediate the release of chromatin DNA into the extracellular space of cancer cells. Here, we report that murine breast cancer 4T1 cells expressing high levels of PADI4 can release cancer extracellular chromatin networks (CECN) in vitro and in vivo. Deletion of Padi4 using CRISPR/Cas9 abolished CECN formation in 4T1 cells. Padi4 deletion from 4T1 cells also reduced the rate of tumor growth in an allograft model, and decreased lung metastasis by 4T1 breast cancers. DNase I treatment, which degrades extracellular DNA including CECNs, also reduced breast to lung metastasis of Padi4 wild-type 4T1 cells in allograft experiments in the Padi4-knockout mice. We further demonstrated that DNase I treatment in this mouse model did not alter circulating tumor cells but decreased metastasis through steps after intravasation. Taken together, our genetic studies show that PAD4 plays a cell autonomous role in cancer metastasis, thus revealing a novel strategy for preventing cancer metastasis by inhibiting cancer cell endogenous PAD4.

The histone demethylase JMJD1A plays a key functional role in spermatogenesis, sex determination, stem cell renewal, and cancer via removing mono- and di-methyl groups from H3K9 to epigenetically control gene expression. However, its role in prostate cancer progression remains unclear. Here, we found JMJD1A was significantly elevated in prostate cancer tissue compared with matched normal tissue. Ectopic JMJD1A expression in prostate cancer cells promoted proliferation, migration, and invasion in vitro, and tumorigenesis in vivo; JMJD1A knockdown exhibited the opposite effects. Mechanically, we revealed that JMJD1A directly interacted with the Snail gene promoter and regulated its transcriptional activity, promoting prostate cancer progression both in vitro and in vivo. Furthermore, we found that JMJD1A transcriptionally activated Snail expression via H3K9me1 and H3K9me2 demethylation at its special promoter region. In summary, our studies reveal JMJD1A plays an important role in regulating proliferation and progression of prostate cancer cells though Snail, and thus highlight JMJD1A as potential therapeutic target for advanced prostate cancer.

We recently reported that restoring the CYP27A1–27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti–prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6–JAK–STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6–JAK–STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation.

Pre-eclampsia (PE)-induced fetal programming predisposes offspring to health hazards in adult life. Here, we tested the hypothesis that pre-eclamptic fetal programming elicits sexually dimorphic inflammatory and cardiovascular complications to endotoxemia in adult rat offspring. PE was induced by oral administration of L-NAME (50 mg/kg per day for seven consecutive days) starting from day 14 of conception. Cardiovascular studies were performed in conscious adult male and female offspring preinstrumented with femoral indwelling catheters. Compared with non-PE male counterparts, intravenous administration of lipopolysaccharide (LPS, 5 mg/kg) to PE male offspring caused significantly greater 1) falls in blood pressure, 2) increases in heart rate, 3) rises in arterial dP/dtmax, a correlate of left ventricular contractility, and 4) decreases in time- and frequency-domain indices of heart rate variability (HRV). By contrast, the hypotensive and tachycardic actions of LPS in female offspring were independent of the pre-eclamptic state and no clear changes in HRV or dP/dtmax were noted. Measurement of arterial baroreflex activity by vasoactive method revealed no sex specificity in baroreflex dysfunction induced by LPS. Immunohistochemical studies showed increased protein expression of toll-like receptor 4 in heart as well as in brainstem neuronal pools of the nucleus of solitary tract and rostral ventrolateral medulla in endotoxic PE male, but not female, offspring. Enhanced myocardial, but not neuronal, expression of monocyte chemoattractant protein-1 was also demonstrated in LPS-treated male offspring. Together, pre-eclamptic fetal programming aggravates endotoxic manifestations of hypotension and autonomic dysfunction in male offspring via exacerbating myocardial and neuromedullary inflammatory pathways.

Cholangiocarcinoma (CCA) is a malignant tumor that arises from the epithelial cells of the bile duct and is notorious for its poor prognosis. The clinical outcome remains disappointing, and thus more effective therapeutic options are urgently required. Cordycepin, a traditional Chinese medicine, provides multiple pharmacological strategies in antitumors, but its mechanisms have not been fully elucidated. In this study, we reported that cordycepin inhibited the viability and proliferation capacity of CCA cells in a time- and dose-dependent manner determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Flow cytometry and Hoechst dye showed that cordycepin induced cancer cell apoptosis via extracellular signal-regulated kinase (ERK) 1/2 deactivation. Moreover, cordycepin significantly reduced the angiogenetic capabilities of CCA in vitro as examined by tube formation assay. We also discovered that cordycepin inhibited DEK expression by using Western blot assay. DEK serves as an oncogenic protein that is overexpressed in various gastrointestinal tumors. DEK silencing inhibited CCA cell viability and angiogenesis but not apoptosis induction determined by Western blot and flow cytometry. Furthermore, cordycepin significantly inhibited tumor growth and angiogenic capacities in a xenograft model by downregulating the expression of DEK, phosphorylated ERK1/2 CD31 and von Willebrand factor (vWF). Taken together, we demonstrated that cordycepin inhibited CCA cell proliferation and angiogenesis with a DEK interaction via downregulation in ERK signaling. These data indicate that cordycepin may serve as a novel agent for CCA clinical treatment and prognosis improvement.

Dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes and obesity because of their beneficial effects on body weight, blood glucose, insulin sensitivity, and food preference, at least short-term. DACRAs activate the receptors for a prolonged time period, resulting in metabolic effects superior to those of amylin. Because of the prolonged receptor activation, different dosing intervals and, hence, less frequent receptor activation might change the efficacy of DACRA treatment in terms of weight loss and food preference. In this study, we compared daily dosing to dosing every other day with the aim of understanding the optimal balance between efficacy and tolerability. Obese and lean male Sprague-Dawley rats were treated with the DACRA KBP-088, applying two different dosing intervals (1.5 nmol/kg once daily and 3 nmol/kg every other day) to assess the effect on body weight, food intake, glucose tolerance, and food preference when given the choice between chow (13% fat) and a high-fat diet (60% fat). Treatment with KBP-088 induced significant weight loss, reduction in adiposity, improvement in glucose control, and altered food preference toward food that is less calorie-dense. KBP-088 dosed every other day (3 nmol/kg) was superior to KBP-088 once daily (1.5 nmol/kg) in terms of weight loss and improvement of food preference. The beneficial effects were evident in both lean and obese rats. Hence, dosing KBP-088 every other day positively affects overall efficacy on metabolic parameters regardless of the lean/obese state, suggesting that less-frequent dosing with KBP-088 could be feasible.

