The Cryptococcus neoformans STRIPAK complex controls genome stability, sexual development, and virulence.

The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B"' subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in C. neoformans, namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB"'/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest C. neoformans STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that PPH22 is a haploinsufficient gene: heterozygous PPH22/pph22Δ mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants pph22Δ, far8Δ, and mob3Δ exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either PPH22 or FAR8 leads to growth defects at 30°C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. The pph22Δ and far8Δ mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, mob3Δ mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the C. neoformans STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen.

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans.

V-ATPase, which comprises 13-14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.

Genome-wide functional analysis of phosphatases in the pathogenic fungus Cryptococcus neoformans.

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.

Nicotine dependence syndrome scale and craving: comparing nicotine-dependent individuals with and without comorbid alcohol dependence.

INTRODUCTION: Although several studies have explored craving for certain drugs, there is limited data describing the relationship between alcohol and nicotine craving from a multidimensional perspective among individuals with comorbid nicotine dependence (ND) and alcohol dependence (AD). METHODS: We compared a group of male patients diagnosed with ND and AD (n = 160) to a group of male patients diagnosed with ND only (n = 235). Smoking- and drinking-related clinical features were measured, including craving levels and the Nicotine Dependence Syndrome Scale (NDSS), which is a multidimensional questionnaire measuring ND. Subsequently, we studied factors that influenced smoking and alcohol craving in the ND and AD group. RESULTS: Regarding the NDSS, the sum, priority and tolerance scores were significantly higher in the ND and AD group compared with the ND only group (P < 0.000, P < 0.000 and P = 0.001, respectively). In the comorbid group, regression analyses revealed that alcohol craving and Fagerstrom Test for Nicotine Dependence (FTND) scores contributed to nicotine craving (beta coefficient = 0.37, P = 0.005 and beta coefficient = 0.35, P = 0.026, respectively) and these two factors explained 36% of the variance). Nicotine craving appeared to be the only factor that contributed to alcohol craving (beta coefficient = 0.35, P = 0.002), and nicotine craving explained 16% of the variance. DISCUSSION: This study may help clarify the clinical relationship between comorbid alcohol and nicotine dependence and help guide the development of effective treatment strategies for ND and AD patients.

Home visitation program for detecting, evaluating and treating socially withdrawn youth in Korea.

AIM: The problems of youth social withdrawal (or hikikomori) became a hot-button social issue in Japan in the 1990s. Unfortunately, current nosology in the DSM-IV may not adequately capture the concept of socially withdrawn youth (SWY) or hikikomori. This study aimed to investigate core SWY issues, evaluate SWY's psychopathologies, and approach them therapeutically through a home visitation program. METHODS: Participants were 65 youth referred by community mental health centers and psychiatric clinics around Seoul and Kyongki-Do province. Among them, only 41 participants (31 male, 10 female, mean age 15 ± 3.6 years) fit our SWY criteria. In addition, 248 middle and high school students in Seoul were recruited as a baseline control group. Caseworkers interviewed the SWY participants and their parents in their homes, using our structured interview manual and a number of psychiatric scales. Caseworkers also approached the participants therapeutically. RESULTS: Participants' Depression Inventory, Trait Anxiety Inventory, Social Anxiety Scale, and Internet Addiction Scale scores were significantly higher than those of baseline controls. Participants' mean number of psychotherapeutic sessions was 2.8, and the mean number of parental interview sessions was 3.4. After the therapeutic sessions, Global Assessment Functioning scores and social activities had improved somewhat in 68.3% of participants. CONCLUSION: These findings suggest that SWY is a complex phenomenon, so an individual psychopathologic process is very important for treatment. The most difficult problem in SWY treatment was therapeutic access. Hence, the home visit approach with a structured manual may be a good gateway for solving this problem.

Terrorism in South Korea.

South Korea has experienced > 30 suspected terrorism-related events since 1958, including attacks against South Korean citizens in foreign countries. The most common types of terrorism used have included bombings, shootings, hijackings, and kidnappings. Prior to 1990, North Korea was responsible for almost all terrorism-related events inside of South Korea, including multiple assassination attempts on its presidents, regular kidnappings of South Korean fisherman, and several high-profile bombings. Since 1990, most of the terrorist attacks against South Korean citizens have occurred abroad and have been related to the emerging worldwide pattern of terrorism by international terrorist organizations or deranged individuals. The 1988 Seoul Olympic Games provided a major stimulus for South Korea to develop a national emergency response system for terrorism-related events based on the participation of multiple ministries. The 11 September 2001 World Trade Center and Pentagon attacks and the 2001 United States of America (US) anthrax letter attacks prompted South Korea to organize a new national system of emergency response for terrorism-related events. The system is based on five divisions for the response to specific types of terrorist events, involving conventional terrorism, bioterrorism, chemical terrorism, radiological terrorism, and cyber-terrorism. No terrorism-related events occurred during the 2002 World Cup and Asian Games held in South Korea. The emergency management of terrorism-related events in South Korea is adapting to the changing risk of terrorism in the new century.