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N6-Methyladenosine (m6A) regulates oocyte-to-embryo transition and the reprogramming of somatic cells into induced pluripotent stem cells. However, the role of m6A methylation in porcine early embryonic development and its reprogramming characteristics in somatic cell nuclear transfer (SCNT) embryos are yet to be known. Here, we showed that m6A methylation was essential for normal early embryonic development and its aberrant reprogramming in SCNT embryos. We identified a persistent occurrence of m6A methylation in embryos between 1-cell to blastocyst stages and m6A levels abruptly increased during the morula-to-blastocyst transition. Cycloleucine (methylation inhibitor, 20 mM) treatment efficiently reduced m6A levels, significantly decreased the rates of 4-cell embryos and blastocysts, and disrupted normal lineage allocation. Moreover, cycloleucine treatment also led to higher levels in both apoptosis and autophagy in blastocysts. Furthermore, m6A levels in SCNT embryos at the 4-cell and 8-cell stages were significantly lower than that in parthenogenetic activation (PA) embryos, suggesting an abnormal reprogramming of m6A methylation in SCNT embryos. Correspondingly, expression levels of m6A writers (METTL3 and METTL14) and eraser (FTO) were apparently higher in SCNT 8-cell embryos compared with their PA counterparts. Taken together, these results indicated that aberrant nuclear transfer-mediated reprogramming of m6A methylation was involved in regulating porcine early embryonic development.
This study compared dementia knowledge between older Chinese adults in Melbourne, Australia, and Beijing, China, and explored factors associated with dementia knowledge between these two groups. Ultimately, this study aimed to inform the development of tailored dementia education programs for older Chinese adults.
A cross-sectional design was employed in this study.
Participants were recruited from 5 Chinese community senior groups in Melbourne and 10 community health centers in Beijing from March to May 2019.
A total of 379 older Chinese adults aged 50 and over completed the questionnaire, including 153 from Melbourne and 226 from Beijing.
Dementia knowledge was assessed using the Alzheimer’s Disease Knowledge Scale (ADKS). Demographic characteristics, dementia-related experience, and the mental health status of participants were collected. Stepwise linear regression was used to analyze the factors associated with dementia knowledge.
In general, older Chinese adults in Melbourne and Beijing reported similar levels of dementia knowledge for both the overall ADKS scale (mean ± SD: 17.2 ± 2.9 in Melbourne vs. 17.5 ± 2.9 in Beijing, p > 0.05) and the seven subdomains. Of the subdomains, the highest correct response rates were observed in the life impact of the dementia subdomain, and the lowest rates were observed in the caregiving subdomain. Stepwise linear regression analysis revealed that younger age and self-reported dementia worry were significantly associated with higher levels of dementia knowledge in the Melbourne group, whereas a positive family history of dementia was significantly associated with higher levels of dementia knowledge in the Beijing group.
Older Chinese adults living in Melbourne and Beijing share similar levels of dementia knowledge, but factors associated with their knowledge are different. These findings will inform the development of culturally and socially appropriate dementia education programs for older Chinese populations in different countries.
Based on a cohort from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), we aimed to evaluate the relationship between sleep duration and the incidence of cognitive impairment among older Chinese adults.
We conducted a prospective analysis based on 3692 participants from the CLHLS at baseline (in 2011), and as a 3-year follow-up (till 2014), 531 participants (14.4%) had cognitive impairment, which was defined as a Mini-Mental State Examination score <24. Sleep duration was classified into three groups: short (≤5 hours/day), normal (>5 but <10 hours), and long (≥10 hours/day). A logistic regression model was used to examine the association between baseline sleep duration and cognitive impairment after adjusting for sociodemographic data, living habits, and health conditions.
Five hundred sixty-two participants (15.2%) were in the short-duration group, and 608 participants (16.5%) were in the long-duration group. After adjusting for multiple potential confounders, compared with normal sleep duration, long sleep duration was associated with the incidence of cognitive impairment (OR = 1.309, 95% CI: 1.019–1.683), especially among men (OR = 1.527, 95% CI: 1.041–2.240) and those having a primary and above education level (OR = 1.559, 95% CI: 1.029–2.361). No significant association was observed between short sleep duration and cognitive impairment (OR = 0.860, 95% CI: 0.646–1.145).