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography–tandem mass spectrometry–based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration–based DDIs caused by unexpected regulation of hepatic transporters by NR modulators.

It has been identified that arginine vasopressin (AVP), vasopressin receptor 2(V2R), and the aquaporin 2 (AQP2) signaling pathway in the inner ear play important roles in hearing and balance functions through regulating the endolymph equilibrium; however, the contributions of this signaling pathway to the development of motion sickness are unclear. The present study was designed to investigate whether the activation of the AVP-V2R-AQP2 signaling pathway in the inner ear is involved in the induction of motion sickness and whether mozavaptan, a V2R antagonist, could reduce motion sickness. We found that both rotatory stimulus and intraperitoneal AVP injection induced conditioned taste aversion (a confirmed behavioral index for motion sickness) in rats and activated the AVP-V2R-AQP2 signaling pathway with a responsive V2R downregulation in the inner ears, and AVP perfusion in cultured epithelial cells from rat endolymphatic sacs induced similar changes in this pathway signaling. Vestibular training, V2R antagonist mozavaptan, or PKA inhibitor H89 blunted these changes in the V2R-AQP2 pathway signaling while reducing rotatory stimulus– or DDAVP (a V2R agonist)-induced motion sickness in rats and dogs. Therefore, our results suggest that activation of the inner ear AVP-V2R-AQP2 signaling pathway is potentially involved in the development of motion sickness; thus, mozavaptan targeting AVP V2Rs in the inner ear may provide us with a new application option to reduce motion sickness.

PF06821497 has been identified as an orally available small-molecule enhancer of zeste homolog 2 inhibitor. The objectives of the present study were to characterize pharmacokinetic-pharmacodynamic-disease relationships of PF06821497 in xenograft mouse models with diffuse large B-cell lymphoma (Karpas422). An indirect-response model reasonably fit dose-dependent pharmacodynamic responses [histone H3 on lysine 27 (H3K27) me3 inhibition] with an unbound EC₅₀ of 76 nM, whereas a signal-transduction model sufficiently fit dose-dependent disease responses (tumor growth inhibition) with an unbound tumor stasis concentration (T_sc) of 168 nM. Thus, effective concentration for 70% of maximal effect (EC₇₀) for H3K27me3 inhibition was roughly comparable to T_sc, suggesting that 70% H3K27me3 inhibition could be required for tumor stasis. Consistently, an integrated pharmacokinetic-pharmacodynamic-disease model adequately describing tumor growth inhibition also suggested that ~70% H3K27me3 inhibition was associated with tumor stasis. Based on these results, we would propose that an EC₇₀ estimate for H3K27me3 inhibition corresponding to tumor stasis could be considered a minimum target efficacious concentration of PF06821497 in cancer patients.

Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS^–/–), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS^–/– mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity.

Despite the progress in the development of novel treatment modalities, a significant portion of patients with psoriasis remains undertreated relative to the severity of their disease. Recent evidence points to targeting the glucose transporter 1 and sugar metabolism as a novel therapeutic strategy for the treatment of psoriasis and other hyperproliferative skin diseases. In this review, we discuss glycoconjugation, an approach that facilitates the pharmacokinetics of cytotoxic molecules and ensures their preferential influx through glucose transporters. We propose pathways of glycoconjugate synthesis to increase effectiveness, cellular selectivity, and tolerability of widely used antipsoriatic drugs. The presented approach exploiting the heightened glucose requirement of proliferating keratinocytes bears the potential to revolutionize the management of psoriasis.

Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33–amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS.

In the olfactory bulb, a cAMP/PKA/CREB-dependent form of learning occurs in the first week of life that provides a unique mammalian model for defining the epigenetic role of this evolutionarily ancient plasticity cascade. Odor preference learning in the week-old rat pup is rapidly induced by a 10-min pairing of odor and stroking. Memory is demonstrable at 24 h, but not 48 h, posttraining. Using this paradigm, pups that showed peppermint preference 30 min posttraining were sacrificed 20 min later for laser microdissection of odor-encoding mitral cells. Controls were given odor only. Microarray analysis revealed that 13 nonprotein-coding mRNAs linked to mRNA translation and splicing and 11 protein-coding mRNAs linked to transcription differed with odor preference training. MicroRNA23b, a translation inhibitor of multiple plasticity-related mRNAs, was down-regulated. Protein-coding transcription was up-regulated for Sec23b, Clic2, Rpp14, Dcbld1, Magee2, Mstn, Fam229b, RGD1566265, and Mgst2. Gng12 and Srcg1 mRNAs were down-regulated. Increases in Sec23b, Clic2, and Dcbld1 proteins were confirmed in mitral cells in situ at the same time point following training. The protein-coding changes are consistent with extracellular matrix remodeling and ryanodine receptor involvement in odor preference learning. A role for CREB and AP1 as triggers of memory-related mRNA regulation is supported. The small number of gene changes identified in the mitral cell input/output link for 24 h memory will facilitate investigation of the nature, and reversibility, of changes supporting temporally restricted long-term memory.

In obsessive–compulsive disorder (OCD), functional behaviors such as checking that a door is locked become dysfunctional, maladaptive, and debilitating. However, it is currently unknown how aversive and appetitive motivations interact to produce functional and dysfunctional behavior in OCD. Here we show a double dissociation in the effects of anxiogenic cues and sensitivity to rewarding stimuli on the propensity to develop functional and dysfunctional checking behavior in a rodent analog of OCD, the observing response task (ORT). While anxiogenic manipulations of perceived threat (presentation of threat-associated contextual cues) and actual threat (punishment of incorrect responding on the ORT) enhanced functional checking, dysfunctional checking was unaffected. In contrast, rats that had previously been identified as "sign-trackers" on an autoshaping task—and therefore were highly sensitive to the incentive salience of appetitive environmental cues—selectively showed elevated levels of dysfunctional checking under a range of conditions, but particularly so under conditions of uncertainty. These data indicate that functional and dysfunctional checking are dissociable and supported by aversive and appetitive motivational processes, respectively. While functional checking is modulated by perceived and actual threat, dysfunctional checking recruits appetitive motivational processes, possibly akin to the "incentive habits" that contribute to drug-seeking in addiction.

