Exercise and Reduced Nicotine Content Cigarettes in Adult Female Smokers: A Pilot Trial.

Background: Although Reduced Nicotine Cigarettes (RNC) are suggested to improve smoking cessation and cardiometabolic health in relation to cancer risk, the effectiveness of exercise training with RNC on smoking cessation and cardiometabolic health is unknown. Methods: Female smokers (N = 27) were randomized to: (1) usual nicotine cigarettes (i.e., control), (2) RNC or (3) RNC plus exercise treatment for 12 weeks. Smoking withdrawal symptoms (e.g., Wisconsin Smoking Withdrawal Scale) and cardiometabolic health (e.g., weight, VO2max, resting respiratory exchange ratio (RER), glucose, HOMA-IR) were examined before and after treatment. Results: Treatments had no differential effect on weight (p = 0.80; partial η2 = 0.29), VO2max (p = 0.20, partial η2 = 0.18), or total cholesterol/HDL ratios (p = 0.59, partial η2 = 0.06). However, RNC + Exercise tended to maintain RER (i.e., fat oxidation; p = 0.10, partial η2 = 0.10) as well as insulin resistance (p = 0.13, partial η2 = 0.25) and cortisol compared (p = 0.06, partial η2 = 0.30) with control and RNC. Increased VO2max was also associated with lower nicotine dependence scores (r = −0.50, p < 0.05). Conclusion: In this pilot study, improved fitness was associated with lower nicotine dependence. Additional work is warranted to examine the effects of exercise in smokers as a tool to improving smoking cessation and lower disease risk.

Role of Blood Pressure Responses to Exercise and Vascular Insulin Sensitivity with Nocturnal Blood Pressure Dipping in Metabolic Syndrome.

INTRODUCTION: Nocturnal systolic blood pressure (SBP) dipping is independently related to cardiovascular disease risk, but it is unclear if vascular insulin sensitivity associates with SBP dipping in patients with metabolic syndrome (MetS). METHODS: Eighteen adults with MetS (ATP III criteria 3.3 ± 0.6; 53.2 ± 6.5 years; body mass index 35.8 ± 4.5 kg/m2) were categorized as "dippers" (≥10% change in SBP; n = 4 F/3 M) or "non-dippers" (<10%; n = 9 F/2 M). Twenty-four-hour ambulatory blood pressure was recorded to assess SBP dipping. A euglycemic-hyperinsulinemic clamp (40 mU/m2/min, 90 mg/dL) with ultrasound (flow mediated dilation) was performed to test vascular insulin sensitivity. A graded, incremental exercise test was conducted to estimate sympathetic activity. Heart rate (HR) recovery after exercise was then used to determine parasympathetic activity. Metabolic panels and body composition (DXA) were also tested. RESULTS: Dippers had greater drops in SBP (16.63 ± 5.2 vs. 1.83 ± 5.6%, p < 0.01) and experienced an attenuated rise in both SBPslope (4.7 ± 2.3 vs. 7.2 ± 2.5 mm Hg/min, p = 0.05) and HRslope to the incremental exercise test compared to non-dippers (6.5 ± 0.9 vs. 8.2 ± 1.7 bpm/min, p = 0.03). SBP dipping correlated with higher insulin-stimulated flow-mediated dilation (r = 0.52, p = 0.03), although the relationship was no longer significant after covarying for HRslope (r = 0.42, p = 0.09). CONCLUSION: Attenuated rises in blood pressure and HR to exercise appear to play a larger role than vascular insulin sensitivity in SBP dipping in adults with MetS.

Insulin stimulation reduces aortic wave reflection in adults with metabolic syndrome.

