Manipulating Diastereomeric Bicyclononynes to Sensitively Determine Enzyme Activity and Facilitate Macromolecule Conjugations.

Bicyclo[6.1.0]nonyne (BCN) is one of the most commonly used cycloalkynes in strain-promoted azide-alkyne cycloaddition (SPAAC). The synthesis of BCN produces two diastereomers, exo-BCN and endo-BCN. The potential significance of the different steric structures of the tricyclic fused rings in SPAAC products synthesized from the BCN diastereomers has not been previously studied. We first demonstrated that only endo-BCN could reduce the level of fluorescence quenching in SPAAC reaction products. The reduction was likely due to the presence of extended tricyclic fused ring systems. This hypothesis was supported by the synthesis of a fluorescence always-on construct by substituting endo-BCN for exo-BCN in a previously reported chemical probe that was characterized with good contact fluorescence quenching. We also synthesized bis-BCN derivatives to enhance the steric structural differences in the corresponding SPAAC products. A constitutional isomer of the azido-derivatized 5(6)-carboxyfluorescein [5(6)-FAM] was reacted with both bis-exo-BCN and bis-endo-BCN compounds. However, one form of the bis-exo-BCN-based product did not augment contact fluorescence quenching, while a second bis-exo-BCN product could not further reduce contact fluorescence quenching. Nevertheless, a new fluorescence turn-on chemical probe was employed to determine the activities of two serum biomarkers, butyrylcholinesterase and paraoxonase 1. Moreover, bis-endo-BCN was exploited to successfully conjugate BSA with a 5-FAM derivative compound.

Inspiratory Muscle Training Improves Aerobic Fitness in Active Children.

Research on the effect of inspiratory muscle training (IMT) on exercise performance is inconsistent. IMT has not been applied to fit child athletes, who are more likely to develop inspiratory muscle fatigue, and, consequently, to benefit from IMT. Methods: Thirty children (mean age: 10.7 ± 1.2 years) were recruited and randomly assigned to a high-intensity (HG), a low-intensity (LG), or a control group (CG). For both training groups, a double-blind procedure was applied. In the HG, 80% of maximal inspiratory pressure (MIP) was used as the level of training intensity. The LG used 30% MIP. Training groups were trained at 6 breaths a set, 4 sets a day, totaling 24 breaths a day for 6 weeks. Exercise capacity outcomes include maximal and submaximal aerobic capacity, as measured as VO2max and distance from six-minute walk test (6MWD). Results show improvement in MIP, VO2max, and 6MWD only in the HG. MIP in the HG significantly increases from 108.7 (100.8-143.3) to 144.4 (130.0-175.6) cmH2O. VO2max in the HG increases from 43.0 (40.5-45) to 53.0 (46-63) mL·kg-1·min-1. The 6MWD increases from 792.0 (737.5-818.0) to 862.0 (798.5-953.5) m. Data are presented as median (interquartile range). No difference is found in the LG or CG. Conclusion: high-intensity IMT increases MIP, maximal, and submaximal exercise capacity in the HG, but no difference is found in the LG or CG. Therefore, high-intensity type of IMT improves aerobic fitness in fit children by appropriately applying inspiratory muscle strength training.

Time-dependent effects of storage at -80 °C on the stability of butyrylcholinesterase activity in human serum.

Objectives: Butyrylcholinesterase (BChE) is an important biomarker in serum, and aberrant BChE activity indicates onset and progression of human diseases. The duration of serum storage at -80 °C may introduce variability into and compromise the reproducibility of BChE activity measurements. Design and Methods: We collected serum samples from eight healthy volunteers and determined serum BChE activity in these samples using a sensitive fluorescence assay at various time points during a six-month storage period at -80 °C. Changes in averaged BChE activity over storage time were assessed by repeated measures analysis of variance (ANOVA). Sidak multiple comparisons test was also used to perform post-hoc analysis. Results: Almost all determined BChE activity values lay within the normal physiological range of BChE activity. However, repeated measures ANOVA using mean BChE activity vs. storage time showed that BChE activity values from two time points were significantly different. Analysis by Sidak multiple comparisons test provided no substantial change of BChE activity during the first 90 days of storage, but BChE activity noticeably decreased after 90 days. Conclusions: Serum samples stored in -80 °C for up to 90 days can be exploited to accurately determine BChE activity.

Sensitive Assay for the Lactonase Activity of Serum Paraoxonase 1 (PON1) by Harnessing the Fluorescence Turn-On Characteristics of Bioorthogonally Synthesized and Geometrically Controlled Chemical Probes.

