Development of diagnostic SNP markers and a novel SNP genotyping assay for distinguishing opium poppies.

The opium poppy acts as an important natural pain reliever but is also responsible for increased rates of severe drug abuse and addiction owing to its characteristic psychoactive effect. Non-medical illicit use of the poppy plant is markedly increasing worldwide, thereby highlighting the need for a robust species identification strategy. In this study, we identified SNPs within the region of two universal DNA barcodes, matK (maturase K) and the trnL-trnF (tRNA-Leu [3'exon]-tRNA-Phe [exon] intergenic spacer, that are forensically applicable for distinguishing opium poppy species based on a genetic analysis of 164 samples of family Papaveraceae obtained from locations spanning Jeolla-do and Jeju Island, Republic of Korea. A comparative analysis of the DNA barcode sequences for two narcotic types of the Papaver species (Papaver somniferum, Papaver somniferum subs. setigerum) to eight non-narcotic species revealed three unique nucleotide substitution events. Newly identified SNPs were located at position 255 of matK and at positions 305 and 306 of trnL-trnF; the narcotic species contained C, A, and T, whereas non-narcotic species contained T, G, and C at these positions. Phylogenetic analysis demonstrated that newly identified SNPs, which we named PsMAT255 and PsLF305/306, could be used to clearly differentiate between the narcotic and non-narcotic types of Papaver species based on the patterns of nucleotide variation. These results indicate that the nucleotide differences between the narcotic and non-narcotic species may influence genetic markers. We, therefore, developed a novel SNP-based allelic genotyping assay using the RT-PCR system that can reliably differentiate the narcotic type of the Papaver species. In summary, our findings suggest that the newly identified species-specific SNPs of both matK and trnL-trnF can be used as identification markers of narcotic Papaver species. Furthermore, a newly developed TaqMan allelic discrimination assay may be used as a practically applicable diagnostic method to survey several illicit narcotic specimens carrying the type-specific SNP.

A new minisatellite VNTR marker, Pscp1, discovered for the identification of opium poppy.

Opium poppy, a member of the Papaveraceae family, is an ancient herbaceous plant and well-known medical resource in the pharmaceutical industry. However, opium poppies are grown worldwide for producing illicit drugs, significantly increasing the incidence of narcotic drug abuse. Since the narcotic poppy has not yet been genetically investigated, we characterized a novel variable number tandem repeat (VNTR) marker of forensically important poppy species based on the genetic analysis of 164 samples collected from two locations spanning the Jeolla province and Jeju island of South Korea. Comparing analysis of the chloroplast (cp) genome sequences for four representative species of Papaver (Papaver somniferum, Papaver somniferum subs. setigerum, Papaver orientale, and Papaver rhoeas) revealed a unique region with 1-3 repeats for 16 nucleotide motifs in the genome inverted repeat A (IRA, positions 128,651 to 128,698) region. For 16 nucleotide motifs, 3 repeats were found in P. somniferum, and 2 repeats were found in P. somniferum subs. setigerum. Therefore, 10 known and the 133 unknown, seized Papaver species were compared to determine whether the species could be identified via variations in the repeat units. The sizes of a novel VNTR ranged from 181 to 252 bp between the species. Phylogenetic analysis confirmed that a novel VNTR, which we named Pscp1, could clearly distinguish between the narcotic and non-narcotic types of Papaver species based on the patterns of sequence variation. Interestingly, we found that Pscp1 could also distinguish between P. somniferum and P. somniferum subs. setigerum. The regions of eight non-narcotic species displayed similar patterns and also differences were found due to the nucleotide substitution and deletion events. The structural differences of Pscp1 were observed within the two narcotic species or between the narcotic and non-narcotic species, suggesting that these variations may act as a genetic marker. We, therefore, developed a new Pscp1 PCR-capillary electrophoresis (CE) method that can reliably identify the narcotic type of Papaver species. Taken together, our findings suggest that the newly developed Pscp1 can be used as an identification marker of opium poppy, and establish that the Pscp1 genotyping method by PCR-CE is an effective primary screening tool that can also contribute to species discrimination in the field of forensic diagnosis and applications.