Adults with metabolic syndrome (MetS) have increased fasting arterial stiffness and altered central hemodynamics that contribute, partly, to increased cardiovascular disease (CVD) risk. Although insulin affects aortic wave reflections in healthy adults, the effects in individuals with MetS are unclear. We hypothesized that insulin stimulation would reduce measures of pressure waveforms and hemodynamics in people with MetS. Thirty-five adults with obesity (27 women; 54.2 ± 6.0 yr; 37.1 ± 4.8 kg/m2) were selected for MetS (ATP III criteria) following an overnight fast. Pulse wave analysis was assessed using applanation tonometry before and after a 2-h euglycemic-hyperinsulinemic clamp (90 mg/dL, 40 mU/m2/min). Deconvolution analysis was used to decompose the aortic waveform [augmentation index corrected to heart rate of 75 beats/min (AIx@75); augmentation pressure (AP)] into backward and forward pressure components. Aerobic fitness (V̇o2max), body composition (DXA), and blood biochemistries were also assessed. Insulin significantly reduced augmentation index (AIx@75, 28.0 ± 9.6 vs. 23.0 ± 9.9%, P < 0.01), augmentation pressure (14.8 ± 6.4 vs. 12.0 ± 5.7 mmHg, P < 0.01), pulse pressure amplification (1.26 ± 0.01 vs. 0.03 ± 0.01, P = 0.01), and inflammation [high-sensitivity C-reactive protein (hsCRP): P = 0.02; matrix metallopeptidase 7 (MMP-7): P = 0.03] compared to fasting. In subgroup analyses to understand HTN influence, there were no insulin stimulation differences on any outcome. V̇o2max, visceral fat, and blood potassium correlated with fasting AIx@75 (r = -0.39, P = 0.02; r = 0.41, P = 0.03; r = -0.53, P = 0.002). Potassium levels were also associated with insulin-mediated reductions in AP (r = 0.52, P = 0.002). Our results suggest insulin stimulation improves indices of aortic reflection in adults with MetS.NEW & NOTEWORTHY This study is one of the first to investigate the effects of insulin on central and peripheral hemodynamics in adults with metabolic syndrome. We provide evidence that insulin infusion reduces aortic wave reflection, potentially through a reduction in inflammation and/or via a potassium-mediated vascular response.

Endothelial function following interval exercise plus low-calorie diet treatment in obese females.

We determined if interval exercise plus a low-calorie diet (LCD + INT) increases endothelial function more than an energy-matched LCD. Obese women (47.2 ± 2.6y, 37.5 ± 1.3kg/m2 ) were randomized to 13 days of a LCD (n = 12; mixed meals of ~ 1200kcal/d) or LCD + INT (n = 13; 12 supervised 60-min INT bouts of 3 min at 90% and 50% HRpeak ). LCD + INT subjects received 350kcal postexercise to equate energy availability with LCD. Fitness (VO2 peak) and body composition (BodPod) were determined and a 120 min, 75 g oral glucose tolerance test was performed to examine fasting and postprandial flow-mediated dilation (FMD, endothelial function), respiratory exchange ratio (RER) via indirect calorimetry as well as glucose and insulin incremental area under the curve (iAUC120min ). LCD + INT increased VO2 peak (P = 0.02) compared with LCD, and both treatments decreased fat mass (P < 0.001) and insulin iAUC120min (P = 0.03). There was no overall treatment effect on fasting or iAUC120min FMD. However, in participants who increased fasting endothelial function after each treatment (Δ > 50%; LCD n = 5, LCD + INT n = 7), LCD + INT increased fasted (P = 0.005) and decreased iAUC120min (P = 0.003) FMD compared with LCD. Enhanced fitness correlated with increased fasting FMD (r = 0.43, P = 0.03) and diminished FMD iAUC120min (r = -0.44, P = 0.03). Decreased FMD iAUC120min correlated with reduced glucose iAUC120min (r = 0.64, P = 0.001) as well as increased 60-min RER (r = -0.42, P = 0.04). Low baseline fasting and iAUC120min FMD was also linked to enhanced fasting and iAUC120min FMD post-treatment (r = -0.71, P < 0.001; r = -0.89, P < 0.001, respectively). In conclusion, increasing fitness via INT may increase the effect of LCD on lowering cardiovascular disease risk in obese women.

From the international space station to the clinic: how prolonged unloading may disrupt lumbar spine stability.