In recent years, there have been intensive debates on whether healthy adults acquire new word knowledge through fast mapping (FM) by a different mechanism from explicit encoding (EE). In this study, we focused on this issue and investigated to what extent retention interval, prior knowledge (PK), and lure type modulated memory after FM and EE. Healthy young participants were asked to learn novel word-picture associations through both FM and EE. Half of the pictures were from familiar categories (i.e., high PK) and the other half were from unfamiliar categories (i.e., low PK). After 10 min and 1 wk, the participants were tested by forced-choice (FC) tasks, with lures from different categories (Experiment 1) or from the same categories of the target pictures (Experiment 2). Pseudowords were used to denote names of the novel pictures and baseline performance was controlled for each task. The results showed that in both Experiments 1 and 2, memory performance remained stable after FM, while it declined after EE from 10 min to 1 wk. Moreover, the effect of PK appeared at 10 min after FM while at 1 wk after EE in Experiment 2. PK enhanced memory of word-picture associations when the lures were from the same categories (Experiment 2), rather than from different categories (Experiment 1). These results were largely confirmed in Experiment 3 when encoding condition was manipulated as a between-subjects factor, while lure type as a within-subjects factor. The findings suggest that different from EE, FM facilitates rapid acquisition and consolidation of word-picture knowledge, and highlight that PK plays an important role in this process by enhancing access to detailed information.

Dalton Buysse, Anna-Katharina Pfitzner, Matt West, Aurelien Roux, and Greg Odorizzi

The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, an ubiquitin hydrolase that deubiquitylates transmembrane proteins sorted into ILVs. We report that the non-catalytic N-terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N-terminus inhibits association of Snf7 with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and also with the ATPase Vps4, which is recruited by Vps2 and Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. Our data suggest a mechanism by which the deubiquitylation machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling.

Monica L. Salgado-Lucio, Danelia Ramirez-Ramirez, Coral Y. Jorge-Cruz, Ana L. Roa-Espitia, and Enrique O. Hernandez-Gonzalez

Actin polymerization is a crucial process during sperm capacitation. We have recently described the participation of FAK during actin polymerization in guinea pig spermatozoa. However, the mechanism by which FAK mediates these processes is unknown. Our previous data have shown that MAPK1 (hereafter referred to as ERK2) is activated during the first minutes of capacitation, and inhibition of ERK2 blocked actin polymerization and the acrosome reaction. In this current study, we found that FAK is involved in ERK2 activation – as FAK was phosphorylated at tyrosine residue 925 and bound to Grb2 – and that inhibition of FAK results in a significant decrease of ERK2 activation. We also confirmed the presence of Rho guanine nucleotide exchange factor 2 (ARHGEF2, hereafter referred to as GEF-H1), which is able to associate with RhoA during capacitation. RhoA activation and its participation in actin polymerization were also analyzed. Inhibition of FAK or ERK1/2 impeded GEF-H1 phosphorylation, RhoA activation, and the association between GEF-H1 and RhoA. Finally, we observed the presence of fibronectin on the sperm surface, its role in sperm–sperm interaction as well as participation of β-integrin in the activation of ERK2. Our results show that the signaling pathway downstream of fibronectin, via integrin, FAK, Grb2, MEK1/2, ERK2, GEF-H1 and RhoA regulates the actin polymerization associated with spermatozoa capacitation.

Annelies Wouters, Jan-Pieter Ploem, Sabine A. S. Langie, Tom Artois, Aziz Aboobaker, and Karen Smeets

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Asja Guzman, Rachel C. Avard, Alexander J. Devanny, Oh Sang Kweon, and Laura J. Kaufman

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional β1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

Kristi McElmurry, Jessica E. Stone, Donghan Ma, Phillip Lamoureux, Yueyun Zhang, Michelle Steidemann, Lucas Fix, Fang Huang, Kyle E. Miller, and Daniel M. Suter

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

Sriram Sudarsanam, Shiri Yaniv, Hagar Meltzer, and Oren Schuldiner

The mechanisms that control intrinsic axon growth potential, and thus axon regeneration following injury, are not well understood. Developmental axon regrowth of Drosophila mushroom body -neurons during neuronal remodeling offers a unique opportunity to study the molecular mechanisms controlling intrinsic growth potential. Motivated by the recently uncovered developmental expression atlas of -neurons, we here focus on the role of the actin-severing protein cofilin during axon regrowth. We show that Twinstar (Tsr), the fly cofilin, is a crucial regulator of both axon growth and branching during developmental remodeling of -neurons. tsr mutant axons demonstrate growth defects both in vivo and in vitro, and also exhibit actin-rich filopodial-like structures at failed branch points in vivo. Our data is inconsistent with Tsr being important for increasing G-actin availability. Furthermore, analysis of microtubule localization suggests that Tsr is required for microtubule infiltration into the axon tips and branch points. Taken together, we show that Tsr promotes axon growth and branching, likely by clearing F-actin to facilitate protrusion of microtubules.