High Quality AlN Layer Grown on Patterned Sapphire Substrates by Hydride Vapor-Phase Epitaxy: A Route for Cost Efficient AlN Templates for Deep Ultraviolet Light Devices.

We report the characteristics of AlN epilayers grown directly on cylindrical-patterned sapphire substrates (CPSS) by hydride vapor-phase epitaxy (HVPE). To evaluate the effect of CPSS, we analyzed the threading dislocation densities (TDDs) of AlN films grown simultaneously on CPSS and flat sapphire substrate (FSS) by transmission electron microscopy (TEM). The corresponding TDD is measured to be 5.69 x 108 cm-2 for the AlN sample grown on the CPSS that is almost an order of magnitude lower than the value of 3.43 × 109 cm-2 on the FSS. The CPSS contributes to reduce the TDs originated from the AlN/sapphire interface via bending the TDs by lateral growth during the coalescence process. In addition, the reduction of direct interface area between AlN and sapphire by CPSS reduce the generation of TDs.

Laparoscopic Ablation System for Complete Circumferential Renal Sympathetic Denervation.

GOAL: The catheter-based renal denervation (RDN) showed promising results for patients in lowering BP, but there were also many non-responders. One of the possible reasons was the incomplete neural ablation due to the ablation of renal nerves at random sites resulting in asymmetric innervation patterns along the renal artery. METHODS: We developed a laparoscopic ablation system that is optimized for complete RDN regardless of renal arterial innervation and size. To demonstrate its effectiveness, we evaluated the system using computational simulation and 28-day survival model using pigs. RESULTS: The ablations were focused around the tunica externa, and the ablation patterns could be predicted numerically during RDN treatment. In the animal study, the mean reduction of systolic BP and diastolic BP in the bilateral main renal arteries was 22.8 mmHg and 14.4 mmHg (P<0.001), respectively. The respond to immunostaining targeting tyrosine hydroxylase was significantly reduced at treatment site (108.2 ± 7.5 (control) vs. 63.4 ± 8.7 (treatment), P<0.001), and an increased degree of sympathetic signals interruption to kidneys was associated with the efficacy of RDN. CONCLUSION: The laparoscopic ablation system achieved complete circumferential RDN at the treatment site and could numerically predict the ablation patterns. SIGNIFICANCE: These findings clearly suggest that the proposed system can significantly improve the RDN effectiveness by reducing the variation to the percentage of injured nerves and open up a new opportunity to treat uncontrolled hypertension.

Laparoscopic Renal Denervation System for Treating Resistant Hypertension: Overcoming Limitations of Catheter-Based Approaches.

GOAL: In a pivotal clinical trial, the percutaneous catheter-based renal denervation system developed to treat resistant hypertension did not show effectiveness in reducing blood pressure because of its fundamental limitation to ablate deeper nerves present around the renal artery. METHODS: We propose a new renal denervation strategy called laparoscopicdenervation system (LDS) based-on laparoscopy procedure to ablate the renal nerves completely but inhibit the thermal arterial damage.The system has flexible electrodes to bend around the arterial wall to ablate nervesThe simulation study using validated in-silico models evaluated the heat distributionon the outer arterial wall,and an acute animal study (swine model) was conducted to demonstrate the feasibility of LDS in vivo. RESULTS: The simulation studyconfirmedthat LDS could localize the heat distributionbetween the electrode and the outer arterial wall. In the animal study, we could maximize nerve denervation by the localizing ablation energy within the renal nerves and achieve nerve denaturationand decrease in neural density by 20.78% (P < 0.001), while maintaining a constant tip temperature of 65 °C for the duration of 70 s treatment. The study confirmed intact lumen artery through histological analysis and acute reduction in systolic blood pressure by 9.55 mmHg (p < 0.001) Conclusion: The LDS presented here has potential to effectively and safely ablate the renal nerves, independent of anatomical variation and nerve distribution, to control hypertension in real clinical conditions. SIGNIFICANCE: LDS approach is innovative, inventive, and presents a novel technique totreat hypertension.

Effects of breathing exercises on resting metabolic rate and maximal oxygen uptake.

[Purpose] The aim of the present study is to examine effects of diaphragm breathing exercise and feedback breathing exercise on maximal oxygen uptake and resting metabolic rate. [Participants and Methods] Thirty-eight healthy participants were randomly assigned to two groups; the diaphragm breathing exercise group and the feedback breathing exercise group. The diaphragm breathing exercise group was asked to perform diaphragm respiration, and the feedback breathing exercise group was asked to breathe with feedback breathing device. Maximal oxygen uptake and resting metabolic rate were measured before and after two breathing exercises. [Results] Significant difference was found in maximal oxygen uptake before and after two breathing exercises. There was also significant difference in resting metabolic rate before and after diaphragm breathing exercise. However, significant difference was not found in resting metabolic rate before and after feedback breathing exercise. There were not significant between-group differences in both maximal oxygen uptake and resting metabolic rate. [Conclusion] Diaphragm breathing exercise and feedback breathing exercise could influence maximal oxygen uptake. Diaphragm breathing exercise could influence resting metabolic rate, but feedback breathing exercise could not.

Effect of diaphragm breathing exercise applied on the basis of overload principle.

[Purpose] The purpose of this study was to examine effects of diaphragm breathing exercise applied on the basis overload principle on respiratory function. [Subjects and Methods] The subjects of this study were 35 normal adults. They were randomly assigned to two group; the maneuver-diaphragm exercise group and self-diaphragm exercise group. The respiratory function was evaluated using the CardioTouch 3000S (BIONET, Korea) as a pulmometry device. [Results] The maneuver-diaphragm exercise was more effective on functional vital capacity and forced expiratory volume at one second when compared to the self-diaphragm exercise. [Conclusion] According to the results of this study, although the self-diaphragm exercise did not show effects as much as the maneuver one, but the self-diaphragm exercise had a similar effects as the maneuver-diaphragm exercise. The self-diaphragmatic respiration applied on the basis of overload principle may be used as an effective respiratory exercise as a part of home respiration program.

Effects of diaphragm breathing exercise and feedback breathing exercise on pulmonary function in healthy adults.

[Purpose] The present study investigated effects of diaphragm breathing exercise and feedback breathing exercise on respiratory function. [Subjects and Methods] Thirty-one subjects were randomly assigned to two groups; the feedback breathing exercise group and the maneuver-diaphragm exercise group. The feedback breathing exercise group was asked to breathe with feedback breathing device, and the maneuver-diaphragm exercise group was asked to perform diaphragm respiration. Respiratory function was evaluated when a subject sat on a chair comfortably. [Results] There was a significant difference in the functional vital capacity and slow vital capacity before and after all breathing exercises. There was a significant between-group difference in functional vital capacity. However, no between-group difference was found in slow vital capacity. [Conclusion] Diaphragm breathing exercise and feedback breathing exercise can affect respiratory function.

Ascorbic acid and vitamin C-containing beverages delay the leucomalachite green reaction to detect latent bloodstains.

The leucomalachite green (LMG) test is one of catalytic tests for the detection of latent bloodstains and generally used in forensic field because of convenience and cost/time-effectiveness. However, contamination of latent bloodstains at crime scenes can interfere with the LMG reaction, resulting in false-negative or false-positive decisions. Herein, we examined if ascorbic acid and vitamin C (l-ascorbic acid or ascorbate)-containing beverages affect the LMG reaction. Ascorbic acid showed the inhibitory activities on the LMG reaction in a dose-dependent manner. Similarly, vitamin C-containing beverages also inhibited the LMG reaction and the inhibitory effects were proportional to the concentrations of vitamin C in beverages. It was also identified that as incubation time after adding LMG reagent to the mixtures of blood and ascorbic acid or beverages was increased, the inhibitory effects of ascorbic acid vitamin C-containing beverages on LMG test were disappeared. These results suggest that the LMG reaction is delayed but not stopped by ascorbic acid and vitamin C-containing beverages. Neither incubation at room temperature around 20-25°C nor the addition of acetic acid affects the inhibitory activity of ascorbic acid on LMG reaction. We also showed that ascorbic acid does not affect DNA stability, allowing us to obtain full short tandem repeat (STR) profiles through amplification of DNA using commercial STR kits. In conclusion, ascorbic acid and vitamin C-containing beverages delayed the LMG reaction, suggesting that it should be considered that negative results of LMG test could be false negative due to contamination of bloodstains with inhibitory factors on LMG test.