BACKGROUND CONTEXT: Prolonged microgravity exposure is associated with localized low back pain and an elevated risk of post-flight disc herniation. Although the mechanisms by which microgravity impairs the spine are unclear, they should be foundational for developing in-flight countermeasures for maintaining astronaut spine health. Because human spine anatomy has adapted to upright posture on Earth, observations of how spaceflight affects the spine should also provide new and potentially important information on spine biomechanics that benefit the general population. PURPOSE: This study compares quantitative measures of lumbar spine anatomy, health, and biomechanics in astronauts before and after 6 months of microgravity exposure on board the International Space Station (ISS). STUDY DESIGN: This is a prospective longitudinal study. SAMPLE: Six astronaut crewmember volunteers from the National Aeronautics and Space Administration (NASA) with 6-month missions aboard the ISS comprised our study sample. OUTCOME MEASURES: For multifidus and erector spinae at L3-L4, measures include cross-sectional area (CSA), functional cross-sectional area (FCSA), and FCSA/CSA. Other measures include supine lumbar lordosis (L1-S1), active (standing) and passive (lying) flexion-extension range of motion (FE ROM) for each lumbar disc segment, disc water content from T2-weighted intensity, Pfirrmann grade, vertebral end plate pathology, and subject-reported incidence of chronic low back pain or disc injuries at 1-year follow-up. METHODS: 3T magnetic resonance imaging and dynamic fluoroscopy of the lumbar spine were collected for each subject at two time points: approximately 30 days before launch (pre-flight) and 1 day following 6 months spaceflight on the ISS (post-flight). Outcome measures were compared between time points using paired t tests and regression analyses. RESULTS: Supine lumbar lordosis decreased (flattened) by an average of 11% (p=.019). Active FE ROM decreased for the middle three lumbar discs (L2-L3: -22.1%, p=.049; L3-L4: -17.3%, p=.016; L4-L5: -30.3%, p=.004). By contrast, no significant passive FE ROM changes in these discs were observed (p>.05). Disc water content did not differ systematically from pre- to post-flight. Multifidus and erector spinae changed variably between subjects, with five of six subjects experiencing an average decrease 20% for FCSA and 8%-9% for CSA in both muscles. For all subjects, changes in multifidus FCSA strongly correlated with changes in lordosis (r2=0.86, p=.008) and active FE ROM at L4-L5 (r2=0.94, p=.007). Additionally, changes in multifidus FCSA/CSA correlated with changes in lordosis (r2=0.69, p=.03). Although multifidus-associated changes in lordosis and ROM were present among all subjects, only those with severe, pre-flight end plate irregularities (two of six subjects) had post-flight lumbar symptoms (including chronic low back pain or disc herniation). CONCLUSIONS: We observed that multifidus atrophy, rather than intervertebral disc swelling, associated strongly with lumbar flattening and increased stiffness. Because these changes have been previously linked with detrimental spine biomechanics and pain in terrestrial populations, when combined with evidence of pre-flight vertebral end plate insufficiency, they may elevate injury risk for astronauts upon return to gravity loading. Our results also have implications for deconditioned spines on Earth. We anticipate that our results will inform new astronaut countermeasures that target the multifidus muscles, and research on the role of muscular stability in relation to chronic low back pain and disc injury.

Pulsed electromagnetic field (PEMF) treatment reduces expression of genes associated with disc degeneration in human intervertebral disc cells.

BACKGROUND CONTEXT: Pulsed electromagnetic field (PEMF) therapies have been applied to stimulate bone healing and to reduce the symptoms of arthritis, but the effects of PEMF on intervertebral disc (IVD) biology is unknown. PURPOSE: The purpose of this study was to determine how PEMF affects gene expression of IVD cells in normal and inflammatory environments. STUDY DESIGN/SETTING: This was an in vitro human cell culture and microarray gene expression study. METHODS: Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were separately encapsulated in alginate beads and exposed to interleukin 1α (IL-1α) (10 ng/mL) to stimulate the inflammatory environment associated with IVD degeneration and/or stimulated by PEMF for 4 hours daily for up to 7 days. RNA was isolated from each treatment group and analyzed via microarray to assess IL-1α- and PEMF-induced changes in gene expression. RESULTS: Although PEMF treatment did not completely inhibit the effects of IL-1α, PEMF treatment lessened the IL-1α-induced upregulation of genes expressed in degenerated IVDs. Consistent with our previous results, after 4 days, PEMF tended to reduce IL-1α-associated gene expression of IL-6 (25%, p=.07) in NP cells and MMP13 (26%, p=.10) in AF cells. Additionally, PEMF treatment significantly diminished IL-1α-induced gene expression of IL-17A (33%, p=.01) and MMP2 (24%, p=.006) in NP cells and NFκB (11%, p=.04) in AF cells. CONCLUSIONS: These results demonstrate that IVD cells are responsive to PEMF and motivate future studies to determine whether PEMF may be helpful for patients with IVD degeneration.

Tibiofemoral contact pressures in radial tears of the meniscus treated with all-inside repair, inside-out repair and partial meniscectomy.

