Corrigendum Compounds Errors and Again Fails to Support the Specificity of Acupoint Tapping.

ABSTRACT: In a prior article (Spielmans, Rosen, Spence-Sing J Nerv Ment Dis 208:628-631, 2020), we demonstrated that Church, Stapleton, Yang, and Gallo's (J Nerv Ment Dis 206:783-793, 2018) meta-analytic finding that acupoint tapping had specific therapeutic benefit was highly flawed, both statistically and methodologically. Our analysis based on corrected effect sizes found no significant benefit for acupoint tapping at study endpoint. Church, Stapleton, Kip, and Gallo (J Nerv Ment Dis 208:632-635, 2020) issued a corrigendum in which they reported a new post hoc analysis using follow-up (rather than study endpoint) measures. Shifting to a post hoc outcome while pooling highly disparate follow-up endpoints is problematic; it ignored the nonsignificant result of the a priori analysis. Here, we clarify these issues and address Church, Stapleton, Kip, and Gallo's (J Nerv Ment Dis 208:632-635, 2020) often irrelevant or confusing responses to our methodological concerns. Considering this recent exchange of articles, and absent meaningful correction to the original incorrect findings, we remain concerned that emotional freedom technique proponents will continue to advance unfounded claims regarding the purported benefits of acupoint tapping.

Tapping Away at a Misleading Meta-analysis: No Evidence for Specificity of Acupoint Tapping.

Church et al.'s meta-analysis of three studies claimed to support the specificity of acupoint tapping as a therapeutic technique in the treatment of mental health problems. However, our critical analysis found substantial methodological problems and inaccurate statistical analyses, which render their results invalid. Specifically, 1) two included studies did not include participants with documented mental health problems; 2) two included studies did not specifically isolate the effect of acupoint tapping; 3) clear rationales for selected measures were not provided; 4) comparison groups were not bona fide therapies; 5) researcher and therapist allegiances were not controlled; and 6) selection of included studies may have been biased. Further, our attempt to replicate their results failed; we found that acupoint tapping fared no better than comparison groups: k = 3 studies, d = -0.38 (95% confidence interval, 0.10 to -0.87), p = 0.12. We conclude that the Church et al.'s meta-analysis actually found no specific mental health benefits for acupoint tapping.

Use of a Probiotic to Enhance Iron Absorption in a Randomized Trial of Pediatric Patients Presenting with Iron Deficiency.

OBJECTIVE: To evaluate the efficacy of low dose ferrous sulfate for the treatment of iron deficiency and if the probiotic Lactobacillus plantarum 299v (LP299v) enhances treatment. STUDY DESIGN: This randomized, double-blinded, controlled trial of the treatment of iron deficiency in children compared the use of low-dose ferrous sulfate (1-3 mg/kg/day), with or without probiotic (LP299v). RESULTS: Serum ferritin level increased in all children from a baseline of 23.7 ng/mL to 45.4 ng/mL after 6-8 weeks of treatment. There was no significant difference in the increase in serum ferritin in children taking the probiotic LP299v compared with controls (23.2 vs 20.0 ng/mL, respectively). Additionally, an increase in ferritin level was not significantly associated with probiotic use when controlling for other factors, including child weight and dosing. Overall, the treatments were well-tolerated, with mild side effects. CONCLUSIONS: Treatment with low-dose ferrous sulfate is well-tolerated and effective in correcting iron deficiency in children. However, the probiotic LP299v did not enhance treatment. Further attention should examine the dose-response effect in children, including an alternate day dosing schedule. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01617044.

In vitro chondrocyte toxicity following long-term, high-dose exposure to Gd-DTPA and a novel cartilage-targeted MR contrast agent.

OBJECTIVE: To determine the concentrations exhibiting toxicity of a cartilage-targeted magnetic resonance imaging contrast agent compared with gadopentetate dimeglumine (Gd-DT-PA) in chondrocyte cultures. MATERIALS AND METHODS: A long-term Swarm rat chondrosarcoma chondrocyte-like cell line was exposed for 48 h to 1.0-20 mM concentrations of diaminobutyl-linked nitroxide (DAB4-DLN) citrate, 1.0-20 mM Gd-DTPA, 1.0 µM staurosporine (positive control), or left untreated. Cell appearance, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of metabolic activity, quantitative PicoGreen assays of DNA content, and calcein-AM viability assays were compared. RESULTS: At 1.0-7.5 mM, minimal decrease in cell proliferation was found for both agents. At all doses of both agents, cell culture appearances were similar after 24 h of treatment. At the higher doses, differences in cell culture appearance were found after 48 h of treatment, with dose-dependent declines in chondrocyte populations for both agents. Concentration-dependent declines in DNA content and calcein fluorescence were found after 48 h of treatment, but beginning at a lower dose of DAB4-DLN citrate than Gd-DTPA. Dose-dependent decreases in MTT staining (cell metabolism) were apparent for both agents, but larger effects were evident at a lower dose for DAB-DLN citrate. Poor MTT staining of cells exposed for 48 h to 20 mM DAB4-DLN citrate probably indicates dead or dying cells. CONCLUSION: The minimal effect of the long-term exposure of model chondrocyte cell cultures to DAB4-DLN citrate and Gd-DTPA concentrations up to 7.5 mM (3x typical arthrographic administration) is supporting evidence that these doses are acceptable for MR arthrography. The findings are reassuring given that the experimental exposure to the contrast agents at sustained concentrations was much longer than when used clinically.

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels.

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100µM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

Designing Molecular Probes To Prolong Intracellular Retention: Application to Nitroxide Spin Probes.

Targeted delivery of molecular probes into cells enables cellular imaging through optical and magnetic modalities. Probe molecules that are well retained by cells can accumulate to higher intracellular concentrations, and thus increase the signal-to-noise ratio of, and widen the temporal window for, imaging. Here we synthesize a paramagnetic spin probe bearing six ionic functional groups and show that it has long intracellular half-life (>12 h) and exceptional biostability in living cells. We demonstrate that judicious incorporation of ionic substituents on probe molecules systematically increases intracellular retention time, and should therefore be beneficial to imaging experiments.

Has DSM-5 saved PTSD from itself?

In 2007, Robert Spitzer considered validity challenges to the diagnosis of post-traumatic stress disorder (PTSD), a construct that originated when he was Chair of DSM-III. Spitzer suggested changes for DSM-5, then in its planning stages, for the purpose of 'Saving PTSD from itself'. With years gone by, it can be asked if DSM-5 followed Spitzer's recommendations to advance our understanding of post-traumatic disorder.

Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy.

Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.

Improved sensitivity for imaging spin trapped hydroxyl radical at 250 MHz.

Radicals, including hydroxyl, superoxide, and nitric oxide, play key signaling roles in vivo. Reaction of these free radicals with a spin trap affords more stable paramagnetic nitroxides, but concentrations in vivo still are so low that detection by electron paramagnetic resonance (EPR) is challenging. Three innovative enabling technologies have been combined to substantially improve sensitivity for imaging spin-trapped radicals at 250 MHz. 1) Spin-trapped adducts of BMPO have lifetimes that are long enough to make imaging by EPR at 250 MHz feasible. 2) The signal-to-noise ratio of rapid-scan EPR is substantially higher than for conventional continuous-wave EPR. 3) An improved algorithm permits image reconstruction with a spectral dimension that encompasses the full 50 G spectrum of the BMPO-OH spin adduct without requiring the wide sweeps that would be needed for filtered backprojection. A 2D spectral-spatial image is shown for a phantom containing ca. 5 µM BMPO-OH.

In vivo electron paramagnetic resonance imaging of differential tumor targeting using cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.

PURPOSE: Electron paramagnetic resonance spectroscopy promises quantitative images of important physiologic markers of animal tumors and normal tissues, such as pO(2), pH, and thiol redox status. These parameters of tissue function are conveniently reported by tailored nitroxides. For defining tumor physiology, it is vital that nitroxides are selectively localized in tumors relative to normal tissue. Furthermore, these paramagnetic species should be specifically taken up by cells of the tumor, thereby reporting on both the site of tumor formation and the physiological status of the tissue. This study investigates the tumor localization of the novel nitroxide, cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidin-yloxyl 3 relative to the corresponding di-acid 4. METHODS: We obtained images of nitroxide 3 infused intravenously into C3H mice bearing 0.5-cm(3) FSa fibrosarcoma on the leg, and compared these with images of similar tumors infused with nitroxide 4. RESULTS: The ratio of spectral intensity from within the tumor-bearing region to that of normal tissue was higher in the mice injected with 3 relative to 4. CONCLUSION: This establishes the possibility of tumor imaging with a nitroxide with intracellular distribution and provides the basis for EPR images of animal models to investigate the relationship between crucial aspects of tumor microenvironment and malignancy and its response to therapy.

