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Abstract Acanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

Background: Second-generation antipsychotics (SGAs) is thought responsible for the metabolic abnormalities of schizophrenic patients, however, some untreated schizophrenic patients had already developed problems with glucose metabolism. The present study examined the hypothesis that schizophrenia itself but not risperidone, an extensively employed SGA, is accountable for metabolic abnormalities. Methods: A 56-day risperidone regimen (1 mg/kg/day) was employed for rats of social isolation rearing (SIR) beginning at different developmental stage (28 or 56 days after weaning, i.e., adolescent and young adulthood, respectively). Metabolic parameters including body weight, systolic blood pressure (SBP), triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol, and plasma glucose were measured at baseline, 28, and 56 days of the regimen. Oral glucose tolerance test (OGTT) was performed at the end of the regimen. Insulin function was evaluated by area under the curve (AUC) of OGTT, homeostasis model assessment-insulin resistance (HOMA-ir), and Matsuda index. Results: Our results demonstrated that: (i) SIR rats presented higher body weight, plasma triglyceride, and HOMA-ir than social controls. (ii) Higher insulin resistance was specifically presented in young adult rather than adolescent SIR rats. (iii) Adolescent drugged rats showed a lower level of LDL in day 28 of the regimen than young adult. Risperidone led to a lower LDL level in only young adult IR rats in day 56 than undrugged rats. (iv) SIR-induced dysregulation of insulin can be reversed by chronic risperidone treatment beginning at adolescence but not young adulthood. Conclusions: Our findings support the primary role of schizophrenia in metabolic abnormalities and risperidone appear beneficial when administered earlier.

Background: Oxidative stress-induced neuronal dysfunction has been considered an essential factor for the development of schizophrenia. However, a longitudinal and causal relation between the impacts of developmental stress and oxidative stress remains unsolved. The present study aimed to examine whether the oxidative stress-relevant dysfunctions of the apoptotic index can be induced in rats of isolation rearing (IR, a rodent model of schizophrenia) and to see if the intervention of antipsychotics can reverse these dysfunctions. Materials and Methods: Pharmacological manipulation (risperidone [RIS] [1 mg/kg/day], olanzapine [OLA] [2.5 mg/kg/day], or saline [SAL] vehicle) was introduced 4 weeks (adolescence) or 8 weeks (young adulthood) after IR (i.e., rats were 7- or 11-week-old). The regime of RIS, OLA, or SAL was continued for 9 weeks. Locomotor activity was employed to validate the IR effect. Rats' hippocampus immediately after sacrifice was removed to measure messenger RNA expression of Bax, Bcl-2, brain-derived neurotrophic factor (BDNF) and the plasma level of nitric oxide (NO). Results: The results showed: (i) IR rats were more hyperactive. (ii) RIS may exert anti-apoptotic effects on IR rats, particularly at their adolescent age (as indexed by increased Bcl-2 and decreased Bax/Bcl-2 ratio). (iii) The therapeutic potential of RIS can be also observed in the change of BDNF in an age-independent manner, in which RIS effectively increased the BDNF level in IR but not social (SOC) rats. (iv) Plasma NO was not altered. Conclusion: The study results support the utility of the IR paradigm in exploring mental disorders with neurodevelopmental origin in which early pharmacological intervention may provide a therapeutic benefit in the overloaded oxidative stress and the dysfunction of BDNF.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytochrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7α, GSK3β and Thr58-Myc, which are involved in c-Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7α and GSK3β expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7α suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7α reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.