The lactonase activity of paraoxonase 1 (PON1) has a crucial antiatherogenic function, and also serves as an important biochemical marker in human blood because the aberrant lactonase activity of PON1 is a key indicator for a number of diverse human diseases. However, no sensitive fluorescence assays that detect PON1 lactonase activity are available. We report the synthesis of two fluorescence turn-on chemical probes 16a and 16b (16) able to quantify PON1 lactonase activity. The chemical probes were constructed utilizing a disulfide-containing bicyclononyne, derivatives of rhodamine B and carboxyfluorescein, and reactions including copper-free azide-alkyne cycloaddition. Fluorescence quenching in 16 was characterized by spectroscopic studies and was mainly attributed to the effect of contact quenching. Kinetic analysis of 16b confirmed the outstanding reactivity and specificity of 16b with thiols in the presence of general base catalysts. The 16b-based assay was employed to determine PON1 lactonase activity, with a linear range of 10.8-232.1 U L-1 and detection limit (LOD) of 10.8 U L-1, to quantify serum PON1 activity in human sera, and to determine the Ki of 20.9 µM for the 2-hydroxyquinoline inhibition of PON1 lactonase. We are employing 16b to develop high-throughput assays for PON1 lactonase activity.

Supervised Phase II Cardiac Exercise Therapy Shortens the Recovery of Exercise Capacity in Patients with Acute Myocardial Infarction.

[Purpose] To investigate the effects of Phase II cardiac exercise therapy (CET) on exercise capacity and changes in coronary risk factors (CRFs) of patients with acute myocardial infarction (AMI). [Subjects] Thirty male subjects with AMI were divided into an experimental group (EG) and a control group (CG). Another 30 age-matched subjects with patent coronary arteries served as a normal-control group (NCG). [Methods] Subjects in EG (n=20) trained using a stationary bicycle for 30 min at their target heart rate twice a week for 8 weeks. Exercise capacity was defined as the maximal metabolic equivalents (METs) that subjects reached during the symptom-limited maximal exercise test. HR, BP and RPP were recorded. Subjects in EG and CG received exercise tests and screening for CRFs at the beginning of, end of, and 3 months after Phase II CET, while subjects in NCG participated only in the 1st test. [Results] METs of CG did not improve until the 3rd test, while RPP at the 2nd test showed a significant increase. However, EG showed increased METs at the 2nd test without increase of RPP, and increased their high density lipoprotein cholesterol (HDL-C) during the follow-up period between the 2nd and 3rd tests. [Conclusion] Phase II CET shortens the recovery time of exercise capacity, helps to maintain the gained exercise capacity and increases HDL-C in phase III.

Sniff nasal inspiratory pressure does not decrease in elderly subjects.

[Purpose] Measurements of inspiratory strength are critical for detecting inspiratory muscle weakness. Sniff nasal inspiratory pressure (SNIP) is a quick, noninvasive measurement of global inspiratory strength; however, it is not clear how many trials are needed for reliable measurements. [Subjects and Methods] One hundred and nineteen subjects (age 39.9±16.5, range 18-69 yrs) completed the study. They were divided into subgroups of different ages and gender. Subjects were asked to take 20 maximal sniffs after normal expiration, with 30 seconds rest in-between. The highest values among the first 10 and last 10 SNIP maneuvers were recorded as SNIP1-10, and SNIP11-20, respectively. The paired t-test was used to compare the differences. Two-way measures ANOVA was used to compare the effects of age and gender on SNIP. [Results] SNIP 11-20 was significantly greater than SNIP1-10, suggesting that 10 trials is not enough to eliminate learning effects. Age did not affect SNIP in either gender, suggesting SNIP is preserved. In stepwise multiple linear regression analysis, the SNIP values were positively related with body mass index in women and positively related with weight in men. [Conclusion] The results suggest that twenty trials are needed for reliable SNIP measurements. The mean value and lower limits of normal SNIP are provided for clinical comparison.

Effects of Combined Phase III and Phase II Cardiac Exercise Therapy for Middle-aged Male Patients with Acute Myocardial Infarction.