Excessive sleep may increase the risk of cognitive impairment in older individuals. It may be a suggestive sign of early neurodegeneration and may be a useful clinical tool to identify those at a higher risk of progressing to cognitive impairment.
The current study evaluated the associations between different forms and sources of Fe and breast cancer risk in Southern Chinese women.
Case–control study. We collected data on the consumption of Fe from different forms and food sources by using a validated FFQ. Multivariable logistic regression and restricted cubic spline (RCS) analysis was used to reveal potential associations between Fe intake and breast cancer risk.
A case-control study of women at three major hospitals in Guangzhou, China.
From June 2007 to March 2019, 1591 breast cancer cases and 1622 age-matched controls were recruited.
In quartile analyses, Fe from plants and Fe from white meat intake were inversely associated with breast cancer risk, with OR of 0·65 (95 % CI 0·47, 0·89, Ptrend = 0·006) and 0·76 (95 % CI 0·61, 0·96, Ptrend = 0·014), respectively, comparing the highest with the lowest quartile. No associations were observed between total dietary Fe, heme or non-heme Fe, Fe from meat or red meat and breast cancer risk. RCS analysis demonstrated J-shaped associations between total dietary Fe, non-heme Fe and breast cancer, and reverse L-shaped associations between heme Fe, Fe from meat and Fe from red meat and breast cancer.
Fe from plants and white meat were inversely associated with breast cancer risk. Significant non-linear J-shaped associations were found between total dietary Fe, non-heme Fe and breast cancer risk, and reverse L-shaped associations were found between heme Fe, Fe from meat or red meat and breast cancer risk.
The objective of this study was to analyze differences in birth weight and overweight/obesity in a Shanghai twin cohort. We also wanted to study their association and explore possible risk factors for the discordance of overweight/obesity within twins. This was an internal case–control study designed for twins. The 2012 Shanghai Twin Registration System baseline survey data of a total of 3417 twin pairs were statistically analyzed using SPSS22 software. Results show that the body mass index (BMI) of the Shanghai twin population increased with age. Twins with a high birth weight had a higher BMI and a higher rate of overweight and obesity; 0- to 6-year-old twins, male twins and dizygotic (DZ) twins had higher rates of overweight/obesity than other groups. The greater the discordant birth weight rate of twins, the more obvious the difference in BMI (p < .05). There was a significant difference in overweight/obesity between twins with a relative difference of birth weight ≥15% in DZ twins (p < .05). DZ twins, male twins and 0- to 6-year-old twins were more likely to be discordant in overweight/obese than others. The discordant birth weight within twins was not a risk factor for discordant overweight/obesity. However, attention should be paid to childhood obesity, and appropriate interventions should be made at the appropriate time. Genetics may play an important role in the occurrence and development of overweight/obesity. In conclusion, discordant growth and development in the uterus early in life may not lead to discordant weight development in the future.
Microglia, the main immune cell of the central nervous system (CNS), categorized into M1-like phenotype and M2-like phenotype, play important roles in phagocytosis, cell migration, antigen presentation, and cytokine production. As a part of CNS, retinal microglial cells (RMC) play an important role in retinal diseases. Diabetic retinopathy (DR) is one of the most common complications of diabetes. Recent studies have demonstrated that DR is not only a microvascular disease but also retinal neurodegeneration. RMC was regarded as a central role in neurodegeneration and neuroinflammation. Therefore, in this review, we will discuss RMC polarization and its possible regulatory factors in early DR, which will provide new targets and insights for early intervention of DR.
Understanding the patterns of treatment response is critical for the treatment of patients with schizophrenia; one way to achieve this is through using a longitudinal dynamic process study design.
This study aims to explore the response trajectory of antipsychotics and compare the treatment responses of seven different antipsychotics over 6 weeks in patients with schizoprenia (trial registration: Chinese Clinical Trials Registry Identifier: ChiCTR-TRC-10000934).
Data were collected from a multicentre, randomised open-label clinical trial. Patients were evaluated with the Positive and Negative Syndrome Scale (PANSS) at baseline and follow-up at weeks 2, 4 and 6. Trajectory groups were classified by the method of k-means cluster modelling for longitudinal data. Trajectory analyses were also employed for the seven antipsychotic groups.
The early treatment response trajectories were classified into a high-trajectory group of better responders and a low-trajectory group of worse responders. The results of trajectory analysis showed differences compared with the classification method characterised by a 50% reduction in PANSS scores at week 6. A total of 349 patients were inconsistently grouped by the two methods, with a significant difference in the composition ratio of treatment response groups using these two methods (χ2 = 43.37, P < 0.001). There was no differential contribution of high- and low trajectories to different drugs (χ2 = 12.52, P = 0.051); olanzapine and risperidone, which had a larger proportion in the >50% reduction at week 6, performed better than aripiprazole, quetiapine, ziprasidone and perphenazine.
The trajectory analysis of treatment response to schizophrenia revealed two distinct trajectories. Comparing the treatment responses to different antipsychotics through longitudinal analysis may offer a new perspective for evaluating antipsychotics.
Little is known about the trend and predictors of 21-year mortality and suicide patterns in persons with schizophrenia.
To explore the trend and predictors of 21-year mortality and suicide in persons with schizophrenia in rural China.
This longitudinal follow-up study included 510 persons with schizophrenia who were identified in a mental health survey of individuals (≥15 years old) in 1994 in six townships of Xinjin County, Chengdu, China, and followed up in three waves until 2015. Kaplan–Meier survival analysis and Cox hazard regressions were conducted.
Of the 510 participants, 196 died (38.4% mortality) between 1994 and 2015; 13.8% of the deaths (n = 27) were due to suicide. Life expectancy was lower for men than for women (50.6 v. 58.5 years). Males consistently showed higher rates of mortality and suicide than females. Older participants had higher mortality (hazard ratio HR = 1.03, 95% CI 1.01–1.05) but lower suicide rates (HR = 0.95, 95% CI 0.93–0.98) than their younger counterparts. Poor family attitudes were associated with all-cause mortality and death due to other causes; no previous hospital admission and a history of suicide attempts independently predicted death by suicide.
Our findings suggest there is a high mortality and suicide rate in persons with schizophrenia in rural China, with different predictive factors for mortality and suicide. It is important to develop culture-specific, demographically tailored and community-based mental healthcare and to strengthen family intervention to improve the long-term outcome of persons with schizophrenia.
Little is known about poverty trends in people with severe mental illness (SMI) over a long time span, especially under conditions of fast socioeconomic development.
This study aims to unravel changes in household poverty levels among people with SMI in a fast-changing rural community in China.
Two mental health surveys, using ICD-10, were conducted in the same six townships of Xinjin county, Chengdu, China. A total of 711 and 1042 people with SMI identified in 1994 and 2015, respectively, participated in the study. The Foster-Greer-Thorbecke poverty index was adopted to measure the changes in household poverty. These changes were decomposed into effects of growth and equity using a static decomposition method. Factors associated with household poverty in 1994 and 2015 were examined and compared by regression analyses.
The proportion of poor households, as measured by the headcount ratio, increased significantly from 29.8% in 1994 to 39.5% in 2015. Decomposition showed that poverty in households containing people with SMI had worsened because of a redistribution effect. Factors associated with household poverty had also changed during the study period. The patient's age, ability to work and family size were of paramount significance in 2015.
This study shows that the levels of poverty faced by households containing people with SMI has become more pressing with China's fast socioeconomic development. It calls for further integration of mental health recovery and targeted antipoverty interventions for people with SMI as a development priority.
In order to elucidate the physical connection between the propulsive performance and the unsteadiness of jet flow, the transient development of the impulse and thrust of laminar starting jets with finite fluid discharged is investigated numerically for cases with different velocity programmes and jet stroke ratios. The simulation quantitatively demonstrates that the impulse and thrust generated are highly sensitive to the jet kinematics and its near-wake dynamics. The momentum flux contribution to the jet impulse is found to be significant and is associated closely with the jet kinematics. On the other hand, although the over pressure effect at the jet initiation stage has been identified previously as the main reason for the enhanced propulsive performance of the starting jet, the current results indicate that its contribution is in fact weakened by the negative local pressure, induced by the formation of the leading vortex ring as well as jet development during the deceleration stage. Contrary to the effects of the leading vortex ring, the stopping vortex formed near the nozzle exit plane during the jet deceleration stage is found to contribute positively to the pressure impulse production, albeit it is relatively small. By augmenting the over pressure effect and mitigating the negative-pressure effect, the cases with the fast acceleration and slow deceleration velocity programme is capable of producing the maximum pressure impulse, leading to additional impulse production over what would be expected from the jet momentum flux alone.
To investigate the cumulative effects of maternal supplementation with nucleotides in the form of uridine (UR) on fatty acid and amino acid constituents of neonatal piglets, fifty-two sows in late gestation were assigned randomly into the control (CON) group (fed a basal diet) or UR group (fed a basal diet with 150 g/t UR). Samples of neonates were collected during farrowing. Results showed that supplementing with UR in sows’ diet significantly decreased the birth mortality of pigs (P = 0·05), and increased serum total cholesterol, HDL and LDL of neonatal piglets (P < 0·05). Moreover, the amino acid profile of serum and liver of neonatal piglets was affected by the addition of UR in sows’ diets (P < 0·05). Furthermore, an up-regulation of mRNA expression of energy metabolism-related genes, including fatty acid elongase 5, fatty acid desaturase 1, hormone-sensitive lipase and cholesterol-7a-hydroxylase, was observed in the liver of neonates from the UR group. Additionally, a decrease in placental gene expression of excitatory amino acid transporters 2, excitatory amino acid transporter 3 and neutral AA transporter 1 in the UR group was concurrently observed (P < 0·05), and higher protein expression of phosphorylated protein kinase B, raptor, PPARα and PPARγ in placenta from the UR group was also observed (P < 0·05). Together, these results showed that maternal UR supplementation could regulate placental nutrient transport, largely in response to an alteration of mTORC1–PPAR signalling, thus regulating the nutrition metabolism of neonatal piglets and improving reproductive performance.
Schizotypy refers to schizophrenia-like traits below the clinical threshold in the general population. The pathological development of schizophrenia has been postulated to evolve from the initial coexistence of ‘brain disconnection’ and ‘brain connectivity compensation’ to ‘brain connectivity decompensation’.
In this study, we examined the brain connectivity changes associated with schizotypy by combining brain white matter structural connectivity, static and dynamic functional connectivity analysis of diffusion tensor imaging data and resting-state functional magnetic resonance imaging data. A total of 87 participants with a high level of schizotypal traits and 122 control participants completed the experiment. Group differences in whole-brain white matter structural connectivity probability, static mean functional connectivity strength, dynamic functional connectivity variability and stability among 264 brain sub-regions of interests were investigated.
We found that individuals with high schizotypy exhibited increased structural connectivity probability within the task control network and within the default mode network; increased variability and decreased stability of functional connectivity within the default mode network and between the auditory network and the subcortical network; and decreased static mean functional connectivity strength mainly associated with the sensorimotor network, the default mode network and the task control network.
These findings highlight the specific changes in brain connectivity associated with schizotypy and indicate that both decompensatory and compensatory changes in structural connectivity within the default mode network and the task control network in the context of whole-brain functional disconnection may be an important neurobiological correlate in individuals with high schizotypy.
The purpose of this study was to construct a glycyrrhetinic acid (GA)-mediated, breakable, intracellular, nanoscale drug-delivery carrier via amide and esterification reactions. The structures were identified by Fourier-transformed infrared (FTIR) and 1H-nuclear magnetic resonance (1H-NMR) spectrophotometry. The compatibility and safety of the carrier were evaluated using hemolysis and cytotoxicity tests. The GA-copolymer micelle was prepared using the solvent evaporation method. FTIR and 1H-NMR detection demonstrated the successful construction of the polymer. No hemolysis occurred in any concentration of polymer within 3 h, and the hemolysis rate was less than 5%. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) experimental results showed that the novel polymer reduced the cell survival rate and had significant cytotoxic effects. The blank nanoparticles were liquid with light blue opalescence. Transmission electron microscopy revealed that the empty micelles were uniform spheres, with an average size of 62 nm and a zeta potential of −13 mV. The novel GA-mediated polymeric carrier material developed here has the potential to effectively kill human SMMC-7721 cancer cells within 3 days when the dose is above 500 ug/mL.
A separately-designable diplexer with multiple transmission zeroes (TZs) using common stub-loaded stepped impedance resonator (SIR) is proposed. The common stub-loaded SIR operating in third harmonic (f3) and fifth harmonic (f5) is used for designing the two diplexer channels. The stub is loaded at the voltage-null point of f3 of the SIR. It can separately control f5 but has no effect on f3 so that the two channels can be separately designed. Meanwhile, the input port is tap-connected to the common stub-loaded SIR, which necessarily produces a TZ between f3 and f5, existing in both channel filtering responses. By properly choosing coupling schemes of the two channels, more TZs are realized at the desired locations. Thanks to the generation of the multiple TZs, both passband selectivity and isolation between the two channels are improved significantly. For demonstration, a diplexer operating at 2.22 and 2.95 GHz is designed, fabricated, and measured. The simulated and measured results are presented, showing good agreement.
The core toroidal plasma intrinsic rotation has been studied by experiments and simulations in the Joint Texas Experimental Tokamak (J-TEXT). The direction of core intrinsic rotation in the J-TEXT plasma is counter-current. As the plasma density ramps up, the rotation velocity increases in the counter-current direction. By comparing four different electron densities, linear local gyrokinetic simulations have been performed by the Gyrokinetic Electromagnetic Numerical Experiment code for the first time on J-TEXT. It is found that the most dominant turbulence is the ion temperature gradient at
is the minor radius of the plasma and this is unchanged during the plasma density ramp up. By scanning the radial wave vectors, it is found that the residual stress term reverses from negative to positive when the plasma density exceeds a certain threshold. The pinch term is larger than the residual stress term at all four electron densities, which means that the pinch term is always dominant in the core of a J-TEXT plasma.
Electrospun membranes have potential applications in the field of waterproof and breathable textile products. However, challenges still exist to improve the breathability, and waterproof and mechanical properties of these microporous membranes. In this paper, a novel hydrophobic microporous nanofiber membrane was prepared via side-by-side electrospinning of fluorosilane-modified silica nanoparticles (F–SiO2) blended with synthesized polyurethane (PU) solution and composited with the polyacrylonitrile (PAN) solution. To prepare F–SiO2, SiO2 nanoparticles were hydrophobically modified by fluorosilane. Composite nanofiber membranes with different blending ratios of PU(F–SiO2)/PAN were fabricated via side-by-side electrospinning by controlling the extruding speed of two spinnerets. Experimental results indicated that regarding F–SiO2 as hydrophobic inorganic particle can improve the hydrophobic properties of PU nanofiber membrane. The prepared PAN/(F–SiO2/PU) nanofiber microporous membranes exhibit relatively excellent waterproof and mechanical properties as that robust tensile strength (19.5 MPa), preferable water vapor permeability [10.3 kg/(m2 d)], favorable water contact angle (137.2°), and superior mechanical properties. It was believed that the reinforced PAN/(F–SiO2/PU) nanofibrous composite membranes have potential applications in chemical protective clothing, army combat uniforms, self-cleaning materials, and other medical products.
Dietary salt intake may vary depending on different lifestyles. We aimed to estimate the different salt intakes and evaluate the knowledge and self-awareness about salt among people speaking the Teochew, Teochew–Hakka and Hakka dialects in the Chaoshan region of southern China.
The study followed a cluster sampling of residents in Chaoshan region. General characteristics, lifestyles, health status as well as knowledge and self-awareness related to salt intake were investigated using a questionnaire. Anthropometric variables as well as Na and K excretion in a 24-h urine collection were measured.
Chaoshan region of China.
Four hundred fifteen adults who spoke only one of these three dialects.
The salt intake of adults who spoke the Teochew, Teochew–Hakka and Hakka dialects was 7·19 (interquartile range (IQR) 5·29–10·17), 9·03 (IQR 6·62–11·54) and 10·12 (IQR 7·61–12·82) g/d, respectively, with significant differences between Teochew and Teochew–Hakka speakers and between Teochew and Hakka speakers (both P < 0·05). The Na:K ratio for adults who spoke the three dialects was 3·00 (IQR 2·00–4·11), 3·50 (IQR 2·64–4·82) and 4·52 (IQR 3·35–5·97), respectively, and differed significantly among the groups (all P < 0·05). Multiple linear regression analysis showed increased Na:K ratio associated with hypertension (β = 0·71, P = 0·043) in Hakka speakers. Knowledge and self-awareness about salt intake were poor in this population.
