In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

The regulation of transposable element (TE) activity by small RNAs is a ubiquitous feature of germlines. However, despite the obvious benefits to the host in terms of ensuring the production of viable gametes and maintaining the integrity of the genomes they carry, it remains controversial whether TE regulation evolves adaptively. We examined the emergence and evolutionary dynamics of repressor alleles after P-elements invaded the Drosophila melanogaster genome in the mid-twentieth century. In many animals including Drosophila, repressor alleles are produced by transpositional insertions into piRNA clusters, genomic regions encoding the Piwi-interacting RNAs (piRNAs) that regulate TEs. We discovered that ~94% of recently collected isofemale lines in the Drosophila melanogaster Genetic Reference Panel (DGRP) contain at least one P-element insertion in a piRNA cluster, indicating that repressor alleles are produced by de novo insertion at an exceptional rate. Furthermore, in our sample of approximately 200 genomes, we uncovered no fewer than 80 unique P-element insertion alleles in at least 15 different piRNA clusters. Finally, we observe no footprint of positive selection on P-element insertions in piRNA clusters, suggesting that the rapid evolution of piRNA-mediated repression in D. melanogaster was driven primarily by mutation. Our results reveal for the first time how the unique genetic architecture of piRNA production, in which numerous piRNA clusters can encode regulatory small RNAs upon transpositional insertion, facilitates the nonadaptive rapid evolution of repression.

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.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation.

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.

Pancreatic cancer is one of the most lethal human malignancies, partly because of its propensity for metastasis. However, the mechanisms of metastasis in pancreatic cancer remain unclear. Oxidized low-density lipoprotein receptor 1 (OLR1), a lectin-like scavenger receptor that recognizes several ligands, such as oxidized low-density lipoprotein, was previously reported in cardiovascular and metabolic diseases. The role and mechanism of OLR1 in pancreatic cancer is unclear. In this study, we found that OLR1 expression was significantly higher in pancreatic cancer tissues than that in adjacent normal tissues and closely associated with reduced overall survival. OLR1 promoted proliferation and metastasis of pancreatic cancer cells in vitro and in vivo. Mechanistically, OLR1 increased HMGA2 transcription by upregulating c-Myc expression to promote the metastasis of pancreatic cancer cells. In addition, patients with pancreatic cancer with high expression of OLR1–c-Myc–HMGA2 axis showed worse prognosis compared with patients with low expression of OLR1–c-Myc–HMGA2 axis.

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.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit ‎CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1β–induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6–induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-B pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1β–induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine–derived, disease-modifying, antirheumatic herbal drug.

Extensive studies have shown that the ₁ receptor (₁R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of ₁R as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates ₁R oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine ₁R oligomerization. In transfected cells, ₁R exhibited as multimers, dimers, and monomers. Overall, ₁R agonists decreased, whereas ₁R antagonists increased ₁R multimers, suggesting that agonists and antagonists differentially affect the stability of ₁R multimers. Endogenous ₁R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N terminus reduced ₁R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of ₁R. The E102Q ₁R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of ₁R multimeric assembly may impair its function. Interestingly, oligomerization of ₁R was pH-dependent and correlated with changes in [³H](+)-pentazocine binding affinity and B_max. Combined with mutational analysis, it is reasoned that ₁R multimers possess high-affinity and high-capacity [³H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that ₁R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated ₁R multimerization may provide novel approaches to modulate the function of ₁R and its interacting proteins.

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.

Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain.

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.

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.