BACKGROUND: To test contact pressures in the knee after treatment of a radial meniscus tear with an all-inside meniscal repair technique and compare the results with inside-out repair and partial meniscectomy. METHODS: Six non-paired cadaveric knees were analyzed with intra-compartment pressures measured at loads of 250 N, 500 N and 1000 N at 0°, eight degrees, 15°, and 30° of knee flexion. Compartmental contact pressures were measured for the intact medial meniscus, radial tear in the posterior horn, all-inside repair using the NovoStitch suture passer device (Ceterix Orthopaedics Inc., Menlo Park, CA), inside-out repair method, and partial meniscectomy. One-way ANOVA was used for statistical analysis. RESULTS: The greatest differences in peak pressures between treatments were observed under 1000 N load at 30° flexion (0.8± (SD) 0.1 MPa (intact meniscus), 0.8± (SD) 0.1 MPa (all-inside), 0.9± (SD) 0.1 MPa (inside-out) and 1.6± (SD) 0.2 MPa (partial meniscectomy)). Treatment with partial meniscectomy resulted in the highest peak pressures compared to all other states (p<0.0001 at each angle). Repair of the radial tear using the all-inside technique as well as the inside-out technique resulted in significantly decreased compartment pressures compared to partial meniscectomies (p<0.0001 at each angle). There were no significant differences between peak pressures in the intact state and after repair with the all-inside or inside-out techniques. CONCLUSION: An all-inside repair technique using the NovoStitch suture passer can decrease contact pressures for a radial meniscus tear similarly to the inside-out repair technique when compared to partial meniscectomy. CLINICAL RELEVANCE: This novel arthroscopic suture passer warrants further analysis in the clinical setting as it may be a reliable method for repair of radial meniscal tears through an arthroscopic all-inside technique.

Corneal foreign body management at a role 1 flight line aid station: risks, benefits, and implications for Special Operations medicine.

Eye injuries are common in forward areas of operations. Definitive diagnosis and care may be limited not by provider skill but rather by available equipment. The ability to treat simple trauma such as corneal foreign bodies at the Role 1 level has advantages including rapid return to duty, decreased cost of treatment, and, most important, decreased risk of delayed care. We propose the device such as a hand-held portable slit lamp should be made available for appropriate Special Operations Medical Forces (SOFMED) or aviation providers.

Osteogenic response to BMP-2 of hMSCs grown on apatite-coated scaffolds.

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to non-mineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration.

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway.

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.

The ubiquitin-proteasome system postsynaptically regulates glutamatergic synaptic function.

The ubiquitin-proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS' roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome beta2 and beta6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors.

Ubiquitin-interacting motifs inhibit aggregation of polyQ-expanded huntingtin.

Expansion of polyglutamine (polyQ) tracts within proteins underlies a number of neurodegenerative diseases, such as Huntington disease, Kennedy disease, and spinocerebellar ataxias. The resulting mutant proteins are unstable, forming insoluble aggregates that are associated with components of the ubiquitin system, including ubiquitin, ubiquitin-like proteins, and proteins that bind to ubiquitin. Given the presence of these ubiquitin-binding proteins in the insoluble aggregates, we examined whether heterologous expression of short motifs that bind ubiquitin, termed ubiquitin-interacting motifs (UIMs), altered the aggregation of polyQ-expanded huntingtin (Htt), the protein product of the Huntington disease gene. We found that a subset of UIMs associated with mutant Htt. The ability to interact with ubiquitin was necessary, but not sufficient, for interaction with mutant Htt. Furthermore, we found that expression of single, isolated UIMs inhibited aggregation of mutant Htt. These data suggest that isolated UIMs might serve as potential inhibitors of polyQ-aggregation in vivo.

The U-box ligase carboxyl-terminus of Hsc 70-interacting protein ubiquitylates Epsin.

Epsin is an endocytic adaptor protein involved in the regulation of clathrin-dependent endocytosis. We and others have demonstrated that Epsin is ubiquitylated in cells and requires its ubiquitin interacting motifs (UIMs) for this modification. To further elucidate the mechanism of Epsin ubiquitylation, we initiated studies to identify the E3 ligase(s) that modifies Epsin. In this study, we discovered that the U-box ubiquitin ligase carboxyl-terminus of Hsc70 interacting protein (CHIP) ubiquitylated Epsin. Using an in vitro ubiquitylation assay, we demonstrate that CHIP specifically ubiquitylated Epsin in a UIM-dependent manner. Furthermore, overexpression of CHIP in cells increased Epsin ubiquitylation also in a UIM-dependent manner. Together, these data provide evidence that CHIP functions to ubiquitylate the endocytic protein Epsin.

Analysis of the role of ubiquitin-interacting motifs in ubiquitin binding and ubiquitylation.

The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIM-dependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.

The ubiquitin-interacting motifs target the endocytic adaptor protein epsin for ubiquitination.

The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.