The microtubule-binding taxanes, docetaxel and cabazitaxel, are administered intravenously for the treatment of castration-resistant prostate cancer (CRPC) as the oral administration of these drugs is largely hampered by their low and highly variable bioavailabilities. Using a simple, rapid, and environmentally friendly microwave-assisted protocol, we have synthesized a number of 3,5-bis(styryl)pyrazoles 2a-l, thus allowing for their screening for antiproliferative activity in the androgen-independent PC3 prostate cancer cell line. Surprisingly, two of these structurally simple 3,5-bis(styryl)pyrazoles (2a and 2l) had concentrations which gave 50% of the maximal inhibition of cell proliferation (GI₅₀) in the low micromolar range in the PC3 cell line and were thus selected for extensive further biologic evaluation (apoptosis and cell cycle analysis, and effects on tubulin and microtubules). Our findings from these studies show that 3,5-bis[(1E)-2(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l 1) caused significant effects on the cell cycle in PC3 cells, with the vast majority of treated cells in the G₂/M phase (89%); 2) induces cell death in PC3 cells even after the removal of the compound; 3) binds to tubulin [dissociation constant (K_d) 0.4 ± 0.1 μM] and inhibits tubulin polymerization in vitro; 4) had no effect upon the polymerization of the bacterial cell division protein FtsZ (a homolog of tubulin); 5) is competitive with paclitaxel for binding to tubulin but not with vinblastine, crocin, or colchicine; and 6) leads to microtubule depolymerization in PC3 cells. Taken together, these results suggest that 3,5-bis(styryl)pyrazoles warrant further investigation as lead compounds for the treatment of CRPC.

PF-05089771 is an aryl sulfonamide Nav1.7 channel blocker that binds to the inactivated state of Nav1.7 channels with high affinity but binds only weakly to channels in the resting state. Such aryl sulfonamide Nav1.7 channel blockers bind to the extracellular surface of the S1-S4 voltage-sensor segment of homologous Domain 4, whose movement is associated with inactivation. This binding site is different from that of classic sodium channel inhibitors like lidocaine, which also bind with higher affinity to the inactivated state than the resting state but bind at a site within the pore of the channel. The common dependence on gating state with distinct binding sites raises the possibility that inhibition by aryl sulfonamides and by classic local anesthetics might show an interaction mediated by their mutual state dependence. We tested this possibility by examining the state-dependent inhibition by PF-05089771 and lidocaine of human Nav1.7 channels expressed in human embryonic kidney 293 cells. At –80 mV, where a small fraction of channels are in an inactivated state under drug-free conditions, inhibition by PF-05089771 was both enhanced and speeded in the presence of lidocaine. The results suggest that lidocaine binding to the channel enhances PF-05089771 inhibition by altering the equilibrium between resting states (with D4S4 in the inner position) and inactivated states (with D4S4 in the outer position). The gating state–mediated interaction between the compounds illustrates a principle applicable to many state-dependent agents.

Proteinase-activated receptors (PARs) are a four-member family of G-protein–coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)–mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα_q/11- and Gα_i-signaling pathways and is dependent on signaling via the β-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function.

Voltage-gated potassium 11.1 (K_v11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated K_v11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal K_v11.1 forward trafficking and thus reduce functional K_v11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a K_v11.1 allosteric modulator/activator, to rescue K_v11.1 trafficking and produce functional K_v11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-K_v11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT K_v11.1 levels was rescued by 10 μM dofetilide or 10 μM dofetilide + 5 μM LUF7244. In trafficking defective G601S-K_v11.1 cells, dofetilide (10 μM) or dofetilide + LUF7244 (10 + 5 μM) also restored K_v11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased I_Kv11.1 despite the presence of dofetilide (1 μM) in WT K_v11.1 cells. In G601S-expressing cells, long-term treatment (24–48 hour) with LUF7244 (10 μM) and dofetilide (1 μM) increased I_Kv11.1 compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues K_v11.1 trafficking and produces functional I_Kv11.1. Thus, combined administration of LUF7244 and an I_Kv11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced K_v11.1 trafficking defects.

Before it was molecularly cloned in 1994, acute-phase response factor or signal transducer and activator of transcription (STAT)3 was the focus of intense research into understanding the mammalian response to injury, particularly the acute-phase response. Although known to be essential for liver production of acute-phase reactant proteins, many of which augment innate immune responses, molecular cloning of acute-phase response factor or STAT3 and the research this enabled helped establish the central function of Janus kinase (JAK) family members in cytokine signaling and identified a multitude of cytokines and peptide hormones, beyond interleukin-6 and its family members, that activate JAKs and STAT3, as well as numerous new programs that their activation drives. Many, like the acute-phase response, are adaptive, whereas several are maladaptive and lead to chronic inflammation and adverse consequences, such as cachexia, fibrosis, organ dysfunction, and cancer. Molecular cloning of STAT3 also enabled the identification of other noncanonical roles for STAT3 in normal physiology, including its contribution to the function of the electron transport chain and oxidative phosphorylation, its basal and stress-related adaptive functions in mitochondria, its function as a scaffold in inflammation-enhanced platelet activation, and its contributions to endothelial permeability and calcium efflux from endoplasmic reticulum. In this review, we will summarize the molecular and cellular biology of JAK/STAT3 signaling and its functions under basal and stress conditions, which are adaptive, and then review maladaptive JAK/STAT3 signaling in animals and humans that lead to disease, as well as recent attempts to modulate them to treat these diseases. In addition, we will discuss how consideration of the noncanonical and stress-related functions of STAT3 cannot be ignored in efforts to target the canonical functions of STAT3, if the goal is to develop drugs that are not only effective but safe.

Recent studies have strived to find an association between rapid antidepressant effects and a specific subset of pharmacological targets and molecular pathways. Here, we propose a broader hypothesis of encoding, consolidation, and renormalization in depression (ENCORE-D), which suggests that, fundamentally, rapid and sustained antidepressant effects rely on intrinsic homeostatic mechanisms evoked as a response to the acute pharmacological or physiologic effects triggered by the treatment. We review evidence that supports the notion that various treatments with a rapid onset of action, such as ketamine, electroconvulsive therapy, and sleep deprivation, share the ability to acutely excite cortical networks, which increases synaptic potentiation, alters patterns of functional connectivity, and ameliorates depressive symptoms. We proceed to examine how the initial effects are short-lived and, as such, require both consolidation during wake and maintenance throughout sleep to remain sustained. Here, we incorporate elements from the synaptic homeostasis hypothesis and theorize that the fundamental mechanisms of synaptic plasticity and sleep, particularly the homeostatic emergence of slow-wave electroencephalogram activity and the renormalization of synaptic strength, are at the center of sustained antidepressant effects. We conclude by discussing the various implications of the ENCORE-D hypothesis and offer several considerations for future experimental and clinical research.

