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ABSTRACT Human toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T. gondii cell cycle mechanism, the G1 restriction checkpoint (R-point), that operates the switch between parasite growth and differentiation. Apicomplexans lack conventional R-point regulators, suggesting adaptation of alternative factors. We showed that Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2, and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. Examination of cyclins verified the correlation of cyclin expression with growth dependence and development capacity of RH and ME49 strains. We demonstrated that rapidly dividing RH tachyzoites were dependent on TgCycP1 expression, which interfered with bradyzoite differentiation. Using the conditional knockdown model, we established that TgCycP2 regulated G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally incompetent RH tachyzoites. We tested the functions of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models. Based on functional and global gene expression analyses, we determined that TgCycP2 also regulated bradyzoite replication, while signal-induced TgCyc5 was critical for efficient tissue cyst maturation. In conclusion, we identified the central machinery of the T. gondii restriction checkpoint comprised of TgCrk2 kinase and three atypical T. gondii cyclins and demonstrated the independent roles of TgCycP1, TgCycP2, and TgCyc5 in parasite growth and development. IMPORTANCE Toxoplasma gondii is a virulent and abundant human pathogen that puts millions of silently infected people at risk of reactivation of the chronic disease. Encysted bradyzoites formed during the chronic stage are resistant to current therapies. Therefore, insights into the mechanism of tissue cyst formation and reactivation are major areas of investigation. The fact that rapidly dividing parasites differentiate poorly strongly suggests that there is a threshold of replication rate that must be crossed to be considered for differentiation. We discovered a cell cycle mechanism that controls the T. gondii growth-rest switch involved in the conversion of dividing tachyzoites into largely quiescent bradyzoites. This switch operates the T. gondii restriction checkpoint using a set of atypical and parasite-specific regulators. Importantly, the novel T. gondii R-point network was not present in the parasite's human and animal hosts, offering a wealth of new and parasite-specific drug targets to explore in the future.

Objectives: Species with a restricted distribution in isolated populations are the most threatened under climate change. Chihuahua spruce (Picea chihuahuana Martínez) is one of those species. The analyses of past and present environmental conditions where these species have occurred allow us to understand the dynamics of their distribution. Therefore, projecting the results to the future under different climatic scenarios provides a basis for conservation purposes. The objective of this study was to evaluate the influence of climatic variables on the availability of suitable habitat for P. chihuahuana under paleoclimatic, current and future scenarios. Methods: Paleoclimate data for the Last Glacial Maximum (LGM ∼ 22,000 years BP), Holocene Medium (HM ∼ 6000 years BP) and climatic data from the 1950–2000 periods were derived from CCSM4 and MIROC-ES models. Carbon dioxide concentration for 2050 and 2070 were derived from CCSM4 y HadGEM2-ES models under four levels of forcing. Data for the different time periods was subjected to the MaxEnt program. Results: The species distribution models showed a good fit with values of AUC> 0.99 for all time periods. Since the LGM, the potential habitat for P. chihuahuana has been declining, most likely due to global warming. Currently, human activities have exacerbated this decline. Climatic variables that most contributed to the models of suitable habitat were the lowest temperature of the coldest month (Bio6), the precipitation of the coldest quarter (Bio19), the precipitation of the warmest quarter (Bio18), and the annual average temperature (Bio 1). Future scenarios predict a reduction of suitable habitat for P. chihuahuana, or even its disappearance. Conclusions: Projections into the future locate possible micro refuges under some climatic change scenarios; however, under the highest atmospheric greenhouse gases scenario, suitable habitats for this species will disappear. Depending upon the response by humans to mitigate climatic change, P. chihuahuana may persist longer in those micro refuges, either naturally or with assisted transplanting. On the other hand, if there are no actions to reduce GHG, it is futile to develop conservation programs because there will be no suitable habitat for this species, and it will become extinct. Keywords: Maxent, Climate change, Species distribution modeling, Chihuahua spruce

The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication, and are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.

Background: Studies investigating leisure noise effect on extended high frequency hearing are insufficient and they have inconsistent results. The aim of this study was to investigate if extended high-frequency hearing threshold shift is related to audiometric notch, and if total leisure noise exposure is associated with extended high-frequency hearing threshold shift. Materials and Methods: A questionnaire of the Ohrkan cohort study was used to collect information on demographics and leisure time activities. Conventional and extended high-frequency audiometry was performed. We did logistic regression between extended high-frequency hearing threshold shift and audiometric notch as well as between total leisure noise exposure and extended high-frequency hearing threshold shift. Potential confounders (sex, school type, and firecrackers) were included. Results: Data from 278 participants (aged 18–23 years, 53.2% female) were analyzed. Associations between hearing threshold shift at 10, 11.2, 12.5, and 14 kHz with audiometric notch were observed with a higher prevalence of threshold shift at the four frequencies, compared to the notch. However, we found no associations between total leisure noise exposure and hearing threshold shift at any extended high frequency. Conclusion: This exploratory analysis suggests that while extended high-frequency hearing threshold shifts are not related to total leisure noise exposure, they are strongly associated with audiometric notch. This leads us to further explore the hypothesis that extended high-frequency threshold shift might be indicative of the appearance of audiometric notch at a later time point, which can be investigated in the future follow-ups of the Ohrkan cohort.

Context: Distortion product otoacoustic emissions (DPOAEs) may indicate preclinical noise-induced hearing loss (NIHL) in adolescents from unsafe personal music player (PMP) use. Aims: The objective, therefore, was to observe preclinical signs of NIHL in 9th grade adolescents with clinically normal hearing by comparing DPOAE signals between different levels of A-weighted equivalent PMP exposure. Settings and Design: Subjects were recruited from all secondary-level schools located in the city of Regensburg, Germany during two academic years 2009/2010 and 2010/2011. Subjects and Methods: A-weighted equivalent sound pressure levels (SPLs) for a 40-hour work week (LAeq,40h) were estimated from questionnaire responses on output and duration of PMP use of the previous week. Subjects were then categorized into four levels of exposure: 85 to

Although there is concern about increasing hearing loss in adolescents caused by leisure noise exposure, prevalence data are scarce. In an US study, about 16-17% of adolescents were affected by audiometric notches. To estimate the prevalence of audiometric notches in adolescents in Germany, baseline data of the cohort study Ohrkan, recruitment during the school years 2009/2010 and 2010/2011 were analyzed. All students in grade 9 visiting any school in the city of Regensburg were eligible for participation. Data was collected via standardized questionnaires from students and their parents. In addition, students were asked to visit the University Clinics of Regensburg for ear examination including a tympanogram and the determination of hearing thresholds in air conduction audiometry. The prevalence of audiometric notches was determined in students with normal tympanogram in both ears and complete audiometry data. Audiometric notches were defined according to criteria used to analyse US data. Overall, 2149 students (1158 girls, 991 boys mainly aged 15-16 years) of the 3846 eligible adolescents (56%) participated. Among the 1843 adolescents with complete audiometry and tympanometry data, the prevalence of audiometric notches was 2.4% (95% confidence interval 1.7-3.1%). We could not confirm the high prevalence of audiometric notches as reported in National Health and Nutrition Examination Surveys for adolescents in the US. Differences in prevalence might be at least partly due to methodical differences in audiometry. Even if empirical evidence is presently ambiguous, it is reasonable to educate young people about the potential risks of high leisure noise exposure.