Salt intake was closely related to lifestyles and was higher than the upper limit (5 g/d) recommended by the WHO in adults of Chaoshan, especially those speaking the Hakka dialect.
Cognitive decline in advanced age is closely related to dementia. The trajectory of cognitive function in older Chinese is yet to be fully investigated. We aimed to investigate the trajectories of cognitive function in a nationally representative sample of older people living in China and to explore the potential determinants of these trajectories.
This study included 2,038 cognitively healthy persons aged 65–104 years at their first observation in the Chinese Longitudinal Healthy Longevity Survey from 2002 to 2014. Cognitive function was measured using the Chinese version of the Mini-Mental State Examination (MMSE). Group-based trajectory modeling was used to identify potential heterogeneity of longitudinal changes over the 12 years and to investigate associations between baseline predictors of group membership and these trajectories.
Three trajectories were identified according to the following types of changes in MMSE scores: slow decline (14.0%), rapid decline (4.5%), and stable function (81.5%). Older age, female gender, having no schooling, a low frequency of leisure activity, and a low baseline MMSE score were associated with the slow decline trajectory. Older age, body mass index (BMI) less than 18.5 kg/m2, and having more than one cardiovascular disease (CVD) were associated with the rapid decline trajectory.
Three trajectories of cognitive function were identified in the older Chinese population. The identified determinants of these trajectories could be targeted for developing prevention and intervention strategies for dementia.
Cruciferous vegetables contain high levels of glucosinolates (GSL) and isothiocyanates (ITC). ITC are known to induce glutathione S-transferases (GST) and thus exert their anticarcinogenic effects. This study explored the combined effects of cruciferous vegetable, GSL and ITC intake and GST polymorphisms on breast cancer risk. A total of 737 breast cancer cases and 756 controls were recruited into this case–control study. OR and 95 % CI were assessed by multivariable logistic regression. Higher cruciferous vegetable, GSL and ITC intakes were inversely associated with breast cancer risk, with adjusted OR of 0·48 (95 % CI 0·35, 0·65), 0·54 (95 % CI 0·40, 0·74) and 0·62 (95 % CI 0·45, 0·84), respectively. Compared with women carrying the GSTP1 rs1695 wild AA genotype and high cruciferous vegetable, GSL or ITC intake, carriers of the AA genotype with low cruciferous vegetable, GSL and ITC intake had greater risk of breast cancer, with adjusted OR of 1·43 (95 % CI 1·01, 1·87), 1·34 (95 % CI 1·02, 1·75) and 1·37 (95 % CI 1·05, 1·80), respectively. Persons with the GSTM1-null genotype and lower intake of cruciferous vegetables, GSL and ITC had higher risk of breast cancer than those with the GSTM1-present genotype and higher intake, with OR of 1·42 (95 % CI 1·04, 1·95), 1·43 (95 % CI 1·05, 1·96) and 1·45 (95 % CI 1·06, 1·98), respectively. Among women possessing the GSTT1-present genotype, low intake of cruciferous vegetables, GSL or ITC was associated with higher risk of breast cancer. But these interactions were non-significant. This study indicated that there were no significant interactions between cruciferous vegetable, GSL or ITC intake and GST polymorphisms on breast cancer risk.
Dysfunctions of glutamatergic and GABAergic neurotransmission are two important hypotheses for the pathogenesis of schizophrenia. Thus, genes in the pathway are candidates for schizophrenia susceptibility. Phosphate-activated glutaminase (GLS), glutamine synthetase (GLUL), glutamic acid decarboxylase (GAD), GABA transaminase (ABAT) and succinic semialdehyde dehydrogenase (ALDH5A1) are five primary enzymes in glutamate and GABA synthetic and degradative pathway. In order to investigate the possible involvement of these genes in the development of paranoid schizophrenia, we genotyped 80 paranoid schizophrenics from northern China and 108 matched controls by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) methods or directly sequencing of PCR product. Seven SNPs were found to be polymorphic in the population investigated. No significant differences in the genotype distributions or allele frequencies between patients and controls were found. Therefore, we conclude the polymorphisms studied in the five genes do not play major roles in pathogenesis of paranoid schizophrenia in the population investigated.