Technology in bioanalysis, -omics, and computation have evolved over the past half century to allow for comprehensive assessments of the molecular to whole body pharmacology of diverse corticosteroids. Such studies have advanced pharmacokinetic and pharmacodynamic (PK/PD) concepts and models that often generalize across various classes of drugs. These models encompass the "pillars" of pharmacology, namely PK and target drug exposure, the mass-law interactions of drugs with receptors/targets, and the consequent turnover and homeostatic control of genes, biomarkers, physiologic responses, and disease symptoms. Pharmacokinetic methodology utilizes noncompartmental, compartmental, reversible, physiologic [full physiologically based pharmacokinetic (PBPK) and minimal PBPK], and target-mediated drug disposition models using a growing array of pharmacometric considerations and software. Basic PK/PD models have emerged (simple direct, biophase, slow receptor binding, indirect response, irreversible, turnover with inactivation, and transduction models) that place emphasis on parsimony, are mechanistic in nature, and serve as highly useful "top-down" methods of quantitating the actions of diverse drugs. These are often components of more complex quantitative systems pharmacology (QSP) models that explain the array of responses to various drugs, including corticosteroids. Progressively deeper mechanistic appreciation of PBPK, drug-target interactions, and systems physiology from the molecular (genomic, proteomic, metabolomic) to cellular to whole body levels provides the foundation for enhanced PK/PD to comprehensive QSP models. Our research based on cell, animal, clinical, and theoretical studies with corticosteroids have provided ideas and quantitative methods that have broadly advanced the fields of PK/PD and QSP modeling and illustrates the transition toward a global, systems understanding of actions of diverse drugs.

Ubiquitin (UB) transfer cascades consisting of E1, E2, and E3 enzymes constitute a complex network that regulates a myriad of biologic processes by modifying protein substrates. Deubiquitinating enzymes (DUBs) reverse UB modifications or trim UB chains of diverse linkages. Additionally, many cellular proteins carry UB-binding domains (UBDs) that translate the signals encoded in UB chains to target proteins for degradation by proteasomes or in autophagosomes, as well as affect nonproteolytic outcomes such as kinase activation, DNA repair, and transcriptional regulation. Dysregulation of the UB transfer pathways and malfunctions of DUBs and UBDs play causative roles in the development of many diseases. A greater understanding of the mechanism of UB chain assembly and the signals encoded in UB chains should aid in our understanding of disease pathogenesis and guide the development of novel therapeutics. The recent flourish of protein-engineering approaches such as unnatural amino acid incorporation, protein semisynthesis by expressed protein ligation, and high throughput selection by phage and yeast cell surface display has generated designer proteins as powerful tools to interrogate cell signaling mediated by protein ubiquitination. In this study, we highlight recent achievements of protein engineering on mapping, probing, and manipulating UB transfer in the cell.

The first Biograph Vision PET/CT system (Siemens Healthineers) was installed at the University Medical Center Groningen. Improved performance of this system could allow for a reduction in activity administration or scan duration. This study evaluated the effects of reduced scan duration in oncologic ¹⁸F-FDG PET imaging on quantitative and subjective imaging parameters and its influence on clinical image interpretation. Methods: Patients referred for a clinical PET/CT scan were enrolled in this study, received a weight-based ¹⁸F-FDG injected activity, and underwent list-mode PET acquisition at 180 s per bed position (s/bp). Acquired PET data were reconstructed using the vendor-recommended clinical reconstruction protocol (hereafter referred to as "clinical"), using the clinical protocol with additional 2-mm gaussian filtering (hereafter referred to as "clinical+G2"), and—in conformance with European Association of Nuclear Medicine Research Ltd. (EARL) specifications—using different scan durations per bed position (180, 120, 60, 30, and 10 s). Reconstructed images were quantitatively assessed for comparison of SUVs and noise. In addition, clinically reconstructed images were qualitatively evaluated by 3 nuclear medicine physicians. Results: In total, 30 oncologic patients (22 men, 8 women; age: 48–88 y [range], 67 ± 9.6 y [mean ± SD]) received a single weight-based (3 MBq/kg) ¹⁸F-FDG injected activity (weight: 45–123 kg [range], 81 ± 15 kg [mean ± SD]; activity: 135–380 MBq [range], 241 ± 47.3 MBq [mean ± SD]). Significant differences in lesion SUV_max were found between the 180-s/bp images and the 30- and 10-s/bp images reconstructed using the clinical protocols, whereas no differences were found in lesion SUV_peak. EARL-compliant images did not show differences in lesion SUV_max or SUV_peak between scan durations. Quantitative parameters showed minimal deviation (~5%) in the 60-s/bp images. Therefore, further subjective image quality assessment was conducted using the 60-s/bp images. Qualitative assessment revealed the influence of personal preference on physicians’ willingness to adopt the 60-s/bp images in clinical practice. Although quantitative PET parameters differed minimally, an increase in noise was observed. Conclusion: With the Biograph Vision PET/CT system for oncologic ¹⁸F-FDG imaging, scan duration or activity administration could be reduced by a factor of 3 or more with the use of the clinical+G2 or the EARL-compliant reconstruction protocol.