In vivo evidence of methamphetamine induced attenuation of brain tissue oxygenation as measured by EPR oximetry.

Abuse of methamphetamine (METH) is a major and significant societal problem in the US, as a number of studies have suggested that METH is associated with increased cerebrovascular events, hemorrhage or vasospasm. Although cellular and molecular mechanisms involved in METH-induced toxicity are not completely understood, changes in brain O2 may play an important role and contribute to METH-induced neurotoxicity including dopaminergic receptor degradation. Given that O2 is the terminal electron acceptor for many enzymes that are important in brain function, the impact of METH on brain tissue pO2 in vivo remains largely uncharacterized. This study investigated striatal tissue pO2 changes in male C57BL/6 mice (16-20 g) following METH administration using EPR oximetry, a highly sensitive modality to measure pO2 in vivo, in situ and in real time. We demonstrate that 20 min after a single injection of METH (8 mg/kg i.v.), the striatal pO2 was reduced to 81% of the pretreatment level and exposure to METH for 3 consecutive days further attenuated striatal pO2 to 64%. More importantly, pO2 did not recover fully to control levels even 24 h after administration of a single dose of METH and continual exposure to METH exacerbates the condition. We also show a reduction in cerebral blood flow associated with a decreased brain pO2 indicating an ischemic condition. Our findings suggests that administration of METH can attenuate brain tissue pO2, which may lead to hypoxic insult, thus a risk factor for METH-induced brain injury and the development of stroke in young adults.

Baeyer-Villiger Rearrangement of a Substituted Pyrrole by Oxone.

Pyrroloxyls have been reported to exhibit very narrow EPR spectral lines, essential for in vivo imaging. En route to pyrroloxyls, we observed an unexpected Baeyer-Villiger rearrangement, leading to loss of aromaticity and formation of a 4,5-dihydro-1H-ketopyrrole.

Dimethyl 5-acetyl-1-hy-droxy-4-methyl-1H-pyrrole-2,3-di-carboxyl-ate, an oxidation-resistant N-hy-droxy-pyrrole.

The title compound, C11H13NO6, exhibits an intra-molecular O-H⋯O=C hydrogen bond between the N-hydroxyl H atom and carbonyl O atom of the neighboring acetyl group. This finding contradicts a previously published model in which the hydrogen bond was postulated to occur with the neighboring carbomethoxy group. This relatively strong hydrogen bond [O-H⋯O: D = 2.5583 (11) Šand θ = 152°] may underlie the resistance of the title compound to oxidation into the corresponding nitroxide.

Use of rapid-scan EPR to improve detection sensitivity for spin-trapped radicals.

The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1-6.0 µM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR.

Ethyl 2,5-di-tert-butyl-5-eth-oxy-4-oxo-4,5-di-hydro-1H-pyrrole-3-carboxyl-ate.

The title compound, C17H29NO4, contains a chiral center and crystallizes as a racemate. The asymmetric unit consists of two non-equivalent mol-ecules, in which the carbeth-oxy groups have markedly different orientations [C(=O)CC(OEt)=O torsion angles = 59.3 (2) and 156.0 (2)°]. In the crystal, mol-ecules form chains along [101] through N-H⋯O hydrogen bonds.

Caveat emptor: Mental health specialty certifications and the public's preferences for clinical care.

Appropriate training and continuing education for mental health professionals are designed to ensure that clinicians provide effective and ethical care. Mental health consumers may depend upon these credentials to judge the level of a professional's competence, but whether these activities and credentials provide a valid indicator of knowledge and skills is subject to debate. The present study was designed to examine preferences for mental health clinicians among potential consumers and factors that may inform these preferences, specifically comparing preferences for doctoral-level mental health clinicians and masters-level clinicians with and without specialty certification for treating anxiety symptoms. Cross-sectional assessment with self-report surveys (clinician preferences, prior mental health diagnosis and treatment, demographic characteristics, generalized anxiety symptoms, mental health literacy, and mental health stigma) was administered in two samples: a college student sample (N = 224; 71.9% female; Mage = 19.1, SD = 1.5) and a sample of adults with chronic pain (N = 116; 74.1% female; Mage = 43.8, SD = 13.8). The present study found that across both samples, therapists with a specialty certification were preferred over those without such credentials within each profession, and that certification status trumped professional standing such that certified masters-level clinicians were rated more highly than noncertified PhD-level clinicians. These findings are indicative of a schism between how the field of clinical psychology conceptualizes itself and how it is seen by its consumers. Implications of our findings for mental health consumers, clinicians, and professional organizations are discussed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Effect of the mammalian arginase inhibitor 2(S)-amino-6-boronohexanoic acid on Bacillus anthracis arginase.

Macrophages, upon phagocytosing endospores of Bacillus anthracis, up-regulate the expression of the immunological isoform of nitric oxide synthase, NOS 2, concomitant with production of nitric oxide (NO•) from metabolism of L -arginine. We have previously demonstrated that macrophages that secrete NO• kill the bacilli of B. anthracis. To circumvent this microbicidal activity of NO•, B. anthracis has evolved pathways that include the enzyme arginase, which metabolizes L: -arginine to ornithine and urea. Compounds that inhibit arginase might, therefore, offer a therapeutic approach to controlling B. anthracis infection. 2(S)-Amino-6-boronohexanoic acid (ABH) has been reported to be an inhibitor of mammalian arginase. In this study, we explore the inhibitory effect of ABH against B. anthracis arginase and its potential for future development, as an effective therapeutic agent against microbial infection. We found that ABH is an inhibitor of bacterial arginase in several different endospore strains of B. anthracis. Further, ABH inhibits neither the phagocytosis of these endospores nor the up-regulation of NOS 2 concomitant with secretion of NO•. These findings set the stage to determine how efficacious ABH will be in promoting NO•-mediating killing of B. anthracis.

Clearance and biodistribution of liposomally encapsulated nitroxides: a model for targeted delivery of electron paramagnetic resonance imaging probes to tumors.

Electron paramagnetic resonance (EPR) imaging using nitroxides as molecular probes is potentially a powerful tool for the detection and physiological characterization of micrometastatic lesions. Encapsulating nitroxides in anti-HER2 immunoliposomes at high concentrations to take advantage of the "self-quenching" phenomenon of nitroxides allows generation of robust EPR signals in HER2-overexpressing breast tumor cells with minimal background from indifferent tissues or circulating liposomes. We investigated the in vivo pharmacological properties of nitroxides encapsulated in sterically stabilized liposomes designed for long circulation times. We show that circulation times of nitroxides can be extended from hours to days; this increases the proportion of liposomes in circulation to enhance tumor targeting. Furthermore, nitroxides encapsulated in sterically stabilized anti-HER2 immunoliposomes can be delivered to HER2-overexpressing tumors at micromolar concentrations, which should be imageable by EPR. Lastly, after in vivo administration, liposomally encapsulated nitroxide signal also appears in the liver, spleen, and kidneys. Although these organs are spatially distinct and would not hinder tumor imaging in our model, understanding nitroxide signal retention in these organs is essential for further improvements in EPR imaging contrast between tumors and other tissues. These results lay the foundation to use liposomally delivered nitroxides and EPR imaging to visualize tumor cells in vivo.

Cellular uptake of electron paramagnetic resonance imaging probes through endocytosis of liposomes.

Electron paramagnetic resonance imaging (EPRI) allows detection and localization of paramagnetic spin probes in vivo and in real time. We have shown that nitroxide spin probes entrapped in the intracellular milieu can be imaged by EPRI. Therefore, with the development of a tumor-targetable vehicle that can efficiently deliver nitroxides into cells, it should be possible to use nitroxide spin probes to label and image cells in a tumor. In this study, we assess the potential of liposomes as a delivery vehicle for imaging probes. We demonstrate that liposomes can stably encapsulate nitroxides at very high concentrations (>100 mM), at which nitroxides exhibit concentration-dependent quenching of their EPR signal-a process analogous to the quenching of fluorescent molecules. The encapsulating liposomes thus appear spectroscopically "dark". When the liposomes are endocytosed and degraded by cells, the encapsulated nitroxides are liberated and diluted into the much larger intracellular volume. The consequent relief of quenching generates a robust intracellular nitroxide signal that can be imaged. We show that through endocytosis of nitroxide-loaded liposomes, CV1 cells can achieve intracellular nitroxide concentrations of approximately 1 mM. By using tissue phantom models, we verify that this concentration is more than sufficient for in vivo EPR imaging.

Anti-HER2 immunoliposomes for selective delivery of electron paramagnetic resonance imaging probes to HER2-overexpressing breast tumor cells.

Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 µM nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals.