HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis.

While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing.

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element.IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis-acting elements regulating HIV-1 splicing.

Youth in Northern Ireland: Linking Violence Exposure, Emotional Insecurity, and the Political Macrosystem.

Growing up in the aftermath of armed conflict puts youth at a higher risk for psychopathology-particularly in societies like Northern Ireland which continue to be characterized by intergroup tension and cyclical violence. This risk may be heightened during adolescence, when youth are beginning to explore their identities and are becoming more aware of intergroup dynamics in both their immediate communities and the broader society. It is also during this stage when youth increasingly witness or engage in antisocial behavior and sectarian activities. A series of studies in Belfast conducted by Cummings et al. (2014, Child Dev Perspect, 12(1), 16-38; 2019, J Clin Child Adolesc Psychol, 48(2), 296-305) showed that adolescents' exposure to sectarian violence resulted in heightened emotional insecurity about the community and subsequent adjustment problems. Though the impact of direct exposure to violence is well documented, few studies have accounted for the influence of sectarianism that occurs outside of one's immediate environment. These influences may include the general climate surrounding events that are not experienced firsthand but are nonetheless salient, such as the overarching levels of tension between groups or societal discourse that is threatening to one's identity. These higher-level influences, often referred to collectively as the macrosystem, are a necessary component to consider for adequately assessing one's socio-developmental environment. Yet, measurement at this level of the social ecology has proven elusive in past work. The current study advances research in this area by using newspaper coding as a method of measuring the political macrosystem in Northern Ireland and assessing whether a tense or threatening climate serves as an added risk factor for youth living in Belfast. In the current study, we measured sectarian violence at the level of the macrosystem by systematically collecting and coding newspaper articles from Northern Ireland that were published between 2006 and 2011 (N = 2,797). Each article was coded according to its level of overall political tension between Catholics and Protestants, threat to Catholics, and threat to Protestants. When aggregated, these assessments reflected the overarching trends in Catholic-Protestant relations during this period. In order to assess the association between these sociopolitical trends and the direct experiences of adolescents, the newspaper coding was linked with five waves of survey data from families (N = 999) in socioeconomically disadvantaged areas of Belfast. Using a series of multilevel moderation analyses, we then tested whether intergroup tension and ingroup threat moderated the relation between adolescents' direct exposure to violence and their emotional insecurity. These analyses were followed by a thematic analysis of the coded newspaper articles in order to provide further context to the findings. The results indicated that adolescents' response to direct exposure to sectarian violence varied based on the political climate at the time of their interview. Overall, the adolescents' emotional insecurity about the community increased with exposure to sectarian violence. During periods when the sociopolitical climate was characterized by high levels of intergroup political tension, this relation was slightly weaker-regardless of the adolescents' ingroup (i.e., Protestant vs. Catholic). During periods when the sociopolitical climate was coded as threatening, this relation was weaker for Catholic adolescents. That is, high levels of macro-level threat-particularly events coded as threatening for Protestants-seemed to be a protective factor for Catholic adolescents. Group differences were also found based on the adolescents' cumulative amount of exposure to sectarian violence. As threat in the macrosystem increased, Catholic adolescents who were directly exposed to higher than average levels of sectarian violence became more emotionally secure, while Catholics with little to no exposure to violence became more insecure. Contrastingly, Protestant adolescents directly exposed to higher than average levels of sectarian violence were more insecure than Protestants with little to no violence exposure. A thematic analysis of the newspaper articles revealed the categories of events that were viewed by coders as politically tense and threatening. Five primary themes emerged: ineffective policing and justice, family and community unrest, memories of violence, destabilized leadership, and organized paramilitary activity. Many of the articles coded as most threatening reported on a spike in attacks organized by dissident republican groups-that is, members of the Catholic community with, particularly hardline views. This may be pertinent to the finding that associations between sectarian violence exposure and emotional insecurity were exacerbated during this time for Protestants but not for Catholics. Findings from the thematic analysis provide a deeper examination of the context of events taking place during the study period, as well as their potential bearing on interpretation of the macro-level effects. In conclusion, these findings illustrate how one's response to the immediate environment can vary based on shifts in the political macrosystem. The current study thus contributes conceptually, empirically, and methodologically to the understanding of process relations between multiple levels of the social ecology and adolescent functioning. These results may further inform the design of future interventions and policies meant to lessen the impact of political violence. The methods used here may also be useful for the study of other contexts in which macrosystem effects are likely to have a salient impact on individual wellbeing.

Reduced insulin sensitivity in young, normoglycaemic subjects alters microvascular tissue oxygenation during postocclusive reactive hyperaemia.

NEW FINDINGS: What is the central question of the study? Are measures of reduced insulin sensitivity in young, normoglycaemic subjects correlated with near-infrared spectroscopy-derived microvascular responsiveness [tissue oxygen saturation reperfusion rate (STO2 upslope)] during postocclusive reactive hyperaemia? What is the main finding and its importance? A sevenfold range of hepatic insulin sensitivity is significantly correlated (r = 0.44, P = 0.02) with STO2 upslope after transient tissue ischaemia. Near-infrared spectroscopy may be an important tool for determining altered microvascular function before onset of hyperglycaemia. Identification of pre-type 2 diabetes much earlier than with the present clinical criteria is important for pre-emptive measures against microvascular deterioration. ABSTRACT: Near-infrared spectroscopy (NIRS) measurement of postocclusive reactive hyperaemia (PORH) tissue oxygen saturation reperfusion rate [STO2 upslope (as a percentage per minute)] has recently been correlated with the percentage of flow-mediated dilatation (%FMD). Cardiovascular disease is associated with impairments in %FMD. Reduced insulin sensitivity may negatively affect the vascular system for many years before prediabetes/type 2 diabetes states. The aim of this study was to determine whether static and dynamic STO2 parameters during PORH are correlated with reduced insulin sensitivity in young, normoglycaemic subjects. Glucose and insulin were measured during an oral glucose tolerance test in 18- to 26-year-old, healthy subjects (11 men and 11 women), and STO2 was measured during PORH of antebrachial muscle. Hepatic (ISIHOMA ) and whole-body (ISICOMP ) insulin sensitivities were calculated. The STO2 upslope was negatively correlated with minimal STO2 (r = -0.5, P = 0.01). The change of STO2 from minimum to baseline (ΔSTO2 ) was significantly negatively correlated with fasting insulin (r = -0.5, P = 0.01) and a positively correlated with ISIHOMA (r = 0.65, P = 0.001). The minimum STO2 was significantly negatively correlated with ISIHOMA , and STO2 upslope was significantly positively correlated with ISIHOMA (r = 0.44, P = 0.02). The minimum STO2 (a measure of O2 extraction while the cuff was inflated), ΔSTO2 (a measure of the amount of reperfusion) and STO2 upslope (a measure of responsiveness of the microcirculation to ischaemia) were all positively correlated with ISIHOMA , one of the longest-used measures of insulin sensitivity. The NIRS-derived STO2 might be a useful tool for assessing how levels of reduced insulin sensitivity in young, normoglycaemic adults affect the microvasculature.

Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells.

Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection.IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked at an early reverse transcription stage in target cells. To explain the basis of this defect, we tracked the fates of multiple viral components in infected cells. Here, we show that the viral RNA genome and IN in eccentric particles are prematurely degraded, whereas reverse transcriptase remains active and stably associated within the capsid lattice. We propose that IN-RNA interactions ensure the packaging of both vRNPs and IN within the protective capsid cores to facilitate subsequent reverse transcription and productive infection in target cells.

Relationship between brachial artery blood flow and total [hemoglobin+myoglobin] during post-occlusive reactive hyperemia.

The associations between macrovascular and microvascular responses reported previously during post-occlusive reactive hyperemia have been inconsistent. The purpose of this study was therefore to determine the temporal relationship between the reactive hyperemic responses within a conduit artery and the downstream microvessels. Conduit artery blood flow was measured in the brachial artery with pulsed Doppler ultrasound. A potential analog of microvascular flow, changes in skeletal muscle total[hemoglobin+myoglobin] (T[Hb+Mb]), was assessed with near-infrared spectroscopy (NIRS). We found a high degree of correlation between these two measures (r=0.91). Cross-correlation analysis revealed two distinct response patterns. In 10 of our 15 subjects there was time displacement between peak brachial artery blood flow (BABF) and T[Hb+Mb] responses; in the remaining 5 the peaks were coincident. Granger causality testing suggested that reactive hyperemia in the macrovessel determined hyperemia in the downstream microvessels in all 15 study subjects. Time constants for the on (τ1) and off (τ2) kinetics of each response were calculated; our initial hypothesis was that τ1 and τ2 for T[Hb+Mb] would correlate with τ1 and τ2 for BABF, respectively. However, only for τ2 was this observed (r=0.52; p<0.05). No similar relationship was observed for τ1. Adipose tissue thickness did not influence either time constant for T[Hb+Mb]. Taken together, our results show that the temporal characteristics of the hyperemic response in the conduit artery are qualitatively reflected in the downstream microvasculature, but mechanisms for quantitative differences remain to be identified.

Characterizing near-infrared spectroscopy responses to forearm post-occlusive reactive hyperemia in healthy subjects.

During post-occlusive reactive hyperemia (PORH) there is a temporary increase in the total hemoglobin + myoglobin (T[Hb+Mb]) signal as measured by near-infrared spectroscopy (NIRS). This transient increase predicts differences in the kinetic responses of deoxy[Hb+Mb] and oxy[Hb+Mb] during PORH. The purpose of this study was to determine whether sigmoidal (Gompertz or logistic) or exponential functions better describe these response curves during PORH. The fit of the three functions (exponential, Gompertz and logistic) to the NIRS responses, as determined from residual sum of squares, was compared using repeated measures ANOVA on Ranks. The Gompertz function provided a better fit to the oxy[Hb+Mb] response curve than did either the exponential or logistic function (χ (2) = 21.7, df = 2, p < 0.001). The logistic function provided a better fit for the deoxy[Hb+Mb] response (χ (2) = 22.9, df = 2, p < 0.001) than did either the Gompertz or exponential functions. For both NIRS signals, the better fitting sigmoidal functions fit the data well, with an average r value of 0.99 or greater. Adipose tissue thickness was correlated with parameters related to signal strength (amplitude, r = 0.86-0.89; baseline, r = 0.67-0.75; all p < 0.001) but was not related to kinetic parameters (time constant and inflection point; p > 0.05 for all comparisons). These results suggest that during PORH distinct sigmoidal mathematical functions best describe the responses of the oxy[Hb+Mb] (Gompertz) and deoxy[Hb+Mb] (logistic) as measured by NIRS. Further, differences in both the kinetic and amplitude aspects for the responses of oxy[Hb+Mb] and deoxy[Hb+Mb] predict the observed transient change in T[Hb+Mb]. Our methods provide a technique to evaluate and quantify NIRS responses during PORH, which may have clinical utility.

Effects of a high-fat meal on pulmonary function in healthy subjects.

Obesity has important health consequences, including elevating risk for heart disease, diabetes, and cancer. A high-fat diet is known to contribute to obesity. Little is known regarding the effect of a high-fat diet on pulmonary function, despite the dramatic increase in the prevalence of respiratory ailments (e.g., asthma). The purpose of our study was to determine whether a high-fat meal (HFM) would increase airway inflammation and decrease pulmonary function in healthy subjects. Pulmonary function tests (PFT) (forced expiratory volume in 1-s, forced vital capacity, forced expiratory flow at 25-75% of vital capacity) and exhaled nitric oxide (eNO; airway inflammation) were performed in 20 healthy (10 men, 10 women), inactive subjects (age 21.9 +/- 0.4 years) pre and 2 h post HFM (1 g fat/1 kg body weight; 74.2 +/- 4.1 g fat). Total cholesterol, triglycerides, and C-reactive protein (CRP; systemic inflammation) were determined via a venous blood sample pre and post HFM. Body composition was measured via dual energy X-ray absorptiometry. The HFM significantly increased total cholesterol by 4 +/- 1%, and triglycerides by 93 +/- 3%. ENO also increased (p < 0.05) due to the HFM by 19 +/- 1% (pre 17.2 +/- 1.6; post 20.6 +/- 1.7 ppb). ENO and triglycerides were significantly related at baseline and post-HFM (r = 0.82, 0.72 respectively). Despite the increased eNO, PFT or CRP did not change (p > 0.05) with the HFM. These results demonstrate that a HFM, which leads to significant increases in total cholesterol, and especially triglycerides, increases exhaled NO. This suggests that a high-fat diet may contribute to chronic inflammatory diseases of the airway and lung.

Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis.

A large number of human immunodeficiency virus 1 (HIV-1) integrase (IN) alterations, referred to as class II substitutions, exhibit pleiotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of the three distinct mechanisms: (i) markedly reducing IN levels thus precluding the formation of IN complexes with viral RNA; (ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; and (iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in the mislocalization of viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

Effect of assuming constant tissue scattering on measured tissue oxygenation values during tissue ischemia and vascular reperfusion.

The purpose of this study was to determine the effects of assuming constant tissue scattering properties on tissue oxygenation measurements during a vascular occlusion test (VOT). Twenty-one subjects (21.8 ± 1.9 yr) completed a VOT [1 min baseline (BL), 5 min of tissue ischemia (TI), and 3 min of vascular reperfusion (VR)]. Absolute concentrations of oxygenated heme (oxy-[heme]), deoxygenated heme (deoxy-[heme]), total heme (total [heme), tissue oxygen saturation (StO2), and heme difference [heme]diff) were measured using frequency domain near-infrared spectroscopy (FD-NIRS) while 1) continuously measuring and incorporating tissue scattering ([Formula: see text]) and 2) assuming scattering remained constant. FD-NIRS measured [Formula: see text] increased during TI at 692 nm (P < 0.001) and decreased at 834 nm (P < 0.001). During VR, [Formula: see text] decreased at 692 nm (P < 0.001) and increased at 834 nm (P < 0.001). When assuming constant scattering, oxy-[heme] was significantly less at TIpeak (P < 0.05) while deoxy-[heme] and StO2 were significantly altered at BL, TIpeak, and VRpeak (all P < 0.001). Total [heme] did not change during the VOT. Absolute changes in deoxy-[heme], oxy-[heme], and StO2 in response to TI and VR were significantly exaggerated (all P < 0.001) and the rates of change during TI (slope 1) and VR (slope 2) in deoxy-[heme], oxy-[heme], StO2, and [heme]diff were significantly increased (all P < 0.05) when constant tissue scattering was assumed. These findings demonstrate the need for caution when interpreting NIRS data without continuously measuring tissue optical properties. Further, assuming tissue optical properties remain constant may have important consequences to experimental data and clinical conclusions made using NIRS.NEW & NOTEWORTHY NIRS measurements provide significant experimental and clinical insight. We demonstrate that absolute changes in tissue oxygenation measurements made with NIRS are overestimated and the kinetic responses of NIRS measurements are exaggerated by varying degrees among individuals if tissue scattering characteristics are assumed to remain constant during vascular occlusion tests.

Muscle microvascular hemoglobin concentration and oxygenation within the contraction-relaxation cycle.

Inability to directly measure microvascular oxygen distribution and extraction in striated muscle during a contraction/relaxation cycle limits our understanding of oxygen transport to and utilization by contracting muscle. We examined muscle microvascular hemoglobin concentration (total [Hb/Mb]) and oxygenation within the contraction-relaxation cycle to determine if microvascular RBC volume would be preserved and if oxygen extraction continued during the actual contraction phase. Eight subjects performed dynamic knee extension exercise (40 contractions/min) at moderate ( approximately 30% of peak work rate) and heavy ( approximately 80% of peak) work rates. Total hemoglobin/myoglobin (total [Hb/Mb]) and deoxy-hemoglobin/myoglobin (deoxy-[Hb/Mb]) were measured in the rectus femoris using NIRS to determine if microvascular total [Hb/Mb] would be preserved during the contraction, and to estimate microvascular oxygen extraction, respectively. Mean values during the relaxation (RP) and contractile phases and the peak values during the contractile phase for both moderate and heavy exercise were calculated. Total [Hb/Mb] increased from rest to steady-state exercise (6.36+/-5.08 microM moderate; 5.72+/-4.46 microM heavy exercise, both P<0.05), but did not change significantly within the contraction/relaxation cycle. Muscle contractions were associated with a significant (1.29+/-0.98 microM moderate; 2.16+/-2.12 microM heavy exercise, P<0.05) increase in deoxy-[Hb/Mb] relative to RP. We conclude that (a) microvascular RBC volume is preserved during muscle contractions (i.e., RBCs are present in the capillaries), and (b) the cyclical pattern of deoxygenation/oxygenation during the respective contraction/relaxation phases of the contraction cycle suggests that oxygen extraction is not restricted to the relaxation phase but continues to occur during muscle contractions.

Insulin resistance and metabolic syndrome criteria in lean, normoglycemic college-age subjects.

AIMS: The goal of this study was to determine insulin sensitivity in a fasted state and during an oral glucose tolerance test (OGTT), in normoglycemic (NGT), lean (L) (n = 35) and, for comparison, overweight/obese (OW/O) (n = 9) college-aged subjects. MATERIALS AND METHODS: Insulin sensitivity for 44 NGT, normotensive subjects, age 18-26 yrs., was determined by homeostasis model assessment (HOMA-IR) and from Matsuda index (ISI Matsuda). RESULTS: Subjects were normoglycemic fasted (4.59 + 0.35 mmol/L) and at two hours post OGTT (4.52  + 1.35 mmol/L). Besides anthropometric measures, there were significant differences between OW/O and L for fasting insulin (P < 0.001) and both measures of insulin sensitivity (P < 0.05). All subjects exhibited a 9-fold range in HOMA-IR (0.88 + 0.51, range 0.3-2.7) and an 8-fold range in ISI Matsuda (11.9 + 4.7, range 3.0-24.2). The latter was inversely correlated with systolic blood pressure (r = 0.35, P = 0.04) even though subjects were normotensive. In lean subjects, 2.3% were IR by HOMA-IR > 2.1, 5.7% by ISI Matsuda < 5.9, and 22.9% had >one criteria for metabolic syndrome (MetS); 28.6% had some negative metabolic biomarker. CONCLUSIONS: Insulin resistance is present in lean, NGT college-age subjects even without MetS criteria and is discernable with an easily applicable OGTT-derived index.

Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication.

Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.

Effects of inspiratory muscle training on exercise responses in normoxia and hypoxia.

The purpose of this study was to determine the effects of inspiratory muscle training (IMT) on exercise in hypoxia (H) and normoxia (N). A 4-week IMT program was implemented with 12 healthy subjects using an inspiratory muscle trainer set at either 15% (C; n=5) or 50% (IMT; n=7) maximal inspiratory mouth pressure (PImax). Two treadmill tests (85% VO2max) to exhaustion and measures of diaphragm thickness (Tdi) and function were completed before and after training in H and N. Significant increases of 8-12% and 24.5+/-3.1% in Tdi and PImax, respectively, were seen in the IMT group. Time to exhaustion remained unchanged in all conditions. Inspiratory muscle fatigue (downward arrowPImax) following exercise was reduced approximately 10% (P<0.05) in IMT after both N and H. During H, IMT reduced (P<0.05) VO2 by 8-12%, cardiac output by 14+/-2%, ventilation by 25+/-3%; and increased arterial oxygen saturation by 4+/-1% and lung diffusing capacity by 22+/-3%. Ratings of perceived exertion and dyspnea were also significantly reduced. These data suggest that IMT significantly improves structural and functional physiologic measures in hypoxic exercise.

Muscle capillary blood flow kinetics estimated from pulmonary O2 uptake and near-infrared spectroscopy.

The near-infrared spectroscopy (NIRS) signal (deoxyhemoglobin concentration; [HHb]) reflects the dynamic balance between muscle capillary blood flow (Q(cap)) and muscle O(2) uptake (Vo(2)(m)) in the microcirculation. The purposes of the present study were to estimate the time course of Q(cap) from the kinetics of the primary component of pulmonary O(2) uptake (Vo(2)(p)) and [HHb] throughout exercise, and compare the Q(cap) kinetics with the Vo(2)(p) kinetics. Nine subjects performed moderate- (M; below lactate threshold) and heavy-intensity (H, above lactate threshold) constant-work-rate tests. Vo(2)(p) (l/min) was measured breath by breath, and [HHb] (muM) was measured by NIRS during the tests. The time course of Q(cap) was estimated from the rearrangement of the Fick equation [Q(cap) = Vo(2)(m)/(a-v)O(2), where (a-v)O(2) is arteriovenous O(2) difference] using Vo(2)(p) (primary component) and [HHb] as proxies of Vo(2)(m) and (a-v)O(2), respectively. The kinetics of [HHb] [time constant (tau) + time delay [HHb]; M = 17.8 +/- 2.3 s and H = 13.7 +/- 1.4 s] were significantly (P < 0.001) faster than the kinetics of Vo(2) [tau of primary component (tau(P)); M = 25.5 +/- 8.8 s and H = 25.6 +/- 7.2 s] and Q(cap) [mean response time (MRT); M = 25.4 +/- 9.1 s and H = 25.7 +/- 7.7 s]. However, there was no significant difference between MRT of Q(cap) and tau(P)-Vo(2) for both intensities (P = 0.99), and these parameters were significantly correlated (M and H; r = 0.99; P < 0.001). In conclusion, we have proposed a new method to noninvasively approximate Q(cap) kinetics in humans during exercise. The resulting overall Q(cap) kinetics appeared to be tightly coupled to the temporal profile of Vo(2)(m).

Muscle contraction-blood flow interactions during upright knee extension exercise in humans.

To test for evidence of a muscle pump effect during steady-state upright submaximal knee extension exercise, seven male subjects performed seven discontinuous, incremental exercise stages (3 min/stage) at 40 contractions/min, at work rates ranging to 60-75% peak aerobic work rate. Cardiac cycle-averaged muscle blood flow (MBF) responses and contraction-averaged blood flow responses were calculated from continuous Doppler sonography of the femoral artery. Net contribution of the muscle pump was estimated by the difference between mean exercise blood flow (MBFM) and early recovery blood flow (MBFR). MBFM rose in proportion with increases in power output with no significant difference between the two methods of calculating MBF. For stages 1 and 5, MBFM was greater than MBFR; for all others, MBFM was similar to MBFR. For the lighter work rates (stages 1-4), there was no significant difference between exercise and early recovery mean arterial pressure (MAP). During stages 5-7, MAP was significantly higher during exercise and fell significantly early in recovery. From these results we conclude that 1) at the lightest work rate, the muscle pump had a net positive effect on MBFM, 2) during steady-state moderate exercise (stages 2-4) the net effect of rhythmic muscle contraction was neutral (i.e., the impedance due to muscle contraction was exactly offset by the potential enhancement during relaxation), and 3) at the three higher work rates tested (stages 5-7), any enhancement to flow during relaxation was insufficient to fully compensate for the contraction-induced impedance to muscle perfusion. This necessitated a higher MAP to achieve the MBFM.

Matching of blood flow to metabolic rate during recovery from moderate exercise in humans.

It is unclear whether measurement of limb or conduit artery blood flow during recovery from exercise provides an accurate representation of flow to the muscle capillaries where gas exchange occurs. To investigate this, we: (a) examined the kinetic responses of femoral artery blood flow (QFA), estimated muscle capillary blood flow (Qcap) and estimated muscle oxygen uptake (VO2m) following cessation of exercise; and (b) compared these responses to verify the adequacy of O2 delivery during recovery. Pulmonary VO2 (VO2p) was measured breath by breath, QFA was measured using Doppler ultrasonography, and deoxy-haemoglobin/myoglobin (deoxy-[Hb/Mb]) was estimated by near-infrared spectroscopy over the rectus femoris in nine healthy subjects during a series of transitions from moderate knee-extension exercise to rest. The time course of Qcap was estimated by rearranging the Fick equation [i.e. Qcap(t) alpha VO2m(t)/deoxy-[Hb/Mb](t)], using the primary component of Vo2p to represent VO2m and deoxy-[Hb/Mb] as a surrogate for arteriovenous O2 difference. There were no significant differences among the overall kinetics of VO2m (tau, 31.4+/-8.2 s), QFA [mean response time (MRT), 34.5+/-20.4 s] and Qcap (MRT, 31.7+/-14.7 s). The VO2m kinetics were also significantly correlated (P<0.05) with those of both QFA and Qcap. Both QFA and Qcap appear to be coupled with VO2m during recovery from moderate knee-extension exercise, such that extraction falls (thus cellular energetic state is not further compromised) throughout recovery.

Effects of pedal frequency on estimated muscle microvascular O2 extraction.

An increase in muscle contraction frequency could limit muscle blood flow QM compromising the matching of QM and muscle oxygen uptake VO2M. This study examined the effects of pedal cadence on skeletal muscle oxygenation at low, moderate and peak exercise. Nine healthy subjects [24.7+/-6.3 years (SD)] performed incremental cycling exercise at 60 and 100 rpm. Pulmonary VO2(VO2P) was measured breath-by-breath and vastus lateralis oxygenation was determined by near-infrared spectroscopy (NIRS). The deoxyhemoglobin signal ([HHb]) from NIRS was used to estimate microvascular O2 extraction (i.e., [HHb] proportional, variant VO2M/QM). The VO2P and [HHb] for low, moderate and at peak exercise were determined. The VO2P at 60 rpm (low=0.64+/-0.13, moderate=2.03+/-0.38 and peak=3.39+/-0.84 l/min) were lower (P<0.01) than at 100 rpm (1.29+/-0.23, 2.14+/-0.39 and 3.54+/-0.88 l/min, respectively). There was a progressive increase in [HHb] from low to peak exercise. However, there was no significant difference (ANOVA, P=0.94) for the 60 (in microM, low=24.0+/-9.5, moderate=30.5+/-13.8 and peak=36.7+/-16.5) and 100 contractions/min (in microM, low=25.7+/-11.6, moderate=32.1+/-14.0 and peak=35.4+/-16.5). We conclude that vastus lateralis O2 extraction was similar at 60 and 100 cpm, suggesting that the VO2M/QM in the microcirculation was not altered and, presumably, no impairment of QM occurred with the increase in pedal frequency.

Human femoral artery and estimated muscle capillary blood flow kinetics following the onset of exercise.

The purpose of this study was to compare the kinetics of estimated capillary blood flow (Qcap) to those of femoral artery blood flow (QFA) and estimated muscle oxygen uptake (VO2m). Nine healthy subjects performed a series of transitions from rest to moderate (below estimated lactate threshold, 6 min bouts) knee extension exercise. Pulmonary oxygen uptake (VO2) was measured breath by breath, (QFA) was measured continuously using Doppler ultrasound, and deoxyhaemoglobin ([HHb]) was estimated by near-infrared spectroscopy over the rectus femoris throughout the tests. The time course of (Qcap) was estimated by rearranging the Fick equation (i.e. Qcap = VO2m/(a-v)O2), (arterio - venous O2 difference) using the primary component of VO2 to represent VO2m and [HHb] as a surrogate for (a - v)O2. The overall kinetics of QFA (mean response time, MRT, 13.7 +/- 7.0 s), VO2m (tau, 27.8 +/- 9.0 s) and Qcap (MRT, 41.4 +/- 19.0 s) were significantly (P < 0.05) different from each other. We conclude that for moderate intensity knee extension exercise, conduit artery blood flow (QFA) kinetics may not be a reasonable approximation of blood flow kinetics in the microcirculation (Qcap), the site of gas exchange. This temporal dissociation suggests that blood flow may be controlled differently at the conduit artery level than in the microcirculation.