Brain-infiltrating leukocytes contribute to multiple sclerosis (MS) and autoimmune encephalomyelitis and likely play a role in traumatic brain injury, seizure, and stroke. Brain-infiltrating leukocytes are also primary targets for MS disease-modifying therapies. However, no method exists for noninvasively visualizing these cells in a living organism. 1-(2'-deoxy-2'-¹⁸F-fluoroarabinofuranosyl) cytosine (¹⁸F-FAC) is a PET radiotracer that measures deoxyribonucleoside salvage and accumulates preferentially in immune cells. We hypothesized that ¹⁸F-FAC PET could noninvasively image brain-infiltrating leukocytes. Methods: Healthy mice were imaged with ¹⁸F-FAC PET to quantify if this radiotracer crosses the blood–brain barrier (BBB). Experimental autoimmune encephalomyelitis (EAE) is a mouse disease model with brain-infiltrating leukocytes. To determine whether ¹⁸F-FAC accumulates in brain-infiltrating leukocytes, EAE mice were analyzed with ¹⁸F-FAC PET, digital autoradiography, and immunohistochemistry, and deoxyribonucleoside salvage activity in brain-infiltrating leukocytes was analyzed ex vivo. Fingolimod-treated EAE mice were imaged with ¹⁸F-FAC PET to assess if this approach can monitor the effect of an immunomodulatory drug on brain-infiltrating leukocytes. PET scans of individuals injected with 2-chloro-2'-deoxy-2'-¹⁸F-fluoro-9-β-d-arabinofuranosyl-adenine (¹⁸F-CFA), a PET radiotracer that measures deoxyribonucleoside salvage in humans, were analyzed to evaluate whether ¹⁸F-CFA crosses the human BBB. Results: ¹⁸F-FAC accumulates in the healthy mouse brain at levels similar to ¹⁸F-FAC in the blood (2.54 ± 0.2 and 3.04 ± 0.3 percentage injected dose per gram, respectively) indicating that ¹⁸F-FAC crosses the BBB. EAE mice accumulate ¹⁸F-FAC in the brain at 180% of the levels of control mice. Brain ¹⁸F-FAC accumulation localizes to periventricular regions with significant leukocyte infiltration, and deoxyribonucleoside salvage activity is present at similar levels in brain-infiltrating T and innate immune cells. These data suggest that ¹⁸F-FAC accumulates in brain-infiltrating leukocytes in this model. Fingolimod-treated EAE mice accumulate ¹⁸F-FAC in the brain at 37% lower levels than control-treated EAE mice, demonstrating that ¹⁸F-FAC PET can monitor therapeutic interventions in this mouse model. ¹⁸F-CFA accumulates in the human brain at 15% of blood levels (0.08 ± 0.01 and 0.54 ± 0.07 SUV, respectively), indicating that ¹⁸F-CFA does not cross the BBB in humans. Conclusion: ¹⁸F-FAC PET can visualize brain-infiltrating leukocytes in a mouse MS model and can monitor the response of these cells to an immunomodulatory drug. Translating this strategy into humans will require exploring additional radiotracers.

C-X-C motif chemokine receptor 4 (CXCR4) is expressed on the surface of various cell types involved in atherosclerosis, with a particularly rich receptor expression on macrophages and T cells. First pilot studies with ⁶⁸Ga-pentixafor, a novel CXCR4-directed PET tracer, have shown promise to noninvasively image inflammation within atherosclerotic plaques. The aim of this retrospective study was to investigate the performance of ⁶⁸Ga-pentixafor PET/CT for imaging atherosclerosis in comparison to ¹⁸F-FDG PET/CT. Methods: Ninety-two patients (37 women and 55 men; mean age, 62 ± 10 y) underwent ⁶⁸Ga-pentixafor and ¹⁸F-FDG PET/CT for staging of oncologic diseases. In these subjects, lesions in the walls of large arteries were identified using morphologic and PET criteria for atherosclerosis (n = 652). Tracer uptake was measured and adjusted for vascular lumen (background) signal by calculation of target-to-background ratios (TBRs) by 2 investigators masked to the other PET scan. On a lesion-to-lesion and patient basis, the TBRs of both PET tracers were compared and additionally correlated to the degree of arterial calcification as quantified in CT. Results: On a lesion-to-lesion basis, ⁶⁸Ga-pentixafor and ¹⁸F-FDG uptake showed a weak correlation (r = 0.28; P < 0.01). ⁶⁸Ga-pentixafor PET identified more lesions (n = 290; TBR ≥ 1.6, P < 0.01) and demonstrated higher uptake than ¹⁸F-FDG PET (1.8 ± 0.5 vs. 1.4 ± 0.4; P < 0.01). The degree of plaque calcification correlated negatively with both ⁶⁸Ga-pentixafor and ¹⁸F-FDG uptake (r = –0.38 vs. –0.31, both P < 0.00001). Conclusion: CXCR4-directed imaging of the arterial wall with ⁶⁸Ga-pentixafor PET/CT identified more lesions than ¹⁸F-FDG PET/CT, with only a weak correlation between tracers. Further studies to elucidate the underlying biologic mechanisms and sources of CXCR4 positivity, and to investigate the clinical utility of chemokine receptor–directed imaging of atherosclerosis, are highly warranted.

Molecular radiotherapy using ¹⁷⁷Lu-DOTATATE is a most effective treatment for somatostatin receptor–expressing neuroendocrine tumors. Despite its frequent and successful use in the clinic, little or no radiobiologic considerations are made at the time of treatment planning or delivery. On positive uptake on octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiologic and DNA damage effects in the tumor. Here, we visualized and quantified the extent of DNA damage response after ¹⁷⁷Lu-DOTATATE therapy using SPECT imaging with ¹¹¹In-anti-H2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with β-emitting therapeutic radiopharmaceuticals. Methods: Six cell lines were exposed to external-beam radiotherapy (EBRT) or ¹⁷⁷Lu-DOTATATE, after which the number of H2AX foci and the clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor–positive tumor xenografts were treated with ¹⁷⁷Lu-DOTATATE or sham-treated and coinjected with ¹¹¹In-anti-H2AX-TAT, ¹¹¹In-IgG-TAT control, or vehicle. Results: Clonogenic survival after external-beam radiotherapy was cell-line–specific, indicating varying levels of intrinsic radiosensitivity. Regarding in vitro cell lines treated with ¹⁷⁷Lu-DOTATATE, clonogenic survival decreased and H2AX foci increased for cells expressing high levels of somatostatin receptor subtype 2. Ex vivo measurements revealed a partial correlation between ¹⁷⁷Lu-DOTATATE uptake and H2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to ¹⁷⁷Lu-DOTATATE irradiation. Conclusion: ¹¹¹In-anti-H2AX-TAT allows monitoring of DNA damage after ¹⁷⁷Lu-DOTATATE therapy and reveals heterogeneous damage responses.

