Calcium-dependent protein kinases (CDPKs) play pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites, and comprise a CaMK-like kinase domain regulated by a calcium-binding domain in the C-terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N-terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate-binding site. This large conformational change constitutes a distinct mechanism in calcium signal transduction pathways.

We recently determined the first structures of inactivated and calcium-activated calcium-dependent protein kinases (CDPKs) from Apicomplexa. Calcium binding triggered a large conformational change that constituted a new mechanism in calcium signaling and a novel EF-hand fold (CAD, for CDPK activation domain). Thus we set out to determine if this mechanism was universal to all CDPKs. We solved additional CDPK structures, including one from the species Plasmodium. We highlight the similarities in sequence and structure across apicomplexan and plant CDPKs, and strengthen our observations that this novel mechanism could be universal to canonical CDPKs. Our new structures demonstrate more detailed steps in the mechanism of calcium activation and possible key players in regulation. Residues involved in making the largest conformational change are the most conserved across Apicomplexa, leading us to propose that the mechanism is indeed conserved. CpCDPK3_CAD and PfCDPK_CAD were captured at a possible intermediate conformation, lending insight into the order of activation steps. PfCDPK3_CAD adopts an activated fold, despite having an inactive EF-hand sequence in the N-terminal lobe. We propose that for most apicomplexan CDPKs, the mode of activation will be similar to that seen in our structures, while specific regulation of the inactive and active forms will require further investigation.

The incidence of and risk factors for Jarisch-Herxheimer (JH) reaction were investigated prospectively among 240 human immunodeficiency virus (HIV)-infected and 115 HIV-uninfected patients with syphilis who received penicillin treatment. The overall rate of JH reaction was 31.5% (34.6% in HIV-infected patients and 25.2% in HIV-uninfected patients). In multivariate analysis, risk factors for JH reaction included high rapid plasma reagin (RPR) titers (per log(2) RPR increase, risk ratio [RR], 1.19; 95% confidence interval [CI], 1.04-1.37), early syphilis (RR, 8.59; 95% CI, 4.75-15.56), and prior penicillin treatment (RR, 0.39; 95% CI, 0.20-0.78).

Invasive fungal infection has become a major cause of morbidity and mortality in immunocompromised patients. Rapid identification of pathogenic fungi to species level is critical for disease treatment. A real-time LightCycler assay aiming at rapid detection and species identification of pathogenic fungi from clinical isolates was developed. Template DNAs of different species were amplified and detected in real time by employing SYBR Green fluorescent dye. The target sequences for species-level detection were located between the 18S and 28S rDNA. Seven fungal species encountered frequently in the clinical setting, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida guilliermondii and Cryptococcus neoformans, could be discriminated by species-specific primers and confirmed by melting-curve analyses. The range of linearity was from 1 ng to 1 pg (ìl À1 water) and the sensitivity was 1 pg fungal DNA ìl À1 . Identification by this real-time PCR method matched biochemical identification for all 58 clinical strains. Therefore, the method is simple, rapid and sensitive enough for detection and identification of several fungal species. INTRODUCTIONInvasive fungal infections have become major causes of morbidity and mortality among immunocompromised patients (Dasbach et al., 2000;Ellis et al., 2001), such as some neutropenic patients with haematological malignancies and recipients of allogenic bone marrow transplants (Denning, 1998) as well as individuals with AIDS (Mitchell & Perfect, 1995). Candida species are now the fourth most frequent cause of nosocomial blood-stream infections in critically ill patients in the United States. At a teaching hospital in Taiwan, yeast infection was identified as the leading cause of nosocomial infection (Chen et al., 1997). The increasing prevalence of yeast infections highlights the need for simple and rapid methods to identify clinically important fungi in a microbiological laboratory.Each Candida species has a different degree of susceptibility to common antifungal agents. For instance, Candida krusei is innately resistant and Candida glabrata, Candida guilliermondii and Candida dubliniensis are less susceptible to fluconazole than other Candida species (Orozco et al., 1998;Piemonte et al., 1996). Emergence of secondary resistance in Candida lusitaniae to amphotericin B has also been observed and monitored closely (Pfaller et al., 2003). Current recommendations suggest that invasive fungal infections, such as candidiasis and aspergillosis, should be treated empirically, because the current diagnoses are difficult and time-consuming (Rex et al., 2000;Stevens et al., 2000). However, there is great concern that such practice would result in the emergence of resistant fungal pathogens.As more and more alternative antifungal agents with various spectra of activities are developed and become available, treatment according to accurate diagnosis has become even more important. Therefore, rapid species identification will be more critical for ...

