PCR diagnosis of Entamoeba histolytica cysts in stool samples.

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

Resistance to Toxoplasma gondii infection in mice treated with silk protein by enhanced immune responses.

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4(+) and CD8(+) T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-γ, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4(+) and CD8(+) T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.

Pulp regeneration with hemostatic matrices as a scaffold in an immature tooth minipig model.

Control of blood clotting in root canal systems is one of the most critical and difficult concerns for regenerative endodontics therapy (RET). The purpose of this study was to investigate the effects of using gelatin- and fibrin-based hemostatic hydrogels as a scaffold on pulp regeneration in a minipig model. Cell viability of human dental pulp stem cells cultured three-dimensionally in gelatin-based and fibrin-based scaffolds was evaluated by MTT and live/dead assay. RET was performed on 24 immature premolars with an autologous blood clot (PC), gelatin-based and fibrin-based hemostatic matrices (GM and FM), or without the insertion of a scaffold (NC). The follow-up period was 12 weeks. Radiographic and histologic assessments for pulp regeneration were performed. Gelatin-based scaffolds exhibited significantly higher cell viability than fibrin-based scaffolds after 15 days (P < 0.05). The PC and GM groups showed favorable root development without inflammation and newly mineralized tissue deposited in the root canal system, while FM group presented inflammatory changes with the continuation of root development. The NC group exhibited internal root resorption with periapical lesions. The application of GM in RET led to favorable clinical outcomes of root development without inflammatory changes compared to conventional RET. Our results suggest that GM may serve as a viable regenerative scaffold for pulp regeneration.

Development of model based on clock gene expression of human hair follicle cells to estimate circadian time.

Considering the effects of circadian misalignment on human pathophysiology and behavior, it is important to be able to detect an individual's endogenous circadian time. We developed an endogenous Clock Estimation Model (eCEM) based on a machine learning process using the expression of 10 circadian genes. Hair follicle cells were collected from 18 healthy subjects at 08:00, 11:00, 15:00, 19:00, and 23:00 h for two consecutive days, and the expression patterns of 10 circadian genes were obtained. The eCEM was designed using the inverse form of the circadian gene rhythm function (i.e., Circadian Time = F(gene)), and the accuracy of eCEM was evaluated by leave-one-out cross-validation (LOOCV). As a result, six genes (PER1, PER3, CLOCK, CRY2, NPAS2, and NR1D2) were selected as the best model, and the error range between actual and predicted time was 3.24 h. The eCEM is simple and applicable in that a single time-point sampling of hair follicle cells at any time of the day is sufficient to estimate the endogenous circadian time.

Comparison of Wearable Activity Tracker with Actigraphy for Sleep Evaluation and Circadian Rest-Activity Rhythm Measurement in Healthy Young Adults.

OBJECTIVE: The purpose of this study was to evaluate the applicability of data obtained from a wearable activity tracker (Fitbit Charge HR) to medical research. This was performed by comparing the wearable activity tracker (Fitbit Charge HR) with actigraphy (Actiwatch 2) for sleep evaluation and circadian rest-activity rhythm measurement. METHODS: Sixteen healthy young adults (female participants, 62.5%; mean age, 22.8 years) wore the Fitbit Charge HR and the Actiwatch 2 on the same wrist; a sleep log was recorded over a 14-day period. We compared the sleep variables and circadian rest-activity rhythm measures with Wilcoxon signed-rank tests and Spearman's correlations. RESULTS: The periods and acrophases of the circadian rest-activity rhythms and the sleep start times did not differ and correlated significantly between the Fitbit Charge HR and the Actiwatch 2. The Fitbit Charge HR tended to overestimate the sleep durations compared with the Actiwatch 2. However, the sleep durations showed high correlation between the two devices for all days. CONCLUSION: We found that the Fitbit Charge HR showed high accuracy in sleep evaluation and circadian rest-activity rhythm measurement when compared with actigraphy for healthy young adults. The results suggest that the Fitbit Charge HR could be applicable on medical research as an alternative tool to actigraphy for sleep evaluation and measurement of the circadian rest-activity rhythm.

A Genome-Wide Association Study Identifies UTRN Gene Polymorphism for Restless Legs Syndrome in a Korean Population.

OBJECTIVE: Restless legs syndrome (RLS) is a highly heritable and common neurological sensorimotor disease disturbing sleep. The objective of study was to investigate significant gene for RLS by performing GWA and replication study in a Korean population. METHODS: We performed a GWA study for RLS symptom group (n=325) and non-RLS group (n=2,603) from the Korea Genome Epidemiology Study. We subsequently performed a replication study in RLS and normal controls (227 RLS and 229 controls) to confirm the present GWA study findings as well as previous GWA study results. RESULTS: In the initial GWA study of RLS, we observed an association of rs11645604 (OR=1.531, p=1.18×10-6) in MPHOSPH6 on chromosome 16q23.3, rs1918752 (OR=0.6582, p=1.93×10-6) and rs9390170 (OR=0.6778, p=7.67×10-6) in UTRN on chromosome 6q24. From the replication samples, we found rs9390170 in UTRN (p=0.036) and rs3923809 and rs9296249 in BTBD9 (p=0.045, p=0.046, respectively) were significantly associated with RLS. Moreover, we found the haplotype polymorphisms of rs9357271, rs3923809, and rs9296249 (overall p=5.69×10-18) in BTBD9 was associated with RLS. CONCLUSION: From our sequential GWA and replication study, we could hypothesize rs9390170 polymorphism in UTRN is a novel genetic marker for susceptibility to RLS. Regarding with utrophin, which is encoded by UTRN, is preferentially expressed in the neuromuscular synapse and myotendinous junctions, we speculate that utrophin is involved in RLS, particularly related to the neuromuscular aspects.

Association of CLOCK, ARNTL, PER2, and GNB3 polymorphisms with diurnal preference in a Korean population.

Polymorphisms in human circadian genes are potential genetic markers that affect diurnal preference in several populations. In this study, we evaluated whether four polymorphisms in circadian genes CLOCK, ARNTL, PER2, and GNB3 were associated with diurnal preference in a Korean population. In all, 499 healthy subjects were genotyped for four functional polymorphisms in CLOCK, ARNTL, PER2, and GNB3. Composite scale of morningness (CSM) was applied to measure phenotype patterns of human diurnal preference. In addition, three subscale scores, i.e. "morningness," "activity planning," and "morning alertness," were extracted from the CSM. No significant associations were observed between CSM scores and CLOCK (rs1801260) genotype or T allele carrier status, CSM scores and ARNTL (rs2278749) C allele carrier status, and CSM scores and GNB3 (rs5443) genotype or C allele carrier status. However, total CSM scores and scores of its subscales were significantly associated with PER2 (rs934945) genotype (p = 0.010, p = 0.018, and p = 0.005 for total, morningness, and activity planning, respectively) and G allele carrier status (p = 0.003, p = 0.005, and p = 0.002 for total, morningness, and activity planning, respectively). The best model result obtained by performing multifactor dimensionality reduction analysis ([Formula: see text]2 = 11.2798, p = 0.0008) indicated that interaction among C/T single nucleotide polymorphism (SNP) in ARNTL, C/T SNP in GNB3, and G/A SNP in PER2 synergistically affected the risk associated with diurnal preference toward eveningness. These results suggest that circadian gene PER2 is associated with diurnal preference in healthy Korean population. Although polymorphisms in ARNTL and GNB3 were not significantly associated with diurnal preference, their interactions with the polymorphism in PER2 may synergistically increase the risk of diurnal preference toward eveningness.

Molecular circadian rhythm shift due to bright light exposure before bedtime is related to subthreshold bipolarity.

This study examined the link between circadian rhythm changes due to bright light exposure and subthreshold bipolarity. Molecular circadian rhythms, polysomnography, and actigraphy data were studied in 25 young, healthy male subjects, divided into high and low mood disorder questionnaire (MDQ) score groups. During the first 2 days of the study, the subjects were exposed to daily-living light (150 lux) for 4 hours before bedtime. Saliva and buccal cells were collected 5 times a day for 2 consecutive days. During the subsequent 5 days, the subjects were exposed to bright light (1,000 lux), and saliva and buccal cell samples were collected in the same way. Molecular circadian rhythms were analyzed using sine regression. Circadian rhythms of cortisol (F = 16.956, p < 0.001) and relative PER1/ARNTL gene expression (F = 122.1, p < 0.001) showed a delayed acrophase in both groups after bright light exposure. The high MDQ score group showed a significant delay in acrophase compared to the low MDQ score group only in salivary cortisol (F = 8.528, p = 0.008). The high MDQ score group showed hypersensitivity in cortisol rhythm shift after bright light exposure, suggesting characteristic molecular circadian rhythm changes in the high MDQ score group may be related to biological processes downstream from core circadian clock gene expression.

Advanced Circadian Phase in Mania and Delayed Circadian Phase in Mixed Mania and Depression Returned to Normal after Treatment of Bipolar Disorder.

Disturbances in circadian rhythms have been suggested as a possible cause of bipolar disorder (BD). Included in this study were 31 mood episodes of 26 BD patients, and 18 controls. Circadian rhythms of BD were evaluated at admission, at 2-week intervals during hospitalization, and at discharge. All participants wore wrist actigraphs during the studies. Saliva and buccal cells were obtained at 8:00, 11:00, 15:00, 19:00, and 23:00 for two consecutive days. Collected saliva and buccal cells were used for analysis of the cortisol and gene circadian rhythm, respectively. Circadian rhythms had different phases during acute mood episodes of BD compared to recovered states. In 23 acute manic episodes, circadian phases were ~7hour advanced (equivalent to ~17hour delayed). Phases of 21 out of these 23 cases returned to normal by ~7hour delay along with treatment, but two out of 23 cases returned to normal by ~17hour advance. In three cases of mixed manic episodes, the phases were ~6-7hour delayed. For five cases of depressive episodes, circadian rhythms phases were ~4-5hour delayed. After treatment, circadian phases resembled those of healthy controls. Circadian misalignment due to circadian rhythm phase shifts might be a pathophysiological mechanism of BD.

Association of CLOCK, ARNTL, and NPAS2 gene polymorphisms and seasonal variations in mood and behavior.

Seasonal affective disorder (SAD) is a condition of seasonal mood changes characterized by recurrent depression in autumn or winter that spontaneously remits in spring or summer. Evidence has suggested that circadian gene variants contribute to the pathogenesis of SAD. In this study, we investigated polymorphisms in the CLOCK, ARNTL, and NPAS2 genes in relation to seasonal variation in 507 healthy young adults. Seasonal variations were assessed with the Seasonality Pattern Assessment Questionnaire. The prevalence of SAD was 12.0% (winter-type 9.3%, summer-type 2.8%). No significant difference was found between the groups in the genotype distribution of ARNTL rs2278749 and NPAS2 rs2305160. The T allele of CLOCK rs1801260 was significantly more frequent in seasonals (SAD + subsyndromal SAD) compared with non-seasonals (p = 0.020, odds ratio = 1.89, 95% confidence interval = 1.09-3.27). Global seasonality score was significantly different among genotypes of CLOCK rs1801260, but not among genotypes of ARNTL rs2278749 and NPAS2 rs2305160. However, statistical difference was observed in the body weight and appetite subscales among genotypes of ARNTL rs2278749 and in the body weight subscale among genotypes of NPAS2 rs2305160. There was synergistic interaction between CLOCK rs1801260 and ARNTL rs2278749 on seasonality. To our knowledge, this study is the first to reveal an association between the CLOCK gene and seasonal variations in mood and behavior in the Korean population. Although we cannot confirm previous findings of an association between SAD and the ARNTL and NPAS2 genes, these genes may influence seasonal variations through metabolic factors such as body weight and appetite. The interaction of the CLOCK and ARNTL genes contributes to susceptibility for SAD.

Association between restless legs syndrome and CLOCK and NPAS2 gene polymorphisms in schizophrenia.

Previous studies have suggested that there is a genetic basis to restless legs syndrome (RLS) development. Occurrence of antipsychotic-induced RLS could also be due to differences in genetic susceptibility. We investigated whether CLOCK and NPAS2 gene polymorphisms are associated with RLS in schizophrenic patients on antipsychotics because RLS symptoms usually manifest during the evening and night. We assessed symptoms of RLS in 190 Korean schizophrenic patients on antipsychotics and divided the subjects into two groups according to the International Restless Legs Syndrome Study Group diagnostic criteria: (i) subjects who met all the criteria and (ii) the remaining subjects who did not meet all the criteria. We found a significant difference in the number of subjects with different genotype and allele carrier frequencies for the CLOCK gene (rs2412646) between the two groups (p = 0.031 and 0.010, respectively). Distribution of CLOCK haplotypes (rs2412646-rs1801260) was significantly different between schizophrenic patients with and without RLS (p = 0.021). However, the distributions of allelic, genotypic, and haplotypic variants of NPAS2 (rs2305160 and rs6725296) were not significantly different between the two groups. Our results suggest that CLOCK polymorphisms are associated with increased susceptibility of schizophrenic patients to RLS. We hypothesize that RLS in schizophrenia patients treated with antipsychotics may be a very mild akathisia that manifests during the night and is under control of circadian oscillation.