In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Tumor-Derived PGE2 Gives NK Cells a Headache.

Tumors use active immunosuppressive mechanisms to evade immune recognition and shape the local inflammatory environment. In this issue of Immunity, Bonavita et al. report that tumor-derived PGE2 blocks early activation of natural killer cells and interferes with subsequent adaptive immune cell recruitment to the tumor.

The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine.

Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes1-3. Recent data have indicated that thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat4,5. This model was based on the super-stoichiometric relationship between the amount of creatine added to mitochondria and the quantity of oxygen consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling activity in mice. Thermogenic fat cells have robust phosphocreatine phosphatase activity, which is attributed to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Unlike in other cells, TNAP in thermogenic fat cells is localized to the mitochondria, where futile creatine cycling occurs. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole-body energy expenditure and leads to rapid-onset obesity in mice, with no change in movement or feeding behaviour. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle.

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

Identity Crisis: CD301b(+) Mononuclear Phagocytes Blur the M1-M2 Macrophage Line.

Obesity shifts the immune phenotype from M2 macrophage polarization to M1, which causes metabolic dysfunction. In this issue of Immunity, Kumamoto et al. (2016) identify a tissue-resident mononuclear phagocyte population that promotes weight gain and glucose intolerance but are defined by the M2 marker CD301b.

Iron deficiency among Japanese whole-blood donors measured by serum ferritin.

BACKGROUND AND OBJECTIVES: A more restrictive blood donation criterion has been applied in Japan, with a maximum volume of whole blood (WB) donation of 400 mL, allowing twice a year for female donors and thrice a year for male donors. However, iron deficiency was as high as 20.5% among female donors prior to donation, increasing to 37.7% after blood donation. More than 20 years have passed since then, so we set out to investigate the present situation. MATERIALS AND METHODS: A total of 2659 (male/female: 1496/1163) donors of 400 mL WB who gave informed consent to join the study were enrolled. Serum ferritin (sFer) of first-time/reactivated (FT/RA) donors were compared with those of repeat donors, according to gender and age; those who returned for subsequent donations during the study period were also followed up. RESULTS: About one-third of FT/RA female donors had iron deficiency, possibly reflecting its high incidence among the general population. Interestingly, although sFer levels were low among pre-menopausal FT/RA female donors, these values were not much different in repeat donors, whereas significant differences were observed between FT/RA and repeat donors among post-menopausal females and in most age groups among males. As expected, donors with a normal initial sFer (≥26 ng/mL) recovered faster than those with a low initial sFer. CONCLUSION: Female donors, especially, have iron deficiency even before donation, and the rate increased compared to what was found previously. Measures to prevent iron deficiency of blood donors is required, and studies are going on in Japan.

Regioselective Transition Metal-Free Catalytic Ring Opening of 2H-Azirines by Phenols and Naphthols; One-Pot Access to Benzo- and Naphthofurans.

Benzofuran and naphthofuran derivatives are synthesized from readily available phenols and naphthols. Regioselective ring openings of 2H-azirine followed by in situ aromatization using a catalytic amount of Brønsted acid have established the novelty of the methodology. The involvement of a series of 2H-azirines with a variety of phenols, 1-naphthols, and 2-naphthols showed the generality of the protocol. In-depth density functional theory calculations revealed the reaction mechanism with the energies of the intermediates and transition states of a model reaction. An alternate pathway of the mechanism has also been proposed with computer modeling.

Regioselection Switch in Nucleophilic Addition to Isoquinolinequinones: Mechanism and Origin of the Regioselectivity in the Total Synthesis of Ellipticine.

Ellipticine was synthesized in six steps and 20% global yield starting from the readily available 2,5-dimethoxy isoquinoline. Unprecedented regioselective control of the nucleophilic attack on the isoquinoline-5,8-dione is first described. Investigation of the possible pathways of this transformation through density functional theory calculations reveals unexpected N-oxide assistance in cascade tautomerizations, which was crucial for directing the nucleophilic attack and hastening the overall process. Using this strategy, we prepared the aniline-isoquinolinedione adduct and submitted it to an intramolecular double C-H cross-coupling activation to furnish ellipticinequinone, which gave ellipticine after a MeLi addition/BH3 reduction sequence.