This report describes the investigation of the genetic profiles of 53 Candida albicans isolates collected from 18 hospitals in Taiwan using three PFGE-based typing methods (PFGE karyotyping, and PFGE of Sfi I and BssHII restriction fragments) and one repetitive-sequence-PCR (rep-PCR) method. All four methods were able to identify clonal related isolates from the same patients. PFGEBssHII exhibited the highest discriminatory power by discriminating 40 genotypes, followed by PFGE-Sfi I (35 genotypes) and then by rep-PCR (31 genotypes), while PFGE karyotyping exhibited the lowest discriminatory power (19 genotypes). High discriminatory power can also be achieved by combining typing methods with different typing mechanisms, such as rep-PCR and PFGE-based typing methods. The results also showed that the genotype of each isolate was patient-specific and not associated with the source of the isolation, geographic origin or antifungal resistance.

BackgroundAmong members of Cryptococcus neoformans- Cryptococcus gattii species complex, C. neoformans is distributed worldwide whereas C. gattii is considered to be more prevalent in the subtropics and tropics including Taiwan. This nationwide study was undertaken to determine the distribution of genotypes, clinical characteristics and outcomes of 219 patients with proven cryptococcosis at 20 hospitals representative of all geographic areas in Taiwan during 1997–2010.Methods and FindingsOf 219 isolates analyzed, C. neoformans accounted for 210 isolates (95.9%); nine isolates were C. gattii (4.1%). The predominant genotype was VNI (206 isolates). The other genotypes included VNII (4 isolates), VGI (3 isolates) and VGII (6 isolates). Antifungal minimal inhibition concentrations higher than epidemiologic cutoff values (ECVs) were found in nine VNI isolates (7 for amphotericin B). HIV infection was the most common underlying condition (54/219, 24.6%). Among HIV-negative patients, liver diseases (HBV carrier or cirrhosis) were common (30.2%) and 15.4% did not have any underlying condition. Meningoencephalitis was the most common presentation (58.9%), followed by pulmonary infection (19.6%) and “others” (predominantly cryptococcemia) (18.7%). The independent risk factors for 10-week mortality, by multivariate analysis, were cirrhosis of liver (P = 0.014) and CSF cryptococcal antigen titer ≥512 (P = 0.020). All except one of 54 HIV-infected patients were infected by VNI genotype (98.1%). Of the 13 isolates of genotypes other than VNI, 12 (92.3%) were isolated from HIV-negative patients. HIV-infected patients compared to HIV-negative patients were more likely to have meningoencephalitis and serum cryptococcal antigen ≥1∶512. Patients infected with C. gattii compared to C. neoformans were younger, more likely to have meningoencephalitis (100% vs. 57%), reside in Central Taiwan (56% vs. 31%), and higher 10-week crude mortality (44.4% vs. 22.2%).ConclusionsCryptococcus neoformans in Taiwan, more prevalent than C. gatii, has a predominant VNI genotype. Isolates with antifungal MIC higher than ECVs were rare.

The p300/CBP‐associated factor (PCAF) and related GCN5 bromodomain‐containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine‐based L‐45 (dubbed L‐Moses) as the first potent, selective, and cell‐active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)‐(−)‐norephedrine furnished L‐45 in enantiopure form. L‐45 was shown to disrupt PCAF‐Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co‐crystal structure of L‐45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L‐45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell‐permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

TERT elevation by AR via integrated HBV and point mutation at TERT promoter region was identified as a new mechanism for the male dominance of HBV-related HCCs. Telomerase and AR thus become new targets for intervention of HCC. This article is protected by copyright. All rights reserved.