Correlation between head posture and proprioceptive function in the cervical region.

[Purpose] The aim of the present study was to investigate correlation between head posture and proprioceptive function in the cervical region. [Subjects and Methods] Seventy-two subjects (35 males and 37 females) participated in this study. For measurement of head posture, the craniovertebral angle was calculated based on the angle between a horizontal line passing through C7 and a line extending from the tragus of the ear to C7. The joint position sense was evaluated using a dual digital inclinometer (Acumar, Lafayette Instrument, Lafayette, IN, USA), which was used to measure the joint position error for cervical flexion and extension. [Results] A significant negative correlation was observed between the craniovertebral angle and position sense error for flexion and extension. [Conclusion] Forward head posture is correlated with greater repositioning error than a more upright posture, and further research is needed to determine whether correction of forward head posture has any impact on repositioning error.

Effects of Kinesio taping on joint position sense of the ankle.

[Purpose] The purpose of this study was to examine the effect of Kinesio taping on the joint position sense of the ankle. [Subjects and Methods] The subjects of this study were 26 nomal adults who had experienced ankle sprain. Kinesio taping was applied over the ankle medial ligament and ankle lateral ligament with eight pattern reinforcement taping. Joint position sense was measured using isokinetic equipment (Biodex System 4 pro dynamometer, Biodex Medical systems Inc., USA) during dorsiflexion/plantarflexion and inversion/eversion, before and after taping. Statistical analyses were performed using SPSS 21.0 for Windows. [Results] Joint position sense after Kinesio taping was improved in the dorsiflexion and inversion positions. [Conclusion] According to the results of this study, Kinesio taping of the ankle is effective for the prevention of ankle sprain.

Effects of increased standing balance on pain in patients with knee osteoarthritis.

[Purpose] The purpose of this study was to examine the relationship between standing balance increased through muscle-strengthening exercises and pain in knee osteoarthritis patients. [Subjects and Methods] Thirty knee osteoarthritis patients were equally divided into a strengthening exercise group and an unstable exercise group. Before and after the six-week experiment, the visual analogue scale was measured, and bilateral one-leg standing tests were performed. [Results] In both the strengthening exercise group and unstable exercise group, the bilateral one-leg standing time significantly increased after the six-week experiment. Regarding the visual analogue scale, a pain measurement scale, the strengthening exercise group had significantly decreased pain. The unstable exercise group also had decreased pain, but the decrease was not statistically significant. [Conclusion] In knee osteoarthritis patients, exercises using an unstable base of support and knee-extensor strengthening exercises with gradually increased loads had a positive effect on improving balance ability and decreasing pain.

The effect of cervical stabilization exercise on active joint position sense: A randomized controlled trial.

We attempted to investigate the effect of cervical stabilization exercise for a period of 5 weeks on active joint position sense in healthy young adults. Twenty-four subjects were randomly assigned to either the exercise group or the control group. The exercise group performed cervical stabilization exercise during the 5-week period. Cervical active joint position sense was measured using the head repositioning accuracy test. The result showed a positive interaction between time and group in the left and right rotation, flexion, and extension. Cervical stabilization exercise significantly improved the sense for four position senses in the exercise group (p< 0.05). However, there was no difference in the four position senses in the control group (p> 0.05). In conclusion, we suggested that cervical stabilization exercise leads to improvement in accuracy of the joint position sense.

Effects of craniocervical flexion exercise on upper-limb postural stability during a goal-directed pointing task.