¹⁸F-prostate-specific membrane antigen (PSMA)-1007 is excreted mainly through the liver. We benchmarked the performance of ¹⁸F-PSMA-1007 against 3 renally excreted PSMA tracers. Methods: Among 668 patients, we selected 27 in whom PET/CT results obtained with ⁶⁸Ga-PSMA-11, ¹⁸F-DCFPyL (2-(3-(1-carboxy-5-[(6-[¹⁸F]fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid), or ¹⁸F-JK-PSMA-7 (JK, Juelich-Koeln) were interpreted as equivocal or negative or as oligometastatic disease (PET-1). Within 3 wk, a second PET scan with ¹⁸F-PSMA-1007 was performed (PET-2). The confidence in the interpretation of PSMA-positive locoregional findings was scored on a 5-point scale, first in routine diagnostics (reader 1) and then by an independent second evaluation (reader 2). Discordant PSMA-positive skeletal findings were examined by contrast-enhanced MRI. Results: For both readers, ¹⁸F-PSMA-1007 facilitated the interpretability of 27 locoregional lesions. In PET-2, the clinical readout led to a significantly lower number of equivocal locoregional lesions (P = 0.024), and reader 2 reported a significantly higher rate of suspected lesions that were falsely interpreted as probably benign in PET-1 (P = 0.023). Exclusively in PET-2, we observed a total of 15 PSMA-positive spots in the bone marrow of 6 patients (22%). None of the 15 discordant spots had a morphologic correlate on the corresponding CT scan or on the subsequent MRI scan. Thus, ¹⁸F-PSMA-1007 exhibits a significantly higher rate of unspecific medullary spots (P = 0.0006). Conclusion: ¹⁸F-PSMA-1007 may increase confidence in interpreting small locoregional lesions adjacent to the urinary tract but may decrease the interpretability of skeletal lesions.

The aim of this study was to evaluate the efficacy of ¹⁷⁷Lu-prostate-specific membrane antigen (PSMA)-617 (¹⁷⁷Lu-PSMA) and selective internal radiation therapy (SIRT) for the treatment of liver metastases of castration-resistant prostate cancer. Methods: Safety and survival of patients with metastatic castration-resistant prostate cancer and liver metastases assigned to ¹⁷⁷Lu-PSMA alone (n = 31) or in combination with SIRT (n = 5) were retrospectively analyzed. Additionally, a subgroup (n = 10) was analyzed using morphologic and molecular response criteria. Results: Median estimated survival was 5.7 mo for ¹⁷⁷Lu-PSMA alone and 8.4 mo for combined sequential ¹⁷⁷Lu-PSMA and SIRT. ¹⁷⁷Lu-PSMA achieved discordant therapy responses with both regressive and progressive liver metastases in the same patient (best vs. worst responding metastases per patient: –35% vs. +63% diameter change; P < 0.05). SIRT was superior to ¹⁷⁷Lu-PSMA for the treatment of liver metastases (0% vs. 56% progression). Conclusion: The combination of ¹⁷⁷Lu-PSMA and SIRT is efficient and feasible for the treatment of advanced prostate cancer. ¹⁷⁷Lu-PSMA alone seems to have limited response rates in the treatment of liver metastases.

¹⁸F-rhPSMA-7 (radiohybrid prostate-specific membrane antigen [PSMA]) is a novel ligand for PET imaging. Here, we present data from a retrospective analysis using PET/CT and PET/MRI examinations to investigate the efficacy of ¹⁸F-rhPSMA-7 PET for primary N-staging of patients with prostate cancer (PC) compared with morphologic imaging (CT or MRI) and validated by histopathology. Methods: Data from 58 patients with high-risk PC (according to the D’Amico criteria) who were staged with ¹⁸F-rhPSMA-7 PET/CT or PET/MRI at our institution between July 2017 and June 2018 were reviewed. The patients had a median prescan prostate-specific antigen value of 12.2 ng/mL (range, 1.2–81.6 ng/mL). The median injected activity of ¹⁸F-rhPSMA-7 was 327 MBq (range, 132–410 MBq), with a median uptake time of 79.5 min (range, 60–153 min). All patients underwent subsequent radical prostatectomy and extended pelvic lymph node dissection. The presence of lymph node metastases was determined by an experienced reader independently for both the PET and the morphologic datasets using a template-based analysis on a 5-point scale. Patient-level and template-based results were both compared with histopathologic findings. Results: Lymph node metastases were present in 18 patients (31.0%) and were located in 52 of 375 templates (13.9%). Receiver-operating-characteristic analyses showed ¹⁸F-rhPSMA-7 PET to perform significantly better than morphologic imaging on both patient-based and template-based analyses (areas under curve, 0.858 vs. 0.649 [P = 0.012] and 0.765 vs. 0.589 [P < 0.001], respectively). On patient-based analyses, the sensitivity, specificity, and accuracy of ¹⁸F-rhPSMA-7 PET were 72.2%, 92.5%, and 86.2%, respectively, and those of morphologic imaging were 50.0%, 72.5%, and 65.5%, respectively. On template-based analyses, the sensitivity, specificity, and accuracy of ¹⁸F-rhPSMA-7 PET were 53.8%, 96.9%, and 90.9%, respectively, and those of morphologic imaging were 9.6%, 95.0%, and 83.2%, respectively. Conclusion: ¹⁸F-rhPSMA-7 PET is superior to morphologic imaging for N-staging of high-risk primary PC. The efficacy of ¹⁸F-rhPSMA-7 is similar to published data for ⁶⁸Ga-PSMA-11.

Neuroendocrinelike transdifferentiation of prostate cancer adenocarcinomas correlates with serum levels of chromogranin A (CgA) and drives treatment resistance. The aim of this work was to evaluate whether CgA can serve as a response predictor for ¹⁷⁷Lu-prostate-specific membrane antigen 617 (PSMA) radioligand therapy (RLT) in comparison with the established tumor markers. Methods: One hundred consecutive patients with metastasized castration-resistant prostate cancer scheduled for PSMA RLT were evaluated for prostate-specific antigen (PSA), lactate dehydrogenase (LDH), and CgA at baseline and in follow-up of PSMA RLT. Tumor uptake of PSMA ligand, a known predictive marker for response, was assessed as a control variable. Results: From the 100 evaluated patients, 35 had partial remission, 16 stable disease, 15 mixed response, and 36 progression of disease. Tumor uptake above salivary gland uptake translated into partial remission, with an odds ratio (OR) of 60.265 (95% confidence interval [CI], 5.038–720.922). Elevated LDH implied a reduced chance for partial remission, with an OR of 0.094 (95% CI, 0.017–0.518), but increased the frequency of progressive disease (OR, 2.717; 95% CI, 1.391–5.304). All patients who achieved partial remission had a normal baseline LDH. Factor-2 elevation of CgA increased the risk for progression, with an OR of 3.089 (95% CI, 1.302–7.332). Baseline PSA had no prognostic value for response prediction. Conclusion: In our cohort, baseline PSA had no prognostic value for response prediction. LDH was the marker with the strongest prognostic value, and elevated LDH increased the risk for progression of disease under PSMA RLT. Elevated CgA demonstrated a moderate impact as a negative prognostic marker in general but was explicitly related to the presence of liver metastases. Well in line with the literature, sufficient tumor uptake is a prerequisite to achieve tumor response.

Prostate-specific membrane antigen (PSMA)–targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT–nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with ²²⁵Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair–associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0–6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair–targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

In around 20% of men with prostate cancer, metastasis develops during the course of their disease. Accordingly, discovering and developing new potent treatment strategies for patients with metastatic prostate cancer has been a major research focus during the last few decades. Identifying disease progression, especially within clinical trials, is essential in determining drug effectiveness. One major remaining question is how best to define disease progression. The criteria of the Prostate Cancer Clinical Trials Working Group (PCWG2) include clinical and laboratory parameters, as well as conventional imaging modalities such as MRI, CT, and bone scan findings, but advanced molecular imaging techniques, especially prostate-specific membrane antigen (PSMA) PET findings, are not considered. This is a problem because PSMA PET is used not only for detecting biochemical recurrence but also for restaging and as an intermediate-endpoint biomarker in ongoing clinical trials. Therefore, response criteria and PSMA PET progression (PPP) criteria need to be established with some urgency. The intent of this article is therefore to define prostate cancer progression by PSMA PET criteria. Our PPP proposal is based on the same principles as were applied for the PCGW2 criteria but adds value by including PSMA PET criteria. PPP defines PSMA treatment response using 3 different criteria. The first is the appearance of 2 or more new PSMA-positive distant lesions. The second is the appearance of 1 new PSMA-positive lesion plus consistent clinical or laboratory data and recommended confirmation by biopsy or correlative imaging within 3 mo of PSMA PET. The third is an increase in size or PSMA uptake of 1 or more existing lesions by at least 30%, plus consistent clinical or laboratory data or confirmation by biopsy or correlative imaging within 3 mo of PSMA PET.

For effective clinical management of patients being treated with ²²³Ra, there is a need for radiographic response biomarkers to minimize disease progression and to stratify patients for subsequent treatment options. The objective of this study was to evaluate an automated bone scan index (aBSI) as a quantitative assessment of bone scans for radiographic response in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods: In a multicenter retrospective study, bone scans from patients with mCRPC treated with monthly injections of ²²³Ra were collected from 7 hospitals in Sweden. Patients with available bone scans before treatment with ²²³Ra and at treatment discontinuation were eligible for the study. The aBSI was generated at baseline and at treatment discontinuation. The Spearman rank correlation was used to correlate aBSI with the baseline covariates: alkaline phosphatase (ALP) and prostate-specific antigen (PSA). The Cox proportional-hazards model and Kaplan–Meier curve were used to evaluate the association of covariates at baseline and their change at treatment discontinuation with overall survival (OS). The concordance index (C-index) was used to evaluate the discriminating strength of covariates in predicting OS. Results: Bone scan images at baseline were available from 156 patients, and 67 patients had both a baseline and a treatment discontinuation bone scan (median, 5 doses; interquartile range, 3–6 doses). Baseline aBSI (median, 4.5; interquartile range, 2.4–6.5) was moderately correlated with ALP (r = 0.60, P < 0.0001) and with PSA (r = 0.38, P = 0.003). Among baseline covariates, aBSI (P = 0.01) and ALP (P = 0.001) were significantly associated with OS, whereas PSA values were not (P = 0.059). After treatment discontinuation, 36% (24/67), 80% (54/67), and 13% (9/67) of patients demonstrated a decline in aBSI, ALP, and PSA, respectively. As a continuous variable, the relative change in aBSI after treatment, compared with baseline, was significantly associated with OS (P < 0.0001), with a C-index of 0.67. Median OS in patients with both aBSI and ALP decline (median, 134 wk) was significantly longer than in patients with ALP decline only (median, 77 wk; P = 0.029). Conclusion: Both aBSI at baseline and its change at treatment discontinuation were significant parameters associated with OS. The study warrants prospective validation of aBSI as a quantitative imaging response biomarker to predict OS in patients with mCRPC treated with ²²³Ra.

The incidence of abnormal cerebrospinal fluid (CSF) flow dynamics in children with central nervous system (CNS) tumors before intraventricular therapy has not been described. Methods: We performed a single-institution, retrospective review of patients with primary or metastatic CNS tumors treated between 2003 and 2018 (15 y). Patients underwent ¹¹¹In-diethylenetriaminepentaacetic acid injection into the CSF intraventricular space followed by nuclear medicine imaging at 90 min, 4 h, 24 h, and 48 h (if required). CSF flow was classified as normal, delayed, asymmetric, or obstructed. Results: In total, 278 CSF flow studies were performed on 224 patients, 202 of whom (90%) were less than 18 y old. Of these, 116 patients (52%) had metastatic CNS neuroblastoma, 57 (25%) had medulloblastoma, and 51 (23%) had other histologic types of CNS tumors. Of the 278 studies, 237 (85%) were normal, 9 (3%) required neurosurgical intervention, 25 (9%) were delayed, and 7 (3%) were asymmetric. Conclusion: Abnormal CSF flow and the necessity for neurosurgical intervention must be considered when attempting to ensure appropriate intraventricular therapy in the pediatric population.

Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2–3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence-guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 ± 2.5 (mean ± SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM.