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Prior studies of behavior therapy for trichotillomania (TTM) have shown that response is variable, and relapse after treatment discontinuation is common. Little information is available concerning prognostic factors capable of predicting individual differences in response or maintenance of improvement. The present study is a secondary analysis of a randomized controlled trial (N = 36) of the Comprehensive Behavioral (ComB) model of treatment for TTM (Carlson et al., 2021). We investigated age, disorder history, pre-treatment symptom severity, longest prior period of abstinence from pulling, and Emotion and Intention hair pulling styles as predictors of initial response. We studied age, disorder history, pre-treatment symptom severity, longest prior period of abstinence from pulling, and post-treatment symptom severity or hair-pulling abstinence as predictors of relapse following treatment. Older age significantly predicted lower TTM severity following treatment. Lower pre-treatment severity significantly predicted lower severity of TTM at the 3-month follow-up.

Sickle cell disease (SCD) is associated with increased risk of neurocognitive deficits. However, whether functioning changes following nonmyeloablative hematopoietic stem cell transplant (HSCT) remains unclear. This study aimed to examine changes in neuropsychological functioning pre- to post-transplant among patients with SCD and compare patients and siblings. Adults with SCD ( n  = 47; M age  = 31.8 ± 8.9) and their sibling stem cell donors ( n  = 22; M age  = 30.5± 9.2) enrolled on a nonmyeloablative HCST protocol completed cognitive and patient-reported outcome assessments at baseline and 12 months post-transplant. Path analyses were used to assess associations between pre-transplant variables and sibling/patient group status and post-transplant function. Mean patient cognitive scores were average at both timepoints. Patient processing speed and somatic complaints improved from baseline to follow-up. Baseline performance predicted follow-up performance across cognitive variables; patient/sibling status predicted follow-up performance on some processing speed measures. Results suggest that patients with SCD demonstrate slower processing speed than siblings. Processing speed increased pre- to post-HSCT among patients and siblings, and on some measures patients demonstrated greater improvement. Thus, HSCT may improve processing speed in patients, although further confirmation is needed. Findings provide promising evidence that neurocognitive functioning remains stable without detrimental effects from pre- to 12-months post nonmyeloablative HSCT in individuals with SCD.

Based on early evidence, risk communication scholars have come to believe that longer (360-character maximum) mobile public warning messages generate more compliance than shorter (90-character maximum) messages. This study used an experimental design to test that premise. The study measured participants' ( N = 481) likelihood of compliance in response to a mock Wireless Emergency Alert (WEA) message, as well as alternatives to immediate compliance: seeking additional information, taking non-recommended action, or ignoring the message. The study found that both longer and shorter messages generated relatively high levels of compliance, but longer messages did not generate higher levels of compliance. Rather than message length, risk personalization and hazard experience were stronger differentiators of WEA message response outcomes. Results included a moderation effect: Shorter messages produced slightly greater compliance than longer messages among people who reported lower levels of risk personalization. The study concluded that 90-character messages may be more effective than previously believed. Consequently, the authors recommend renewed focus on public safety communication related to risk personalization and hazard experience. (169 words).