[Purpose] To investigate the effects of cardiac exercise therapy (CET) on exercise capacity and coronary risk factors (CRFs) of patients with acute myocardial infarction (AMI). [Methods] Patients who participated in an 8-week supervised, hospital-based phase II and 6-month home-based phase III CET with monthly telephone and/or home visits were defined as the exercise group (EG) (n=20), while those who did not receive phase II or phase III CET were defined as the no-exercise group (NEG) (n=10). CRFs were evaluated pre- and post-phase II and eight months after discharge. One and two-way repeated measures ANOVA were used to perform intra- and inter-group comparisons. [Results] Thirty men with AMI aged 49.3 ± 8.3 years were studied. EG increased their exercise capacity (METs) (6.8 ± 1.6 vs.10.0 ± 1.9) after phase II CET and was able to maintain it at 8-month follow-up. Both groups had significantly fewer persons who kept on smoking compared to the first examination. High density lipoprotein cholesterol (HDL-C) increased from 38.1 ± 11.0 to 43.7 ± 8.7 mg/dl at follow-up in EG while no significant difference was noted in NEG. [Conclusion] After phase III CET subjects had maintained the therapeutic effects of smoking cessation, and increasing exercise capacity obtained in phase II CET. HDL-C in EG continued to improve during phase III CET.

Relationship between Respiratory Load Perception and Perception of Nonrespiratory Sensory Modalities in Subjects with Life-Threatening Asthma.

Subjects with life-threatening asthma (LTA) have reported decreased sensitivity to inspiratory resistive (R) loads. It is unknown if decreased sensitivity is specific for inspiratory R loads, other types of respiratory loads, or a general deficit affecting sensory modalities. This study hypothesized that impairment is specific to respiratory stimuli. This study tested perceptual sensitivity of LTA, asthmatic (A), and nonasthmatic (NA) subjects to 4 sensory modalities: respiratory, somatosensory, auditory, visual. Perceptual sensitivity was measured with magnitude estimation (ME): respiratory loads ME, determined using inspiratory R and pressure threshold (PT) loads; somatosensory ME, determined using weight ranges of 2-20 kg; auditory ME, determined using graded magnitudes of 1 kHz tones delivered for 3 seconds bilaterally; visual ME, determined using gray-to-white disk intensity gradations on black background. ME for inspiratory R loads lessened for LTA over A and NA subjects. There was no significant difference between the 3 groups in ME for PT inspiratory loads, weight, sound, and visual trials. These results demonstrate that LTA subjects are poor perceivers of inspiratory R loads. This deficit in respiratory perception is specific to inspiratory R loads and is not due to perceptual deficits in other types of inspiratory loads, somatosensory, auditory, or visual sensory modalities.

Effect of time ratio of heat to cold on brachial artery blood velocity during contrast baths.

BACKGROUND: Contrast baths have been adopted widely in clinics. However, the time ratio of heat to cold modalities has not been well established. OBJECTIVE: The purpose of this study was to explore the effect of time ratio on brachial artery mean blood velocity (aMBV) and determine the optimal duration in the second heating phase. DESIGN: This was a within-participant, repeated-measures, experimental study. METHODS: Thirty-four young volunteers who were healthy were recruited. Each participant performed 2 kinds of contrast baths within 2 separate sessions. In the first trial with a fixed time ratio, participants immersed their left hands in a 40°C hot bath for 3 minutes and then in an 18°C cold bath for 1 minute. This procedure was repeated 3 times. In the second trial, after the initial 3-minute hot bath and 1-minute cold bath, a 10-minute 40°C hot bath immersion was adopted as the second cycle. A color Doppler ultrasound scanner was used to measure aMBV, which was used to calculate the percentage of change related to the baseline (aMBV%) and the fluctuation in the heating phases (ΔaMBV%). RESULTS: In the first trial, compared with the first heating phase, the ΔaMBV% was significantly lower by 57% and by 46% in the second and third heating phases, respectively. In the second trial, the ΔaMBV% beyond the 7th minute did not reach a significantly lower level. LIMITATIONS: The results cannot be generalized to elderly individuals or patients with medical conditions. CONCLUSION: A longer duration in the second heating phase during contrast baths was required to produce a sufficient fluctuation in blood flow.

Comparison of inspiratory muscle strength training effects between older subjects with and without chronic obstructive pulmonary disease.

BACKGROUND/PURPOSE: Inspiratory muscle strength training (IMST) has been traditionally recommended for patients with chronic obstructive pulmonary disease (COPD) to improve respiratory strength. Respiratory strength is reduced as age increases. However, few studies have focused on the effects of IMST on older adults without COPD. METHODS: Subjects were divided into training non-COPD (TNC, n = 24) and training COPD (TC, n = 12) according to their forced expiratory volume in 1 second (% predicted). Both groups received 6 weeks of IMST, with training at 75-80% of maximal inspiratory pressure using pressure threshold trainers. A second group of COPD subjects served as controls (CC, n = 24), which received no training. Dyspnea was measured using the basic dyspnea index. Health-related quality of life was measured using the SF-36. The SF-36 subcategories, physical component summary and mental component summary were compared. A 6-minute walk test was performed to determine functional status. Two-way repeated measures analysis of variance was used to compare group effects and training effects of IMST. RESULTS: Maximal inspiratory pressure was increased in both training groups (TNC: 59.1 cmH(2)O pre-IMST to 82.5 cmH(2)O post-IMST; TC: 53.2 to 72.6), but not in the CC group. Therefore, the basic dyspnea index was improved in both training groups (TNC: 9.6 to 10.8; TC: 6.2 to 7.3). Functional status was improved in the TNC group (TNC: 392.1 m to 436.3 m), but not in the TC or CC groups. Quality of life was improved in the physical component summary in both training groups. CONCLUSION: IMST increases maximal inspiratory pressure, relieves dyspnea and improves health-related quality of life in older adults. IMST especially improves functional status in subjects without COPD. IMST benefits subjects with COPD and those without COPD. Therefore, IMST as a treatment tool is not confined to patients with COPD.