[Purpose] This study investigated the effects of craniocervical flexion exercise on upper-limb postural stability by measuring upper-limb postural tremor during a goal-directed pointing task. [Subjects and Methods] Twenty-four subjects were randomly assigned to the exercise or control group. The exercise group performed craniocervical flexion exercise four days per week for five weeks. Upper-limb postural tremor was measured by using a three-dimensional electromagnetic motion tracking system (trakSTAR™, Ascension Technology Corporation, Burlington, VT, USA) during a goal-directed pointing task. [Results] In the exercise group, the range and velocity of the trajectories of the shoulder, wrist, and finger in the lateral direction improved significantly. However, no significant changes were observed in the control group. [Conclusion] Craniocervical flexion exercise reduces the range and velocity of upper-limb postural tremor, thereby increasing postural stability.

Effects of the ankle angle of an ankle foot orthosis on foot pressure during the gait in healthy adults.

[Purpose] The purpose of this study was to investigate the effects of the ankle angle of an ankle foot orthosis (AFO) on foot pressure during the gait in healthy adults. [Subjects] Sixteen healthy males with neither orthopedic nor neurological problems participated in this study. [Methods] Subjects walked on a walkway at a self-selected pace with an AFO set at four different ankle angles (-5°, 0°, 5°, and 10°). Foot pressure was measured randomly according to the ankle angle of the AFO using an F-scan system. Three trials were measured and averaged for data analysis. [Results] The peak foot pressure of the hallux, 2nd-5th toes, 2nd and 3rd metatarsal heads, 4th and 5th metatarsal heads, and the heel showed significant differences among the AFO ankle angles: angles of 0° and -5° increased the foot pressure of the lateral legions, and the peak foot pressure of the heel at an AFO ankle angle of 10° was significantly greater than those of the other angles. [Conclusion] The ankle angle of the AFO affected foot pressure and gait patterns during gait. The results suggest that the appropriate angle for an AFO is between 5° and 10° when AFOs are prescribed by clinicians.

Characteristics of cervical position sense in subjects with forward head posture.

[Purpose] The purpose of this study was to investigate the effect of forward head posture (FHP) on proprioception by determining the cervical position-reposition error. [Subjects and Methods] A sample population was divided into two groups in accordance with the craniovertebral angle: the FHP group and the control group. We measured the craniovertebral angle, which is defined as the angle between a horizontal line passing through C7 and a line extending from the tragus of the ear to C7. The error value of the cervical position sense after cervical flexion, extension, and rotation was evaluated using the head repositioning accuracy test. [Results] There were significant differences in the error value of the joint position sense (cervical flexion, extension, and rotation) between the FHP and control groups. In addition, there was an inverse correlation between the craniovertebral angle and error value of the joint position sense. [Conclusion] FHP is associated with reduced proprioception. This result implies that the change in the muscle length caused by FHP decreases the joint position sense. Also, proprioception becomes worse as FHP becomes more severe.

The influence of exercise on an unstable surface on the physical function and muscle strength of patients with osteoarthritis of the knee.

[Purpose] This study investigated the influence of exercise on an unstable surface on ROM of the knee, muscle strength and the physical function of patients with osteoarthritis of the knee. [Subjects] The subjects were 30 patients diagnosed with degenerative osteoarthritis of the knee, they were divided into an experimental group (n=15) and a control group (n=15). [Methods] The experimental group performed exercise using an aero step XL (TOGU, Germany) 3 times a week, for 6 weeks. A control group performed the same exercise on a stable surface and without aero step XL. [Results] After the intervention, the experimental group showed statistically significant improvements in active knee flexion, knee joint manual muscle test (MMT), knee joint MMT hamstring and WOMAC score. [Conclusion] Exercise on the unstable surface improved the symptoms of patient with osteoarthritis. Exercise on an unstable surface might be helpful for improving the muscle strength and alignment of lower extremities as well as improving physical function related to the knee joint.

CD53, a suppressor of inflammatory cytokine production, is associated with population asthma risk via the functional promoter polymorphism -1560 C>T.