How can clinicians choose between conflicting and discordant systematic reviews? A replication study of the Jadad algorithm.

INTRODUCTION: The exponential growth of published systematic reviews (SRs) presents challenges for decision makers seeking to answer clinical, public health or policy questions. In 1997, an algorithm was created by Jadad et al. to choose the best SR across multiple. Our study aims to replicate author assessments using the Jadad algorithm to determine: (i) if we chose the same SR as the authors; and (ii) if we reach the same results. METHODS: We searched MEDLINE, Epistemonikos, and Cochrane Database of SRs. We included any study using the Jadad algorithm. We used consensus building strategies to operationalise the algorithm and to ensure a consistent approach to interpretation. RESULTS: We identified 21 studies that used the Jadad algorithm to choose one or more SRs. In 62% (13/21) of cases, we were unable to replicate the Jadad assessment and ultimately chose a different SR than the authors. Overall, 18 out of the 21 (86%) independent Jadad assessments agreed in direction of the findings despite 13 having chosen a different SR. CONCLUSIONS: Our results suggest that the Jadad algorithm is not reproducible between users as there are no prescriptive instructions about how to operationalise the algorithm. In the absence of a validated algorithm, we recommend that healthcare providers, policy makers, patients and researchers address conflicts between review findings by choosing the SR(s) with meta-analysis of RCTs that most closely resemble their clinical, public health, or policy question, are the most recent, comprehensive (i.e. number of included RCTs), and at the lowest risk of bias.

Theoretical investigation of the cooperative effect of solvent: a case study.

The effect of solvent was investigated at the DFT level, M06-2X/6-31++G(d,p), for the implicit, namely the universal solvent model based on solute electron density (SMD) and hybrid solvation models, and a new pathway for the Markovnikov for the addition of HCl to 1-butene was suggested, incorporating the solvent in the reaction. The results showed that the use of implicit solvent brings greater stabilization for a large part of the reaction coordinates and for the charges of the transition states (TS). Studying the hybrid solvent model, it was shown by quantum mechanics and molecular simulations that although the first solvation shell is composed of approximately 30 solvent molecules, most of the effect comes from just eight solvent molecules explicitly added, with a variation in energy that tends to about -19.3 kJ mol-1. The reaction rates for the hydration of 1-butene were only able to achieve a reasonable accuracy with the addition of three explicit solvent molecules of 5.97 × 10-8 M-1 s-1 and 2.33 × 10-9 M-1 s-1 for the calculated and the experimental values, respectively. This indicates that not only hybrid solvation may be required, but also the number of explicit molecules added may heavily influence the calculated reaction rates. It was found that for the intermediary product of hydration the hydrogen bonds are stronger than average, suggesting a partial covalent characteristic.

Comparison of 2 Weekly Frequencies of Resistance Training on Muscular Strength, Body Composition, and Metabolic Biomarkers in Resistance-Trained Older Women: Effects of Detraining and Retraining.

ABSTRACT: Amarante do Nascimento, M, Nunes, JPA, Pina, FLC, Ribeiro, AS, Carneiro, NH, Venturini, D, Barbosa, DS, Mayhew, JL, and Cyrino, ES. Comparison of 2 weekly frequencies of resistance training on muscular strength, body composition, and metabolic biomarkers in resistance-trained older women: Effects of detraining and retraining. J Strength Cond Res 36(5): 1437-1444, 2022-This study aimed to compare the effects of 2 weekly frequencies of resistance training (RT) on muscular strength, body composition, and metabolic biomarkers in previously resistance-trained older women after detraining and retraining. Forty subjects (>60 years) performed RT (8 exercises, 1 set of 10-15 repetitions maximum) 2 (G2x) or 3 (G3x) times per week over 12 weeks of training and retraining. After training, subjects were detrained for 12 weeks. After detraining, there were significant decreases (p < 0.05) in upper-body (∼12%) and lower-body (∼14%) muscular strength, fat-free mass (FFM) (∼2%), and testosterone (∼26%), whereas increases were revealed for fat mass (FM) (∼4%), relative body fat (∼3%), fasting glucose (∼8%), low-density lipoprotein cholesterol (LDL-C) (∼21%), and triglycerides (∼24%), with no differences between groups (p > 0.05). Following retraining, there were significant increases (p < 0.05) for upper (∼7%) and lower (∼10%) muscular strength, FFM (∼2%), and testosterone (∼20%). In contrast, decreases were found for FM (∼7%), relative body fat (∼3%), fasting glucose (∼6%), LDL-C (∼14%), and triglycerides (∼21%), also with no differences between groups (p > 0.05). Gains after retraining were lower than after training (p < 0.05) only for upper- and lower-body muscular strength (∼6%) and testosterone (∼11%). Total cholesterol, high-density lipoprotein cholesterol, IGF-1, and C-reactive protein did not change at any point in the study for either group (p > 0.05). Our results suggest that older women can regain previous RT program benefits following detraining, regardless of the weekly training frequency. However, some fitness components may take longer to reestablish than the initial training level.

Mitogenomic phylogeny of Callithrix with special focus on human transferred taxa.

BACKGROUND: Callithrix marmosets are a relatively young primate radiation, whose phylogeny is not yet fully resolved. These primates are naturally para- and allopatric, but three species with highly invasive potential have been introduced into the southeastern Brazilian Atlantic Forest by the pet trade. There, these species hybridize with each other and endangered, native congeners. We aimed here to reconstruct a robust Callithrix phylogeny and divergence time estimates, and identify the biogeographic origins of autochthonous and allochthonous Callithrix mitogenome lineages. We sequenced 49 mitogenomes from four species (C. aurita, C. geoffroyi, C. jacchus, C. penicillata) and anthropogenic hybrids (C. aurita x Callithrix sp., C. penicillata x C. jacchus, Callithrix sp. x Callithrix sp., C. penicillata x C. geoffroyi) via Sanger and whole genome sequencing. We combined these data with previously published Callithrix mitogenomes to analyze five Callithrix species in total. RESULTS: We report the complete sequence and organization of the C. aurita mitogenome. Phylogenetic analyses showed that C. aurita was the first to diverge within Callithrix 3.54 million years ago (Ma), while C. jacchus and C. penicillata lineages diverged most recently 0.5 Ma as sister clades. MtDNA clades of C. aurita, C. geoffroyi, and C. penicillata show intraspecific geographic structure, but C. penicillata clades appear polyphyletic. Hybrids, which were identified by phenotype, possessed mainly C. penicillata or C. jacchus mtDNA haplotypes. The biogeographic origins of mtDNA haplotypes from hybrid and allochthonous Callithrix were broadly distributed across natural Callithrix ranges. Our phylogenetic results also evidence introgression of C. jacchus mtDNA into C. aurita. CONCLUSION: Our robust Callithrix mitogenome phylogeny shows C. aurita lineages as basal and C. jacchus lineages among the most recent within Callithrix. We provide the first evidence that parental mtDNA lineages of anthropogenic hybrid and allochthonous marmosets are broadly distributed inside and outside of the Atlantic Forest. We also show evidence of cryptic hybridization between allochthonous Callithrix and autochthonous C. aurita. Our results encouragingly show that further development of genomic resources will allow to more clearly elucidate Callithrix evolutionary relationships and understand the dynamics of Callithrix anthropogenic introductions into the Brazilian Atlantic Forest.

Nonclassical Applications of closo-Carborane Anions: From Main Group Chemistry and Catalysis to Energy Storage.

Classically closo-carborane anions, particularly [HCB11H11]- and [HCB9H9]-, and their derivatives have primarily been used as weakly coordinating anions to isolate reactive intermediates, platforms for stoichiometric and catalytic functionalization, counteranions for simple Lewis acid catalysis, and components of materials like liquid crystals. The aim of this article is to educate the reader on the contemporary nonclassical applications of these anions. Specifically, this review will cover new directions in main group catalysis utilized to achieve some of the most challenging catalytic reactions such as C-F, C-H, and C-C functionalizations that are difficult or impossible to realize with transition metals. In addition, the review will cover the utilization of the clusters as dianionic C σ-bound ligands for coordination chemistry, ligand substituents for coordination chemistry and advanced catalyst design, and covalently bound spectator substituents to stabilize radicals. Furthermore, their applications as solution-based and solid-state electrolytes for Li, Na, and Mg batteries will be discussed.

Intramuscular oxytocin versus Syntometrine® versus carbetocin for prevention of primary postpartum haemorrhage after vaginal birth: a randomised double-blinded clinical trial of effectiveness, side effects and quality of life.

OBJECTIVE: To compare intramuscular oxytocin, Syntometrine® and carbetocin for prevention of postpartum haemorrhage after vaginal birth. DESIGN: Randomised double-blinded clinical trial. SETTING: Six hospitals in England. POPULATION: A total of 5929 normotensive women having a singleton vaginal birth. METHODS: Randomisation when birth was imminent. MAIN OUTCOME MEASURES: Primary: use of additional uterotonic agents. Secondary: weighed blood loss, transfusion, manual removal of placenta, adverse effects, quality of life. RESULTS: Participants receiving additional uterotonics: 368 (19.5%) oxytocin, 298 (15.6%) Syntometrine and 364 (19.1%) carbetocin. When pairwise comparisons were made: women receiving carbetocin were significantly more likely to receive additional uterotonics than those receiving Syntometrine (odds ratio [OR] 1.28, 95% CI 1.08-1.51, P = 0.004); the difference between carbetocin and oxytocin was non-significant (P = 0.78); Participants receiving Syntometrine were significantly less likely to receive additional uterotonics than those receiving oxytocin (OR 0.75, 95% CI 0.65-0.91, P = 0.002). Non-inferiority between carbetocin and Syntometrine was not shown. Use of Syntometrine reduced non-drug PPH treatments compared with oxytocin (OR 0.64, 95% CI 0.42-0.97) but not carbetocin (P = 0.64). Rates of PPH and blood transfusion were not different. Syntometrine was associated with an increase in maternal adverse effects and reduced ability of the mother to bond with her baby. CONCLUSIONS: Non-inferiority of carbetocin to Syntometrine was not shown. Carbetocin is not significantly different to oxytocin for use of additional uterotonics. Use of Syntometrine reduced use of additional uterotonics and need for non-drug PPH treatments compared with oxytocin. Increased maternal adverse effects are a disadvantage of Syntometrine. TWEETABLE ABSTRACT: IM carbetocin does not reduce additional uterotonic use compared with IM Syntometrine or oxytocin.

Selenomethionine administration decreases the oxidative stress induced by post mortem ischemia in the heart, liver and kidneys of rats.

Selenium is an essential element in human and animal metabolism integrated into the catalytic site of glutathione peroxidase (GPX1), an antioxidant enzyme that protects cells from damage caused by reactive oxygen species (ROS). Oxidative stress refers the imbalance between ROS and antioxidant defense systems. It generates alterations of DNA, proteins and lipid peroxidation. The imbalance occurs particularly during ischemia and lack of postmortem perfusion. This mechanism is of relevance in transplant organs, affecting their survival. The aim of this research is to evaluate the effect of seleno-methionine (SeMet) as a protective agent against postmortem ischemia injury in transplant organs. Wistar rats were orally administered with SeMet. After sacrifice, liver, heart and kidney samples were collected at different postmortem intervals (PMIs). SeMet administration produced a significant increase of Se concentration in the liver (65%, p < 0.001), heart (40%, p < 0.01) and kidneys (45%, p < 0.05). Levels of the oxidative stress marker malondialdehyde (MDA) decreased significantly compared to control in the heart (0.21 ± 0.04 vs. 0.12 ± 0.02 mmol g-1) and kidneys (0.41 ± 0.02 vs. 0.24 ± 0.03 mmol g-1) in a PMI of 1-12 h (p < 0.01). After SeMet administration for 21 days, a significant increase in GPX1 activity was observed in the liver (80%, p < 0.001), kidneys (74%, p < 0.01) and heart (35%, p < 0.05). SeMet administration to rats significantly decreased the oxidative stress in the heart, liver and kidneys of rats generated by postmortem ischemia.

Menopause, hormone therapy and cognition: maximizing translation from preclinical research.

Menopause-associated and hormone-associated cognitive research has a rich history built from varied disciplines and species. This review discusses landmark rodent and human work addressing cognitive outcomes associated with varied experiences of menopause and hormone therapy. Critical variables in menopause and cognitive aging research are considered, including menopause etiology, background hormone milieu and parameters of exposure to estrogens and progestogens. Recent preclinical research has identified that menopause and ovarian hormone fluctuations across many neurobiological systems affect cognitive aging, mapping novel avenues for future research. Preclinical models provide insight into complex interdisciplinary relationships in a systematic and highly controlled fashion. We highlight that acknowledging the strengths and weaknesses for both preclinical and clinical research approaches is vital to accurate interpretation, optimal translation and the direction of future research. There is great value in collaboration and communication across preclinical and clinical realms, especially regarding reciprocal feedback of findings to advance preclinical models, improve experimental designs and enrich basic science translation to the clinic. In searching for biological mechanisms underlying the cognitive consequences of menopause and hormone therapies, it is noteworthy that clinical and preclinical scientists are grounded in the same fundamental goal of optimizing health outcomes for women across the lifespan.

Shattered thyroid gland from blunt neck trauma in a child.

Thyroid gland rupture in a child secondary to blunt trauma is an extremely rare occurrence. The number of published cases of thyroid gland injuries in children is very limited in the research literature. A case report of shattered thyroid gland in a child is presented. This case highlights potential life-threatening complications and reviews management of thyroid gland injuries. Based on this case and other published pediatric case reports, conservative management may be a reasonable approach in thyroid gland injuries from blunt neck trauma in children.

Responsiveness to muscle mass gain following 12 and 24 weeks of resistance training in older women.

BACKGROUND: Many factors may influence the magnitude of individual responses to resistance training (RT). How the manipulation of training volume and frequency affects responsiveness level for muscle mass gain in older women has not been investigated. AIMS: This study had the objective of identifying responders (RP) and non-responders (N-RP) older women for skeletal muscle mass (SMM) gain from a 12-week resistance training (RT) program. Additionally, we analyzed whether the N-RP could gain SMM with an increase in weekly training volume over 12 additional weeks of training. METHODS: Thirty-nine older women (aged ≥ 60 years) completed 24 weeks of a whole-body RT intervention (eight exercises, 2-3×/week, 1-2 sets of 10-15 repetitions). SMM was estimated by DXA, and the responsive cut-off value was set at two times the standard error of measurement. Participants were considered as RP if they exceeded the cut-off value after a 12-week RT phase, while the N-RP were those who failed to reach the SMM cut-off. RESULTS: Of the 22 participants considered to be N-RP, only 3 accumulated SMM gains (P = 0.250) that exceeded the cut-off point for responsiveness following 12 additional weeks of training, while 19 maintained or presented negative SMM changes. Of the 17 participants considered to be RP, all continued to gain SMM after the second 12-week RT phase. No significant correlation was observed between the changes in SMM and any baseline aspect of the participants. CONCLUSIONS: Our results suggest that some older women are RP, while others are N-RP to SMM gains resulting from RT. Furthermore, the non-responsiveness condition was not altered by an increase of training volume and intervention duration while RP participants continue to increase SMM; it appears that RP continue to be RP, and N-RP continue to be N-RP.