Myeloid-derived suppressor cells (MDSC) have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species and the production of inhibitory cytokines. Our group has shown that signaling through Bruton's tyrosine kinase (BTK) is important for MDSC function. Ibrutinib is an orally administered targeted agent that inhibits BTK activation and is currently used for the treatment of B cell malignancies. Using a syngeneic murine model of melanoma, the effect of BTK inhibition with ibrutinib on the therapeutic response to systemic PD-L1 blockade was studied. BTK was expressed by murine MDSC and their activation was inhibited by ibrutinib. Ibrutinib was not directly cytotoxic to cancer cells in vitro, but it inhibited BTK activation in MDSC and reduced expression of inducible nitric oxide synthase (NOS2) and production of nitric oxide. Ibrutinib treatments decreased the levels of circulating MDSC in vivo and increased the therapeutic efficacy of anti-PD-L1 antibody treatment. Gene expression profiling showed that ibrutinib decreased Cybb (NOX2) signaling, and increased IL-17 signaling (upregulating downstream targets Mmp9, Ptgs2, and S100a8). These results suggest that further exploration of MDSC inhibition could enhance the immunotherapy of advanced melanoma.PrécisInhibition of Bruton's tyrosine kinase, a key enzyme in myeloid cellular function, improves therapeutic response to an anti-PD-L1 antibody in an otherwise fairly resistant murine melanoma model., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Adequate sleep is essential for healthy physical, emotional, and cognitive functioning, including memory. However, sleep ability worsens with increasing age. Older adults on average have shorter sleep durations and more disrupted sleep compared with younger adults. Age-related sleep changes are thought to contribute to age-related deficits in episodic memory. Nonetheless, the nature of the relationship between sleep and episodic memory deficits in older adults is still unclear. Further complicating this relationship are age-related changes in circadian rhythms such as the shift in chronotype toward morningness and decreased circadian stability, which may influence memory abilities as well. Most sleep and cognitive aging studies do not account for circadian factors, making it unclear whether age-related and sleep-related episodic memory deficits are partly driven by interactions with circadian rhythms. This review will focus on age-related changes in sleep and circadian rhythms and evidence that these factors interact to affect episodic memory, specifically encoding and retrieval. Open questions, methodological considerations, and clinical implications for diagnosis and monitoring of age-related memory impairments are discussed., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that expand dramatically in many cancer patients. This expansion contributes to immunosuppression in cancer and reduces the efficacy of immune-based cancer therapies. One mechanism of immunosuppression mediated by MDSCs involves production of the reactive nitrogen species peroxynitrite (PNT), where this strong oxidant inactivates immune effector cells through destructive nitration of tyrosine residues in immune signal transduction pathways. As an alternative to analysis of nitrotyrosines indirectly generated by PNT, we used an endoplasmic reticulum (ER)-targeted fluorescent sensor termed PS3 that allows direct detection of PNT produced by MDSCs. When the MDSC-like cell line MSC2 and primary MDSCs from mice and humans were treated with PS3 and antibody-opsonized TentaGel microspheres, phagocytosis of these beads led to production of PNT and generation of a highly fluorescent product. Using this method, we show that splenocytes from a EMT6 mouse model of cancer, but not normal control mice, produce high levels of PNT due to elevated numbers of granulocytic (PMN) MDSCs. Similarly, peripheral blood mononuclear cells (PBMCs) isolated from blood of human melanoma patients produced substantially higher levels of PNT than healthy human volunteers, coincident with higher peripheral MDSC levels. The kinase inhibitor dasatinib was found to potently block the production of PNT both by inhibiting phagocytosis in vitro and by reducing the number of granulocytic MDSCs in mice in vivo, providing a chemical tool to modulate the production of this reactive nitrogen species (RNS) in the tumor microenvironment., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Objective: Fragments of fibrillin-1 and fibrillin-2 will be detectable in the plasma of patients with aortic dissections and aneurysms. We sought to determine whether the plasma fibrillin fragment levels (PFFLs) differ between patients with thoracic aortic pathology and those presenting with nonaortic chest pain., Methods: PFFLs were measured in patients with thoracic aortic aneurysm (n = 27) or dissection (n = 28). For comparison, patients without aortic pathology who had presented to the emergency department with acute chest pain (n = 281) were categorized into three groups according to the cause of the chest pain: ischemic cardiac chest pain; nonischemic cardiac chest pain; and noncardiac chest pain. The PFFLs were measured using a sandwich enzyme-linked immunosorbent assay., Results: Fibrillin-1 fragments were detectable in all patients and were lowest in the ischemic cardiac chest pain group. Age, sex, and the presence of hypertension were associated with differences in fibrillin-1 fragment levels. Fibrillin-2 fragments were detected more often in the thoracic aneurysm and dissection groups than in the emergency department chest pain group ( P  < .0001). Patients with aortic dissection demonstrated a trend toward increased detectability ( P  = .051) and concentrations ( P  = .06) of fibrillin-2 fragments compared with patients with aortic aneurysms. Analysis of specific antibody pairs identified fibrillin-1 B15-HRP26 and fibrillin-2 B205-HRP143 as the most informative in distinguishing between the emergency department and aortic pathology groups., Conclusions: Patients with thoracic aortic dissections demonstrated elevated plasma fibrillin-2 fragment levels (B205-HRP143) compared with patients presenting with ischemic or nonischemic cardiac chest pain and increased fibrillin-1 levels (B15-HRP26) compared with patients with ischemic cardiac chest pain. Investigation of fibrillin-1 and fibrillin-2 fragment generation might lead to diagnostic, therapeutic, and prognostic advances for patients with thoracic aortic dissection.

The cation channel of sperm (CatSper) is a validated target for nonhormonal male contraception, but it lacks selective blockers, hindering studies to establish its role in both motility and capacitation. Via an innovative calcium uptake assay utilizing human sperm we discovered novel inhibitors of CatSper function from a high-throughput screening campaign of 72,000 compounds. Preliminary SAR was established for seven hit series. HTS hits or their more potent analogs blocked potassium-induced depolarization and noncompetitively inhibited progesterone-induced CatSper activation. CatSper channel blockade was confirmed by patch clamp electrophysiology and these compounds inhibited progesterone- and prostaglandin E1-induced hyperactivated sperm motility. One of the hit compounds is a potent CatSper inhibitor with high selectivity for CatSper over hCav1.2, hNav1.5, moderate selectivity over hSlo3 and hERG, and low cytotoxicity and is therefore the most promising inhibitor identified in this study. These new CatSper blockers serve as useful starting points for chemical probe development and drug discovery efforts., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The cation channel of sperm (CatSper) is the principal entry point for calcium in human spermatozoa and its proper function is essential for successful fertilization. As CatSper is potently activated by progesterone, we evaluated a range of steroids to define the structure-activity relationships for channel activation and found that CatSper is activated by a broad range of steroids with diverse structural modifications. By testing steroids that failed to elicit calcium influx as inhibitors of channel activation, we discovered that medroxyprogesterone acetate, levonorgestrel, and aldosterone inhibited calcium influx produced by progesterone, prostaglandin E 1 , and the fungal natural product l -sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K + and pH. In contrast to these steroid antagonists, we demonstrated for the first time that the T-type calcium channel blocker ML218 acts similarly to mibefradil, blocking CatSper channels activated by both ligands and alkalinization/depolarization. These T-type calcium channel blockers produced an insurmountable blockade of CatSper, whereas the three steroids produced antagonism that was surmountable by increasing concentrations of each activator, indicating that the steroids selectively antagonize ligand-induced activation of CatSper rather than blocking channel function. Both the channel blockers and the steroid antagonists markedly reduced hyperactivated motility of human sperm assessed by computer-aided sperm analysis, consistent with inhibition of CatSper activation. Unlike the channel blockers mibefradil and ML218, which reduced total and progressive motility, medroxyprogesterone acetate, levonorgestrel, and aldosterone had little effect on these motility parameters, indicating that these steroids are selective inhibitors of hyperactivated sperm motility. SIGNIFICANCE STATEMENT: The steroids medroxyprogesterone acetate, levonorgestrel, and aldosterone selectively antagonize progesterone- and prostaglandin E 1 -induced calcium influx through the CatSper cation channel in human sperm. In contrast to T-type calcium channel blockers that prevent all modes of CatSper activation, these steroid CatSper antagonists preferentially reduce hyperactivated sperm motility, which is required for fertilization. The discovery of competitive antagonists of ligand-induced CatSper activation provides starting points for future discovery of male contraceptive agents acting by this unique mechanism., (Copyright © 2021 by The Author(s).)

This study provides the longest follow-up yet for comprehensive behavioral (ComB) treatment of trichotillomania (TTM) (M = 24.59 months after pre-treatment and 15.92 months after the last follow-up point in a recent clinical trial (Carlson et al., 2021), which had shown ComB to be significantly more efficacious than minimal attention at post-treatment). This study also examined changes in TTM severity from before to during the COVID-19 pandemic. Participants ( N  = 23) completed a survey assessing current TTM symptoms, the impact of the pandemic on their coping with TTM, and their experience with ComB treatment. Self-reported symptom severity at this follow-up evaluation fell between the scores obtained at the clinical trial's pre-treatment assessment and at its last follow-up before the pandemic and did not significantly differ from either time point. Most participants (73%) reported some change in their TTM management since onset of the pandemic, with changes to their environment/routine (61%) and in anxiety (32%) being the most common. Pandemic-related changes were associated with variable outcomes, improving symptoms and management for some while worsening them for others. Use of strategies from ComB had declined since the most recent follow-up, but more than half (55%) of participants reported that strategies from ComB remained useful., Competing Interests: All authors declare that they have no conflicts of interest., (© 2021 Elsevier Inc. All rights reserved.)

Objective: This study is the first controlled trial of comprehensive behavioral (ComB) treatment of trichotillomania (TTM). ComB provides individualized treatment based on factors triggering and maintaining hair pulling., Method: Participants (N = 36) were adults (M = 34.08 years old, SD = 12.26) meeting DSM5 criteria for TTM. A majority were female (80%) and Caucasian (75%), whereas 17% were African American and 19% Hispanic/Latinx. In a parallel-group design, participants were randomly assigned to (a) Immediate ComB (12 sessions) or (b) Minimal Attention Control (MAC), followed by delayed ComB after week 12. Follow-up continued through week 38. Primary outcomes were self-report (Massachusetts General Hospital Hair pulling Scale; MGH-HPS) and interviewer-rated (NIMH-Trichotillomania Impact Scale and Trichotillomania Severity Scale; TIS/TSS) TTM symptom severity, as well as diagnosis (Trichotillomania Diagnostic Interview)., Results: Immediate efficacy of ComB (vs. MAC) was statistically significant (p = .03) for self-reported symptoms, with an effect size d = -.78, but not significant for interviewer-rated symptoms or diagnostic status. Immediate ComB was significantly more likely than MAC (27% vs. 0%) to lead to complete abstinence from hair pulling at week 12. Follow-ups showed good maintenance of effects., Conclusions: Efficacy of ComB was established for self-reported symptoms. Future research is needed to establish whether the lack of more widespread effects stems from limitations of the model or to a need for more extensive therapist training, as secondary analyses suggested stronger results among therapists with more TTM experience., (Copyright © 2021. Published by Elsevier Ltd.)

Immunolocalization studies have shown that fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which fibrillin-1 green fluorescence was detectable. Other arteries gained detectable fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Two samples of living blood cells and of cleared blood plasma from the Phlebobranch tunicate Ascidia ceratodes from Bodega Bay, California, and one of fresh Henze solution from A. ceratodes of Monterey Bay, California, have been examined using sulfur K-edge x-ray absorption spectroscopy (XAS). Biological sulfur included sulfate esters, sulfate and bisulfate ions, benzothiazole, thianthrene, epi-sulfide, thiol and disulfide. Glutathione dominated reduced sulfur, from which an average intracellular Voltage of -0.21 V was calculated. Sulfate-bisulfate ratios yielded blood cell pH values of 2.0 and 2.8. Total blood cell [sulfur] was 373±9 mM or 296±73 mM from BaSO 4 gravimetry. Two plasma samples (pH 6.9 or 7.0; [S] = 33±6 mM or 26±4 mM) were dominated by sulfate and disulfide. Fresh Henze solution evidenced a sulfur inventory similar to blood cells, with calculated pH = 2.7. A V(III)-sulfonate fraction varied systematically with intracellular pH across six independent blood cell samples, implying a vanadium mobilization pathway. Bodega Bay and Monterey Bay A. ceratodes appear to maintain alternative suites of low-valent sulfur. The significance of the vanabins to vanadium metabolism is critically examined in terms of known protein - V(IV) biochemistry. Finally, a detailed hypothesis for the reduction of [VO 4 ] 3 - to V(III) in ascidians is introduced. A vanadium oxido-reductase is proposed to span the signet ring membrane and to release V(III) into the inner acidic vacuole. The V(V) to V(III) reduction is predicted require an inner-sphere mechanism, a thiol reductant, 7-coordinate V(III), a biologically accessible Voltage, and proton-facilitated release of V(III)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Peroxynitrite (PNT) is a highly reactive oxidant that plays a key role in the destruction of foreign pathogens by specific phagocytic immune cells such as macrophages. However, when its production is dysregulated, this oxidant can contribute to cardiovascular disease, neurological diseases, and cancer. To facilitate the detection of PNT in living cells, we designed and synthesized a fluorescent sensor termed PS3 that accumulates in membranes of the endoplasmic reticulum (ER). This subcellular targeting enhances the proximity of PS3 to the phagosome of macrophages where PNT is generated. When PS3-treated macrophages are stimulated with 10 µm opsonized tentagel microspheres, antibody-dependent cellular phagocytosis (ADCP) of these particles results in production of endogenous PNT, oxidative cleavage of the fluorescence-quenching phenolic side chain of PS3, and increased fluorescence that can be detected by confocal laser scanning microscopy, flow cytometry, and other assays. We describe methods for the synthesis of PS3 and evaluation of its photophysical properties, selectivity, and reactivity. We further report differential production of PNT during ADCP by the phagocytic cell lines RAW 264.7, J774A.1, and THP-1, as detected by confocal microscopy and changes in fluorescence intensity on 96-well plates. This approach may be useful for identification of modulators of PNT and related studies of ADCP., (© 2020 Elsevier Inc. All rights reserved.)

This study shows for the first time that an iminosugar exerts anti-spermiogenic effect, inducing reversible infertility in a species that is not related to C57BL/6 male mice. In CD rats, N-butyldeoxygalactonojirimycin ( N B-DGJ) caused reversible infertility at 150 mg/kg/day when administered daily as single oral dose. N B-DGJ inhibited CD rat-derived testicular β-glucosidase 2 (GBA2) activity at 10 µM but did not inhibit CD rat-derived testicular ceramide-specific glucosyltransferase (CGT) at doses up to 1000 µM. Pharmacokinetic studies revealed that sufficient plasma levels of N B-DGJ (50 µM) were achieved to inhibit the enzyme. Fertility was blocked after 35 days of treatment and reversed one week after termination of treatment. The rapid return of fertility indicates that the major effect of N B-DGJ may be epididymal rather than testicular. Collectively, our in vitro and in vivo studies in rats suggest that iminosugars should continue to be pursued as potential lead compounds for development of oral, non-hormonal male contraceptives. The study also adds evidence that GBA2, and not CGT, is the major target for the contraceptive effect of iminosugars., Competing Interests: The authors declare no conflict of interest. The research for this article was conducted while Dr. Hild was employed at Bioqual Inc. Her current affiliation is Office of Research Infrastructure Programs, Office of the Director, National Institutes of Health (NIH), Bethesda, MD. The opinions expressed in this article are the author’s own and do not reflect the views of NIH or the Department of Health and Human Services.

[This corrects the article DOI: 10.1371/journal.pone.0181166.].

Analogues of N-butyl-1-deoxynojirimycin (NB-DNJ) were prepared and assayed for inhibition of ceramide-specific glucosyltransferase (CGT), non-lysosomal β-glucosidase 2 (GBA2) and the lysosomal β-glucosidase 1 (GBA1). Compounds 5 a-6 f, which carry sterically demanding nitrogen substituents, and compound 13, devoid of the C3 and C5 hydroxy groups present in DNJ/NB-DGJ (N-butyldeoxygalactojirimycin) showed no inhibitory activity for CGT or GBA2. Inversion of stereochemistry at C4 of N-(n-butyl)- and N-(n-nonyl)-DGJ (compounds 24) also led to a loss of activity in these assays. The aminocyclopentitols N-(n-butyl)- (35 a), N-(n-nonyl)-4-amino-5-(hydroxymethyl)cyclopentane- (35 b), and N-(1-(pentyloxy)methyl)adamantan-1-yl)-1,2,3-triol (35 f), were found to be selective inhibitors of GBA1 and GBA2 that did not inhibit CGT (>1 mm), with the exception of 35 f, which inhibited CGT with an IC 50 value of 1 mm. The N-butyl analogue 35 a was 100-fold selective for inhibiting GBA1 over GBA2 (K i values of 32 nm and 3.3 μm for GBA1 and GBA2, respectively). The N-nonyl analogue 35 b displayed a K i value of ≪14 nm for GBA1 inhibition and a K i of 43 nm for GBA2. The N-(1-(pentyloxy)methyl)adamantan-1-yl) derivative 35 f had K i values of ≈16 and 14 nm for GBA1 and GBA2, respectively. The related N-bis-substituted aminocyclopentitols were found to be significantly less potent inhibitors than their mono-substituted analogues. The aminocyclopentitol scaffold should hold promise for further inhibitor development., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Background: Sex-related differences as well as the adverse effect of pregnancy on aortic disease outcome are well-established phenomena in humans with Marfan syndrome (MFS). The underlying mechanisms of these observations are largely unknown., Objectives: In an initial (pilot) step we aimed to confirm the differences between male and female MFS patients as well as between females with and without previous pregnancy. We then sought to evaluate whether these findings are recapitulated in a pre-clinical model and performed in-depth cardiovascular phenotyping of mutant male and both nulliparous and multiparous female Marfan mice. The effect of 17β-estradiol on fibrillin-1 protein synthesis was compared in vitro using human aortic smooth muscle cells and fibroblasts., Results: Our small retrospective study of aortic dimensions in a cohort of 10 men and 20 women with MFS (10 pregnant and 10 non-pregnant) confirmed that aortic root growth was significantly increased in the pregnant group compared to the non-pregnant group (0.64mm/year vs. 0.12mm/year, p = 0.018). Male MFS patients had significantly larger aortic root diameters compared to the non-pregnant and pregnant females at baseline and follow-up (p = 0.002 and p = 0.007, respectively), but no significant increase in aortic root growth was observed compared to the females after follow-up (p = 0.559 and p = 0.352). In the GT-8/+ MFS mouse model, multiparous female Marfan mice showed increased aortic diameters when compared to nulliparous females. Aortic dilatation in multiparous females was comparable to Marfan male mice. Moreover, increased aortic diameters were associated with more severe fragmentation of the elastic lamellae. In addition, 17β-estradiol was found to promote fibrillin-1 production by human aortic smooth muscle cells., Conclusions: Pregnancy-related changes influence aortic disease severity in otherwise protected female MFS mice and patients. There may be a role for estrogen in the female sex protective effect.

The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

Background Treatment of scaphoid nonunion is challenging, leading clinicians to pursue innovation in surgical technique and adjunctive therapies to improve union rates. Purpose The purpose of this study was to investigate the use of low-intensity pulsed ultrasound as an adjunctive treatment modality following surgical treatment of scaphoid nonunion in adolescent patients, for whom this therapy has not yet been FDA-approved. Patients and Methods We performed a retrospective review of adolescent patients with scaphoid nonunion treated surgically followed by adjunctive low-intensity pulsed ultrasound therapy. All patients underwent 20 minutes of daily ultrasound therapy postoperatively until there was evidence of bony healing, based on both clinical and radiographic criteria. Final healing was confirmed by > 50% bone bridging on CT scan. Results Thirteen of fourteen (93%) patients healed at a mean interval of 113 days (range 61-217 days). There were no surgical or postoperative complications. One patient developed heterotopic bone formation about the scaphoid. Conclusions Our study suggests that low-intensity pulsed ultrasound therapy can safely be utilized as an adjunctive modality in adolescents to augment scaphoid healing following surgical intervention. Level of Evidence Level IV, Case series.

We present 2 cases that demonstrate the potential for tendon involvement in the presence of a carpal boss. In the first, a patient presented with tendon rupture without antecedent pain. In the second, pain and tendon irritation prompted magnetic resonance imaging that revealed tendon fraying, which was confirmed at surgery. These cases illustrate the potential for tendinous sequelae of a carpal boss. Advanced imaging may be considered when tendon irritation is clinically suspected. Attention to the possibility of tendon rupture in the setting of an otherwise asymptomatic carpal boss is advised., (Copyright © 2014 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Swan neck deformity in patients with cerebral palsy can result from hand intrinsic muscle spasticity or overpull of the digital extensors. After accurate identification of the etiology of the deformity, surgical treatment is directed at correcting the underlying muscle imbalance. Intrinsic lengthening can be used to treat intrinsic muscle spasticity, whereas central slip tenotomy is employed when digital extensor overpull is the deforming force. Accurate diagnosis and application of the proper surgical technique are essential when treating swan neck deformity in patients with cerebral palsy., (Copyright © 2014 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Background: Our goal was to determine 3-dimensional interfragmentary motions due to simulated transverse fracture and volar wedge osteotomy of the scaphoid during physiologic flexion-extension of a cadaveric wrist model., Methods: The model consisted of a cadaveric wrist (n = 8) from the metacarpals through the distal radius and ulna with load applied through the major flexor-extensor tendons. Flexibility tests in flexion-extension were performed in the following 3 test conditions: intact and following transverse fracture and wedge osteotomy of the scaphoid. Scaphoid interfragmentary motions were measured using optoelectronic motion tracking markers. Average peak scaphoid interfragmentary motions due to transverse fracture and wedge osteotomy were statistically compared (P<0.05) to intact., Findings: The accuracy of our computed interfragmentary motions was ± 0.24 mm for translation and ± 0.54° for rotation. Average peak interfragmentary motions due to fracture ranged between 0.9 mm to 1.9 mm for translation and 5.3° to 10.8° for rotation. Significant increases in interfragmentary motions were observed in volar/dorsal translations and flexion/extension due to transverse fracture and in separation and rotations in all 3 motion planes due to wedge osteotomy., Interpretation: Comparison of our results with data from previous in vitro and in vivo biomechanical studies indicates a wide range of peak interfragmentary rotations due to scaphoid fracture, from 4.6° up to 30°, with peak interfragmentary translations on the order of several millimeters. Significant interfragmentary motions, indicating clinical instability, likely occur due to physiologic flexion-extension of the wrist in those with transverse scaphoid fracture with or without volar bone loss., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rationale: Mutations in fibrillin-1 are associated with thoracic aortic aneurysm (TAA) in Marfan syndrome. Genome-wide association studies also implicate fibrillin-1 in sporadic TAA. Fragmentation of the aortic elastic lamellae is characteristic of TAA., Objective: Immunoassays were generated to test whether circulating fragments of fibrillin-1, or other microfibril fragments, are associated with TAA and dissection., Methods and Results: Plasma samples were obtained from 1265 patients with aortic aneurysm or dissection and from 125 control subjects. Concentrations of fibrillin-1, fibrillin-2, and fibulin-4 were measured with novel immunoassays. One hundred and seventy-four patients (13%) had aneurysms with only abdominal aortic involvement (abdominal aortic aneurysm), and 1091 (86%) had TAA. Of those with TAA, 300 patients (27%) had chronic dissection and 109 (10%) had acute or subacute dissection. Associations of fragment concentrations with TAA (versus abdominal aortic aneurysm) or with dissection (versus no dissection) were estimated with odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, and smoking. Compared with controls, significantly higher percentages of aneurysm patients had detectable levels of fibrillin fragments. TAA was significantly more common (than abdominal aortic aneurysm) in the highest compared with lowest quartile of fibrillin-1 concentration (OR=2.9; 95% CI, 1.6-5.0). Relative to TAA without dissection, acute or subacute dissection (OR=2.9; 95% CI, 1.6-5.3), but not chronic dissection, was more frequent in the highest compared with lowest quartile of fibrillin-1 concentration. Neither TAA nor dissection was associated with fibrillin-2 or fibulin-4., Conclusions: Circulating fibrillin-1 fragments represent a new potential biomarker for TAA and acute aortic dissection.

Study Design: Retrospective case series., Objective: To characterize the clinical utility of imaging in the postanesthesia care unit (PACU) after anterior cervical decompression and fusion (ACDF) procedures., Summary of Background Data: Two sets of imaging are often taken at the end of ACDF procedures: one intraoperatively and the other in the PACU. The latter may have low clinical utility., Materials and Methods: One hundred four patients who underwent ACDF procedures with anterior plate/screw constructs were identified. A panel assessed intraoperative and PACU series for adequacy of images to detect potential issues with placement of the surgical construct and for any actual visible issues with placement of the surgical construct., Results: Intraoperative series were adequate to detect potential issues with construct placement for 78.8% of cases, whereas PACU series were adequate for only 58.7% of cases (significant difference, P<0.001). For both series, nearly all inadequacies were because of the shoulders obstructing the lateral view. Accordingly, cases with lower inferior operative levels were much more likely to have inadequate intraoperative and PACU series than cases with higher inferior operative levels (significant differences, P<0.001 for both). In no case was an issue with construct placement visible on a PACU series that was not also visible on an intraoperative series., Conclusions: This study demonstrates that PACU images are inferior to intraoperative images and offer little or no incremental clinical utility for detecting issues with surgical construct placement after ACDF procedures. PACU imaging after ACDF procedures might be discontinued to realize savings in time, cost, and radiation exposure.

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes., Competing Interests: The authors have declared that no competing interests exist.

In genetic studies, many interesting traits, including growth curves and skeletal shape, have temporal or spatial structure. They are better treated as curves or function-valued traits. Identification of genetic loci contributing to such traits is facilitated by specialized methods that explicitly address the function-valued nature of the data. Current methods for mapping function-valued traits are mostly likelihood-based, requiring specification of the distribution and error structure. However, such specification is difficult or impractical in many scenarios. We propose a general functional regression approach based on estimating equations that is robust to misspecification of the covariance structure. Estimation is based on a two-step least-squares algorithm, which is fast and applicable even when the number of time points exceeds the number of samples. It is also flexible due to a general linear functional model; changing the number of covariates does not necessitate a new set of formulas and programs. In addition, many meaningful extensions are straightforward. For example, we can accommodate incomplete genotype data, and the algorithm can be trivially parallelized. The framework is an attractive alternative to likelihood-based methods when the covariance structure of the data is not known. It provides a good compromise between model simplicity, statistical efficiency, and computational speed. We illustrate our method and its advantages using circadian mouse behavioral data.

We present the use of direct bonded copper (DBC) for the straightforward fabrication of high power atom chips. Atom chips using DBC have several benefits: excellent copper/substrate adhesion, high purity, thick (>100 μm) copper layers, high substrate thermal conductivity, high aspect ratio wires, the potential for rapid (<8 h) fabrication, and three-dimensional atom chip structures. Two mask options for DBC atom chip fabrication are presented, as well as two methods for etching wire patterns into the copper layer. A test chip, able to support 100 A of current for 2 s without failing, is used to determine the thermal impedance of the DBC. An assembly using two DBC atom chips is used to magnetically trap laser cooled (87)Rb atoms. The wire aspect ratio that optimizes the magnetic field gradient as a function of power dissipation is determined to be 0.84:1 (height:width).

Apical reabsorption from the urine has been shown to be important for such processes as the maintenance of critical metabolites in the blood and the excretion of nephrotoxic compounds. The solute carrier (SLC) transporter OAT4 (SLC22A11) is expressed on the apical membrane of renal proximal tubule cells and is known to mediate the transport of a variety of xenobiotic and endogenous organic anions. Functional characterization of genetic variants of apical transporters thought to mediate reabsorption, such as OAT4, may provide insight into the genetic factors influencing the complex pathways involved in drug elimination and metabolite reclamation occurring in the kidney. Naturally occurring genetic variants of OAT4 were identified in public databases and by resequencing DNA samples from 272 individuals comprising 4 distinct ethnic groups. Nine total nonsynonymous variants were identified and functionally assessed using uptake of three radiolabeled substrates. A nonsense variant, R48Stop, and three other variants (R121C, V155G, and V155M) were found at frequencies of at least 2% in an ethnic group specific fashion. The L29P, R48Stop, and H469R variants displayed a complete loss of function, and kinetic analysis identified a reduced V(max) in the common nonsynonymous variants. Plasma membrane levels of OAT4 protein were absent or reduced in the nonfunctional variants, providing a mechanistic reason for the observed loss of function. Characterization of the genetic variants of reabsorptive transporters such as OAT4 is an important step in understanding variability in tubular reabsorption with important implications in innate homeostatic processes and drug disposition.

In humans, mutations in fibrillin-1 result in a variety of genetic disorders with distinct clinical phenotypes. While most of the known mutations in fibrillin-1 cause Marfan syndrome, a number of other mutations lead to clinical features unrelated to Marfan syndrome. Pathogenesis of Marfan syndrome is currently thought to be driven by mechanisms due to haploinsufficiency of wild-type fibrillin-1. However, haploinsufficiency-driven mechanisms cannot explain the distinct phenotypes found in other fibrillinopathies. To test the hypothesis that mutations in fibrillin-1 cause disorders through primary effects on microfibril structure, two different mutations were generated in Fbn1 in mice. One mutation leads to a truncated fibrillin-1 molecule that is tagged with green fluorescent protein, allowing visualization of mutant fibrillin-1 incorporated into microfibrils. In heterozygosity, these mutant mice demonstrate progressive fragmentation of the aortic elastic lamellae and also display fragmentation of microfibrils in other tissues. Fibrillin-2 epitopes are also progressively revealed in these mice, suggesting that fibrillin-2 immunoreactivity can serve as a marker for microfibril degradation. In contrast, a second mutation (in-frame deletion of the first hybrid domain) in fibrillin-1 results in stable microfibrils, demonstrating that fibrillin-1 molecules are not required to be in perfect register for microfibril structure and function and that the first hybrid domain is dispensable for microfibril assembly. Taken together, these results suggest that perturbation of microfibril structure may underlie one of the major features of the Marfan syndrome: fragmentation of aortic elastic lamellae.

A new class of potent NK(1) receptor antagonists with a tetrahydroindolizinone core has been identified. This series of compounds demonstrated improved functional activities as compared to previously identified 5,5-fused pyrrolidine lead structures. SAR at the 7-position of the tetrahydroindolizinone core is discussed in detail. A number of compounds displayed high NK(1) receptor occupancy at both 1 h and 24 h in a gerbil foot tapping model. Compound 40 has high NK(1) binding affinity, good selectivity for other NK receptors and promising in vivo properties. It also has clean P(450) inhibition and hPXR induction profiles., (2010 Elsevier Ltd. All rights reserved.)

3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy}-4-(4-fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (17) is a high affinity, brain-penetrant, hydroisoindoline-based neurokinin-1 (NK(1)) receptor antagonist with a long central duration of action in preclinical species and a minimal drug-drug interaction profile. Positron emission tomography (PET) studies in rhesus showed that this compound provides 90% NK(1) receptor blockade in rhesus brain at a plasma level of 67 nM, which is about 10-fold more potent than aprepitant, an NK(1) antagonist marketed for the prevention of chemotherapy-induced and postoperative nausea and vomiting (CINV and PONV). The synthesis of this enantiomerically pure compound containing five stereocenters includes a Diels-Alder condensation, one chiral separation of the cyclohexanol intermediate, an ether formation using a trichloroacetimidate intermediate, and bis-alkylation to form the cyclic amine.

Vanadium K-edge X-ray absorption spectroscopy (XAS) has been used to track the uptake and fate of VO(2+) ion in blood cells from Ascidia ceratodes, following exposure to dithiothreitol (DTT) or to DTT plus VO(2+). The full range of endogenous vanadium was queried by fitting the XAS of blood cells with the XAS spectra of model vanadium complexes. In cells exposed only to DTT, approximately 0.4% of a new V(III) species was found in a site similar to Na[V(edta)(H(2)O)]. With exposure to DTT and VO(2+), average intracellular [VO(aq)](2+) increased from 3% to 5%, and 6% of a new complexed form of vanadyl ion appeared evidencing a ligand array similar to [VO(edta)](2-). At the same time, the relative ratio of blood cell [V(H(2)O)(6)](3+) increased at the expense of [V(H(2)O)(5)(SO(4))](+) in a manner consistent with a significant increase in endogenous acidity. In new UV/Visible experiments, VO(2+) could be reduced to 7-coordinate [V(nta)(H(2)O)(3)] or [V(nta)(ida)](2-) with cysteine methyl ester in pH 6.5 solution. Ascorbate reduced [VO(edta)](2-) to 7-coordinate [V(edta)(H(2)O)](-), while [VO(trdta)](2-) was unreactive. These results corroborate the finding that the reductive EMF of VO(2+) is increased by the availability of a 7-coordinate V(III) product. Finally, a new and complete hypothesis is proposed for an ascidian vanadate reductase. The structure of the enzyme active site, the vanadate-vanadyl-vanadic reduction mechanism, the cellular locale, and elements of the regulatory machinery governing the biological reduction of vanadate and vanadyl ion by ascidians are all predicted. Together these constitute the new field of vanadium redox enzymology.

Background: Previous clinical studies have identified the cervical facet joint, including the capsular ligaments, as sources of pain in whiplash patients. The goal of this study was to determine whether whiplash caused increased capsular ligament laxity by applying quasi-static loading to whiplash-exposed and control capsular ligaments., Methods: A total of 66 capsular ligament specimens (C2/3 to C7/T1) were prepared from 12 cervical spines (6 whiplash-exposed and 6 control). The whiplash-exposed spines had been previously rear impacted at a maximum peak T1 horizontal acceleration of 8 g. Capsular ligaments were elongated at 1mm/s in increments of 0.05 mm until a tensile force of 5 N was achieved and subsequently returned to neutral position. Four pre-conditioning cycles were performed and data from the load phase of the fifth cycle were used for subsequent analyses. Ligament elongation was computed at tensile forces of 0, 0.25, 0.5, 0.75, 1.0, 2.5, and 5.0 N. Two factor, non-repeated measures ANOVA (P<0.05) was performed to determine significant differences in the average ligament elongation at tensile forces of 0 and 5 N between the whiplash-exposed and control groups and between spinal levels., Findings: Average elongation of the whiplash-exposed capsular ligaments was significantly greater than that of the control ligaments at tensile forces of 0 and 5 N. No significant differences between spinal levels were observed., Interpretation: Capsular ligament injuries, in the form of increased laxity, may be one component perpetuating chronic pain and clinical instability in whiplash patients.

Background Context: Most previous studies have investigated ligament mechanical properties at slow elongation rates of less than 25 mm/s., Purpose: To determine the tensile mechanical properties, at a fast elongation rate, of intact human cervical anterior and posterior longitudinal, capsular, and interspinous and supraspinous ligaments, middle-third disc, and ligamentum flavum., Study Design/setting: In vitro biomechanical study., Methods: A total of 97 intact bone-ligament-bone specimens (C2-C3 to C7-T1) were prepared from six cervical spines (average age: 80.6 years, range, 71 to 92 years) and were elongated to complete rupture at an average (SD) peak rate of 723 (106) mm/s using a custom-built apparatus. Nonlinear force versus elongation curves were plotted and peak force, peak elongation, peak energy, and stiffness were statistically compared (p<.05) among ligaments. A mathematical model was developed to determine the quasi-static physiological ligament elongation., Results: Highest average peak force, up to 244.4 and 220.0 N in the ligamentum flavum and capsular ligament, respectively, were significantly greater than in the anterior longitudinal ligament and middle-third disc. Highest peak elongation reached 5.9 mm in the intraspinous and supraspinous ligaments, significantly greater than in the middle-third disc. Highest peak energy of 0.57 J was attained in the capsular ligament, significantly greater than in the anterior longitudinal ligament and middle-third disc. Average stiffness was generally greatest in the ligamentum flavum and least in the intraspinous and supraspinous ligaments. For all ligaments, peak elongation was greater than average physiological elongation computed using the mathematical model., Conclusions: Comparison of the present results with previously reported data indicated that high-speed elongation may cause cervical ligaments to fail at a higher peak force and smaller peak elongation and they may be stiffer and absorb less energy, as compared with a slow elongation rate. These comparisons may be useful to clinicians for diagnosing cervical ligament injuries based upon the specific trauma.

SAR studies on amides, ureas, and vinylogous amides derived from pyrrolidine led to the discovery of several potent hNK(1) antagonists. One particular vinylogous amide (45b) had excellent potency, selectivity, pharmacokinetic profile, and functional activity in vivo. An in vivo rhesus macaque brain receptor occupancy PET study for compound 45b revealed an estimated Occ(90) approximately 300 ng/ml.

Background: OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs., Methods: We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers., Results: Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1., Conclusions: The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.

Background Context: Elongation-induced vertebral artery (VA) injury has been hypothesized to occur during nonphysiological coupled head motions during automobile impacts. Although previous work has investigated VA elongation during head-turned and head-forward rear impacts, no studies have performed similar investigations for frontal or side impacts., Purpose: The present study quantified dynamic VA elongations during simulated frontal and side automotive collisions, and compared these data with corresponding physiological limits., Study Design/setting: In vitro biomechanical study of dynamic VA elongation during simulated impacts., Methods: A biofidelic whole cervical spine model with muscle force replication and surrogate head underwent simulated frontal impacts (n=6) of 4, 6, 8, and 10 g or left side impacts (n=6) of 3.5, 5, 6.5, and 8 g., Results: Average (SD) maximum physiological VA elongation was 7.1 (3.2) mm, measured during intact flexibility testing. Average peak dynamic elongation of right VA during left side impact, up to 17.4 (2.6) mm, was significantly greater (p<.05) than physiological beginning at 6.5 g, whereas the highest average peak VA elongation during frontal impact was 2.5 (2.4) mm, which did not exceed the physiological limit. Side impact, as compared with frontal impact, caused earlier occurrence of average peak VA elongation, 113.8 (13.5) ms versus 155.0 (46.2) ms, and higher average peak VA elongation rate, 608.8 (99.0) mm/s versus 130.0 (62.9) mm/s., Conclusions: Elongation-induced VA injury is more likely to occur during side impact as compared with frontal impact.

The dog is an attractive model for genetic studies of complex disease. With drafts of the canine genome complete, a large number of single-nucleotide polymorphisms (SNPs) that are potentially useful for gene-mapping studies and empirical estimations of canine diversity and linkage disequilibrium (LD) are now available. Unfortunately, most canine SNPs remain uncharacterized, and the amount and quality of DNA available from population-based samples are limited. We assessed how these real-world challenges influence automated SNP genotyping methods such as Illumina's GoldenGate assay. We examined 384 SNPs on canine chromosome 9 and successfully genotyped a minimum of 217 and a maximum of 275 SNPs using buccal swab samples for 181 dogs (86 beagles, 76 border collies, and 15 Australian shepherds). Call rates per SNP and sample averaged 97%, with reproducibility within and between analyses averaging 98%. The majority of these SNPs were polymorphic across all 3 breeds. We observed extensive LD, albeit less than reported for surveys using fewer dogs, consistent between breeds. Analyses of population substructure indicated that beagles are distinct from border collies and Australian shepherds. These results demonstrate the suitability of amplified canine buccal samples for high-throughput multiplex genotyping and confirm extensive LD in the dog.

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.

Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples. Four other microsatellite markers were as sensitive as, and more specific than, commonly used genotyping techniques based on merozoite surface proteins 1 and 2. When applied to samples from 15 patients in Burkina Faso with recurrent parasitemia after treatment with sulphadoxine-pyrimethamine, the addition of these four microsatellite markers to msp1 and msp2 genotyping resulted in a reclassification of outcomes that strengthened the association between dhfr 59R, an anti-folate resistance mutation, and recrudescence (P = 0.31 versus P = 0.03). Four microsatellite markers performed well on polyclonal samples and may provide a valuable addition to genotyping for clinical drug efficacy studies in high transmission areas.

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V(max) for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.

The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.

Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (Km = 457 +/- 118 microM; Vmax = 17.5 +/- 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V(max) for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.

A new class of high affinity hNK1R antagonists based on seven-membered ring cores has been identified. This series, with relatively simple, compact structures, includes compounds with high affinity, good selectivity, and promising in vivo properties.

Sod2-/- mice, which are deficient in the mitochondrial form of superoxide dismutase (MnSOD), have a short survival time that is strongly affected by genetic background. This suggests the existence of genetic modifiers that are capable of modulating the degree of mitochondrial oxidative damage caused by the MnSOD deficiency, thereby altering longevity. To identify these modifier(s), we generated recombinant congenic mice with quantitative trait loci (QTL) containing the putative genetic modifiers on the short-lived C57BL/6J genetic background. MnSOD deficient C57BL/6J mice with a QTL from the distal region of chromosome 13 from DBA/2J were able to survive for as long as those generated on the long-lived DBA/2J background. Within this region, the gene encoding nicotinamide nucleotide transhydrogenase (Nnt) was found to be defective in C57BL/6J mice, and no mature NNT protein could be detected. The forward reaction of NNT, a nuclear-encoded mitochondrial inner membrane protein, couples the generation of NADPH to proton transport and provides NADPH for the regeneration of two important antioxidant compounds, glutathione and thioredoxin, in the mitochondria. This action of NNT could explain its putative protective role in MnSOD-deficient mice.