Abicipar pegol (abicipar) is a novel DARPin therapeutic and highly potent vascular endothelial growth factor (VEGF) inhibitor intended for the treatment of neovascular age-related macular degeneration (nAMD). Here we develop a translational pharmacokinetic/pharmacodynamic (PK/PD) model for abicipar to guide dosing regimens in the clinic. The model incorporated abicipar-VEGF binding kinetics, VEGF expression levels, and VEGF turnover rates to describe the ocular and systemic PK data collected from the vitreous, aqueous humor (AH), choroid, retina, and serum of rabbits after a 1-mg abicipar intravitreal (IVT) dose. The model was translated to humans using human-specific mechanistic parameters and refitted to human serum and AH concentrations from patients with diabetic macular edema and nAMD. The model was then used to simulate 8-, 12- (quarterly), and 16-week dosing intervals in the clinic. Simulations of 2 mg abicipar IVT at 8-week or quarterly dosing in humans indicates minimum steady-state vitreal concentrations are maintained above both in vitro IC₅₀ and in vivo human IC₅₀ values. The model predicted virtually complete VEGF inhibition for the 8-week and quarterly dosing schedule during the 52-week treatment period. In the 16-week schedule, clinically significant VEGF inhibition was maintained during the 52-week period. The model quantitatively described abicipar-VEGF target engagement leading to rapid reduction of VEGF and a long duration of VEGF inhibition demonstrating the clinical feasibility of up to a 16-week dosing interval. Abicipar is predicted to reduce IVT dosing compared with other anti-VEGF therapies with the potential to lessen patient treatment burden.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [¹³C₁₆]-PA was methylated to form [¹³C₁₆]-PAME in incubation medium or the Krebs–Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis.

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.

We investigated whether cycloheximide (CHX) would induce amnesia for the stress-induced impairment of extinction retrieval. First, a single restraint stress session was demonstrated to impair extinction retrieval, but not fear conditioning. A second experiment showed that when CHX was administered immediately after restraint, rats exhibited significant extinction retrieval at test (i.e., retrograde amnesia for the stress). In a third experiment, the stress session impaired various amounts of extinction durations, suggesting that the stress inhibited extinction retrieval rather than enhancing the original fear learning. These results suggest memories for acute stress are susceptible to disruption, which could have clinical implications.

Xiaofei Cao, Sergio Lilla, Zhenbo Cao, Marie Anne Pringle, Ojore B. V. Oka, Philip J. Robinson, Tomasz Szmaja, Marcel van Lith, Sara Zanivan, and Neil J. Bulleid

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.

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.

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.

Yi Liu, Jaeyoun Kim, Reuben Philip, Vaishali Sridhar, Megha Chandrashekhar, Jason Moffat, Mark van Breugel, and Laurence Pelletier

PLK4 has emerged as a prime target for cancer therapeutics, and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind the role of PLK4 in these processes still remains only partly understood. Our previous work has demonstrated that an intact CEP85–STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here, we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL to the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 (also known as ACTR2) phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85–STIL complex in modulating PLK4-driven cancer cell migration.

This article has an associated First Person interview with the first author of the paper.

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the μ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy.

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.

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.

When we critically assess the reason for the current dominance of ⁶⁸Ga-labeled peptides and peptide-like ligands in radiopharmacy and nuclear medicine, we have to conclude that the major advantage of such radiopharmaceuticals is the apparent lack of suitable ¹⁸F-labeling technologies with proven clinical relevance. To prepare and to subsequently perform a clinical proof-of-concept study on the general suitability of silicon-fluoride-acceptor (SiFA)–conjugated radiopharmaceuticals, we developed inhibitors of the prostate-specific membrane antigen (PSMA) that are labeled by isotopic exchange (IE). To compensate for the pronounced lipophilicity of the SiFA unit, we used metal chelates, conjugated in close proximity to SiFA. Six different radiohybrid PSMA ligands (rhPSMA ligands) were evaluated and compared with the commonly used ¹⁸F-PSMA inhibitors ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007. Methods: All inhibitors were synthesized by solid-phase peptide synthesis. Human serum albumin binding was measured by affinity high-performance liquid chromatography, whereas the lipophilicity of each tracer was determined by the n-octanol/buffer method. In vitro studies (IC₅₀, internalization) were performed on LNCaP cells. Biodistribution studies were conducted on LNCaP tumor–bearing male CB-17 SCID mice. Results: On the laboratory scale (starting activities, 0.2–9.0 GBq), labeling of ¹⁸F-rhPSMA-5 to -10 by IE was completed in < 20 min (radiochemical yields, 58% ± 9%; radiochemical purity, >97%) with molar activities of 12–60 GBq/μmol. All rhPSMAs showed low nanomolar affinity and high internalization by PSMA-expressing cells when compared with the reference radiopharmaceuticals, medium-to-low lipophilicity, and high human serum albumin binding. Biodistribution studies in LNCaP tumor–bearing mice revealed high tumor uptake, sufficiently fast clearance kinetics from blood, low hepatobiliary excretion, fast renal excretion, and very low uptake of ¹⁸F activity in bone. Conclusion: The novel ¹⁸F-rhPSMA radiopharmaceuticals developed under the radiohybrid concept show equal or better targeting characteristics than the established ¹⁸F-PSMA tracers ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007. The unparalleled simplicity of production, the possibility to produce the identical ⁶⁸Ga-labeled ¹⁹F-⁶⁸Ga-rhPSMA tracers, and the possibility to extend this concept to true theranostic radiohybrid radiopharmaceuticals, such as F-Lu-rhPSMA, are unique features of these radiopharmaceuticals.

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.

⁶⁸Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as ⁶⁸Ga-PSMA-11, are promising small molecules for targeting prostate cancer (PCa). Although this radiopharmaceutical was produced mostly by means of manual synthesis and automated synthesis modules, a sterile cold kit was recently introduced. The aim of our study was to evaluate the image quality of ⁶⁸Ga-PSMA-11 PET/CT (PSMA-PET) in a population of PCa patients after the injection of comparable activities of ⁶⁸Ga-PSMA-11 obtained with the 2 different synthetic procedures. A secondary aim was to identify secondary factors that may have an impact on image quality and, thus, final interpretation. Methods: Two different groups of 100 consecutive PCa patients who underwent PSMA-PET were included in the study. The first group of patients was imaged with ⁶⁸Ga-PSMA-11 obtained using synthesis modules, whereas the second group’s tracer activity was synthesized using a sterile cold kit. All PET images were independently reviewed by 2 nuclear medicine diagnosticians with at least 2 y of experience in PSMA-based imaging and unaware of the patients’ clinical history. The 2 reviewers independently rated the quality of each PSMA-PET scan using a 3-point Likert-type scale. In cases of discordance, the operators together reviewed the images and reached a consensus. Performance was evaluated on the basis of the expected biodistribution, lesion detection rate, and physiologic background uptake. Results: Overall, 104 of 200 (52%) PSMA-PET scans were positive for PCa-related findings. No significant differences in image quality between cold kits and synthesis modules were found (P = 0.13), although a higher proportion of images was rated as excellent by the observers for kits than for modules (45% vs. 34%). Furthermore, after image quality had been dichotomized as excellent or not excellent, multivariate regression analysis found several factors to be significantly associated with a not-excellent quality: an increase in patient age (+5 y: odds ratio [OR], 1.40; 95% confidence interval [CI], 1.12–1.75), an increase in patient weight (+5 kg: OR, 1.89; 95% CI, 1.53–2.32), an increase in ⁶⁸Ga-PSMA-11 uptake time (+10 min: OR, 1.45; 95% CI, 1.08–1.96), and a decrease in injected activity (–10 MBq: OR, 1.28; 95% CI, 1.07–1.52). Conclusion: No significant differences were identified between the 2 groups of patients undergoing PSMA-PET; therefore, we were not able to ascertain any significant influences of tracer production methodology on final scan quality. However, increased patient age, increased patient weight, decreased injected activity, and increased ⁶⁸Ga-PSMA-11 uptake time were significantly associated with an overall poorer image quality.

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.

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.

Monoclonal antibodies (mAbs) against programmed cell death 1 (PD-1), such as nivolumab and pembrolizumab, are associated with high response rates in patients with relapsed or refractory classic Hodgkin lymphoma (HL). To date, no prognostic factor for overall survival (OS) has been established with these agents in HL. We examined whether the first early response assessment evaluated using ¹⁸F-FDG PET/CT may be associated with OS in this setting. Methods: This retrospective study included 45 patients from 34 institutions. In a masked, centralized review, 3 independent radiologists classified PET/CT scans obtained at a median of 2.0 mo (interquartile range, 1.7–3.7 mo) after nivolumab initiation using existing criteria (i.e., 2014 Lugano classification and 2016 LYRIC). Patients were classified according to 4 possible response categories: complete metabolic response (CMR), partial metabolic response (PMR), no metabolic response (NMR), or progressive metabolic disease (PMD). Because the OS of patients with NMR and PMR was similar, they were grouped together. OS was estimated using the Kaplan–Meier method and compared between groups using log-rank testing. Results: Eleven patients (24%) died after a median follow-up of 21.2 mo. The classification was identical between Lugano and LYRIC because all 16 progression events classified as indeterminate response per LYRIC were confirmed on subsequent evaluations. Both Lugano and LYRIC classified patients as CMR in 13 cases (29%), PMD in 16 (36%), NMR in 4 (9%), and PMR in 12 (27%). The 2-y OS probability was significantly different in patients with PMD (0.53; 95% confidence interval [95%CI], 0.32–0.87), NMR or PMR (0.80; 95%CI, 0.63–1.00), and CMR (1.00; 95%CI, 1.00–1.00) in the overall population (P = 0.02, 45 patients), as well as according to a landmark analysis at 3 mo (P = 0.05, 32 patients). Conclusion: In relapsed or refractory HL patients treated with anti-PD-1 mAbs, the first early PET/CT assessment using either Lugano or LYRIC predicted OS and allowed early risk stratification, suggesting that PET/CT might be used to develop risk-adapted strategies.

Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may remain asymptomatic, leading to under-recognition of the related disease, coronavirus disease, 2019 (COVID-19), and to incidental findings in nuclear imaging procedures performed for standard clinical indications. Here, we report about our local experience in a region with high COVID-19 prevalence and dynamically increasing infection rates. Methods: Within the 8-d period of March 16–24, 2020, hybrid imaging studies of asymptomatic patients who underwent ¹⁸F-FDG PET/CT or ¹³¹I SPECT/CT for standard oncologic indications at our institution in Brescia, Italy, were analyzed for findings suggestive of COVID-19. The presence, radiologic features, and metabolic activity of interstitial pneumonia were identified, correlated with the subsequent short-term clinical course, and described in a case series. Results: Six of 65 patients (9%) who underwent PET/CT for various malignancies showed unexpected signs of interstitial pneumonia on CT and elevated regional ¹⁸F-FDG avidity. Additionally, 1 of 12 patients who received radioiodine for differentiated thyroid carcinoma also showed interstitial pneumonia on SPECT/CT. Five of 7 patients had subsequent proof of COVID-19 by reverse-transcriptase polymerase chain reaction. The remaining 2 patients were not tested immediately but underwent quarantine and careful monitoring. Conclusion: Incidental findings suggestive of COVID-19 may not be infrequent in hybrid imaging of asymptomatic patients in regions with an expansive spread of SARS-CoV-2. Nuclear medicine services should prepare accordingly.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N⁶-methyladenosine (m⁶A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m⁶A in 18S rRNA at position A₁₈₃₂. We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m⁶A in rRNA in stemness, differentiation, development, and diseases.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs’ role in localizing the BLM–TOP3A–RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAF^V600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRAS^Q61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAF^V600E PDX highlighting its effectiveness in combating the advent of drug resistance.