BACKGROUND: In this study, the association of asthma with CD53, a member of the tetraspanin family, was assessed for the first time in a mechanism-based study. METHODS: Genetic polymorphisms of CD53 were analyzed in 591 subjects and confirmed in a replication study of 1001 subjects. CD53 mRNA and protein levels were measured in peripheral blood leukocytes, and the effects of the promoter polymorphisms on nuclear factor binding were examined by electrophoretic mobility shift assay. Cellular functional studies were conducted by siRNA transfections. RESULTS: Among tagging SNPs of CD53, the -1560 C>T in the promoter region was significantly associated with asthma risk. Compared with the CC genotype, the CT and TT genotypes were associated with a higher asthma risk, with odd ratios of 1.74 (P=0.009) and 2.03 (P=0.004), respectively. These findings were confirmed in the replication study with odd ratios of 1.355 (P=0.047) and 1.495 (P=0.039), respectively. The -1560 C>T promoter SNP had functional effects on nuclear protein binding as well as mRNA and protein expression levels in peripheral blood leukocytes. When CD53 was knocked down by siRNA in THP-1 human monocytic cells stimulated with house dust mite, the production of inflammatory cytokines as well as NFκB activity was significantly over-activated, suggesting that CD53 suppresses over-activation of inflammatory responses. CONCLUSIONS: The -1560 C>T SNP is a functional promoter polymorphism that is significantly associated with population asthma risk, and is thought to act by directly modulating nuclear protein binding, thereby altering the expression of CD53, a suppressor of inflammatory cytokine production.

The anti-adipogenic effects of (-)epigallocatechin gallate are dependent on the WNT/ß-catenin pathway.

(-)Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and reportedly has anti-obesity and anti-adipogenic effects. In this study, we determined that the up-regulation of the WNT/ß-catenin pathway is the anti-adipogenic mechanisms of EGCG in 3T3-L1 cells. EGCG treatment down-regulates the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer binding protein (C/EBP)α, fatty acid binding protein (FABP)4 and fatty acid synthase (FASN), while up-regulating the nuclear level of ß-catenin. Knockdown of ß-catenin using small interfering (si) RNA attenuated the inhibitory effects of EGCG on intracellular lipid accumulation. ß-catenin siRNA transfection also recovered terminal adipocyte markers such as FABP4, FASN, lipoprotein lipase and adiponectin, which were down-regulated by EGCG. The DNA binding activities as well as the expression levels of PPARγ and C/EBPα, which were down-regulated by EGCG, were significantly restored by ß-catenin siRNA transfection. In addition, we found that EGCG efficiently up-regulates the WNT/ß-catenin pathway. Among the members of the WNT/ß-catenin pathway, the expressions of low density lipoprotein receptor-related protein (LRP)5, LRP6, disheveled (DVL)2 and DVL3 were significantly up-regulated, while AXIN expression was down-regulated by EGCG, and the phosphorylation of glycogen synthase kinase 3ß was increased. These results suggest that EGCG activates the WNT/ß-catenin pathway, resulting in the up-regulation of ß-catenin, which down-regulates the major genes of the adipogenesis pathway. Taken together, our findings clearly show that the anti-adipogenic effects of EGCG are, at least partially, dependent on the WNT/ß-catenin pathway.

Anti-adipogenic effects of 1,25-dihydroxyvitamin D3 are mediated by the maintenance of the wingless-type MMTV integration site/ß-catenin pathway.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, was found to have anti-adipogenic activity, however, its mechanism of action has not been fully elucidated. In this study, 3T3-L1 preadipocytes were differentiated in the presence and absence of 1,25(OH)2D3, and the expression of the genes and proteins of the wingless-type MMTV integration site (WNT)/ß-catenin pathway were analyzed. While the expression of the members of the WNT/ß-catenin pathway were significantly downregulated during the adipogenesis of untreated 3T3-L1 cells, 1,25(OH)2D3 treatment was found to maintain the WNT/ß-catenin pathway. Among the members of the WNT/ß-catenin pathway, the levels of WNT10B and disheveled (DVL)2 as well as the phosphorylation of glycogen synthase kinase (GSK)3ß were maintained by 1,25(OH)2D3 treatment. The levels of nuclear ß-catenin, which were downregulated during adipogenesis, were also maintained by 1,25(OH)2D3 treatment. The results of this study suggested that the anti-adipogenic effect of 1,25(OH)2D3 was mediated by the maintenance of the WNT/ß-catenin pathway, which was normally downregulated during adipogenesis.