Effects of inspiratory strength training on the detection of inspiratory loads.

Pressure-threshold loads (DeltaPT) are inspiratory force-related loads, which contrast with resistive loads (DeltaR), are airflow-dependent loads. If detection of respiratory loads is a function of the background load, then pressure-threshold type inspiratory muscle strength training (IMST) would affect the detection of DeltaPT but have less effect on detection of DeltaR. DeltaR and DeltaPT detection and ventilatory responses were measured in healthy volunteers. IMST consisted of 4 sets of 6 breaths per day for 4 weeks, at 75% of maximal inspiratory pressure (MIP). MIP increased and a measure of inspiratory dirve, the mouth pressure generated in the initial 100 msec of an occluded inspiration (P(0.1)), decreased after IMST. IMST significantly increased MIP after 4 weeks of training. IMST did not change DeltaR detection threshold and DeltaR-breathing pattern. IMST decreased DeltaPT detection percent and DeltaPT-breathing pattern. Comparing DeltaR and DeltaPT at the same mouth pressure-generating level, the detection percent was different. We conclude that IMST affects the detection of DeltaPT, but not DeltaR. These results also suggest that mouth pressure is not the primary determinant of the inspiratory load detection. The significance of these results is that inspiratory pressure generating capacity can be increased by our pressure threshold training and this increase in respiratory muscle strength increases the ability of pulmonary patients to compensate for increased respiratory load and modulates the threshold for detection of changes in pulmonary mechanics.

Effect of inspiratory muscle strength training on inspiratory motor drive and RREP early peak components.

This study investigated the effect of inspiratory muscle strength training (IMST) on inspiratory motor drive [mouth occlusion pressure at 0.1 s (P(0.1))] and respiratory-related evoked potentials (RREP). It was hypothesized that, if IMST increased inspiratory muscle strength, inspiratory motor drive would decrease. If motor drive were related to the RREP, it was further hypothesized that an IMST-related decrease in drive would change RREP latency and/or amplitude. Twenty-three subjects received IMST at 75% of their maximal inspiratory pressure (Pi(max)) with the use of a pressure threshold valve. IMST consisted of four sets of six breaths daily for 4 wk. P(0.1) and the RREP were recorded before and after IMST. Posttraining, Pi(max) increased significantly by 36.0 +/- 2.7%. P(0.1) decreased significantly by 21.9 +/- 5.2%. The increase in Pi(max) was significantly correlated to the decrease in P(0.1). RREP peaks P(1a), N(f), P(1), and N(1) were identified pre- and post-IMST, and there was no difference in either amplitude or latency for those peaks. These results demonstrate that high-intensity IMST significantly increased Pi(max), decreased P(0.1), but did not change the RREP.

Axonemal dynein expression in human fetal tracheal epithelium.

Ciliogenesis in human fetal airway epithelium occurs from 11 to 24 gestational weeks. Using genetic and antigenic markers specific for human axonemal dynein heavy chain 9, we characterized temporal aspects of axonemal dynein expression associated with large airway epithelial ciliogenesis during human fetal development. Late in the first trimester, an undifferentiated columnar epithelium is characteristic of the large airways, and immunocytochemical studies exhibited focal localization of axonemal dynein antigen on luminal epithelial cell borders at sites consistent with emergent ciliary beds. From 12 to 22 wk, immunocytochemical labeling of new ciliary beds was prominent, and localization within the cytoplasm of epithelial cells suggested avid synthesis of axonemal dynein in advance of ciliogenic events. Quantitative RT-PCR of tracheal RNA and in situ hybridization studies compared favorably with immunocytochemical findings with the earliest expression of axonemal dynein at 9-10 wk gestation. These studies have documented that axonemal dynein is expressed early in human fetal life during airway epithelial maturation and well before histological or ultrastructural evidence of ciliogenesis is apparent.