Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Discovery of ASP6918, a KRAS G12C inhibitor: Synthesis and structure-activity relationships of 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivatives as covalent inhibitors with good potency and oral activity for the treatment of solid tumors.

Although KRAS protein had been classified as an undruggable target, inhibitors of KRAS G12C mutant protein were recently reported to show clinical efficacy in solid tumors. In our previous report, we identified 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivative (1) as a KRAS G12C inhibitor that covalently binds to Cys12 of KRAS G12C protein. Compound 1 exhibited potent cellular pERK inhibition and cell growth inhibition against a KRAS G12C mutation-positive cell line and showed an antitumor effect on subcutaneous administration in an NCI-H1373 (KRAS G12C mutation-positive cell line) xenograft mouse model in a dose-dependent manner. In this report, we further optimized the substituents on the quinazoline scaffold based on the structure-based drug design from the co-crystal structure analysis of compound 1 and KRAS G12C to enhance in vitro activity. As a result, ASP6918 was found to exhibit extremely potent in vitro activity and induce dose-dependent tumor regression in an NCI-H1373 xenograft mouse model after oral administration.

Gliding motility of Plasmodium merozoites.

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways.

Integrative Genetic Manipulation of Plasmodium cynomolgi Reveals Multidrug Resistance-1 Y976F Associated With Increased In Vitro Susceptibility to Mefloquine.

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo.

BACKGROUND: Understanding Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC). METHODS: A total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block 2 and pfmsp2 block 3. RESULTS: Nine allele types were observed in pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele (52%, 152/293) and RO33-type allele (44%, 129/293). Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele (66%, 201/304). Polyclonal infections were detected in 63% (95% CI 56, 69) of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers (2.24 versus 1.69, adjusted b: 0.36, (95% CI 0.01, 0.72), p = 0.046) and parasitaemia > 10,000 parasites/µL compared to parasitaemia < 5000 parasites/µL (2.68 versus 1.63, adjusted b: 0.89, (95% CI 0.46, 1.25), p < 0.001). CONCLUSION: This survey showed low allelic diversity and MOI of P. falciparum, which reflects a moderate intensity of malaria transmission in the study areas. MOIs were more likely to be common in symptomatic infections and increased with the parasitaemia level. Further studies in different transmission zones are needed to understand the epidemiology and parasite complexity in the DRC.

Two New Cytotoxic Sesquiterpene-Amino Acid Conjugates and a Coumarin-Glucoside from Crossostephium chinense.

The Asteraceae family is a promising source of bioactive compounds, such as the famous Asteraceae plants Tanacetum cinerariifolium (pyrethrin) and Artemisia annua (artemisinin). As a result of our series of phytochemical studies of the subtropical plants, two novel sesquiterpenes, named crossoseamines A and B in this study (1 and 2, respectively), one undescribed coumarin-glucoside (3), and eighteen known compounds (4-21) were isolated from the aerial part of Crossostephium chinense (Asteraceae). The structures of isolated compounds were elucidated by spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY), IR spectrum, circular dichroism spectrum (CD), and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). All isolated compounds were evaluated for their cytotoxic activities against Leishmania major, Plasmodium falciparum, Trypanosoma brucei (gambiense and rhodesiense), and human lung cancer cell line A549 because of the high demand for the discovery of new drug leads to overcome the present side effects and emerging drug-resistant strains. As a result, the new compounds (1 and 2) showed significant activities against A549 (IC50, 1: 3.3 ± 0.3; 2: 12.3 ± 1.0 µg/mL), L. major (IC50, 1: 6.9 ± 0.6; 2: 24.9 ± 2.2 µg/mL), and P. falciparum (IC50, 1: 12.1 ± 1.1; 2: 15.6 ± 1.2 µg/mL).

Novel Babesia bovis exported proteins that modify properties of infected red blood cells.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

Discovery and biological evaluation of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as covalent inhibitors of KRAS G12C with favorable metabolic stability and anti-tumor activity.

RAS protein plays a key role in cellular proliferation and differentiation. RAS gene mutation is a known driver of oncogenic alternation in human cancer. RAS inhibition is an effective therapeutic treatment for solid tumors, but RAS protein has been classified as an undruggable target. Recent reports have demonstrated that a covalent binder to KRAS protein at a mutated cysteine residue (G12C) is effective for the treatment of solid tumors. Here, we report a series of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as potent covalent inhibitors against KRAS G12C identified throughout structural optimization of an acryloyl amine moiety to improve in vitro inhibitory activity. From an X-ray complex structural analysis, the 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one moiety binds in the switch-II pocket of KRAS G12C. Further optimization of the lead compound (5c) led to the successful identification of 1-[7-[6-chloro-8-fluoro-7-(5-methyl-1H-indazol-4-yl)-2-[(1-methylpiperidin-4-yl)amino]quinazolin-4-yl]-2,7-diazaspiro[3.5]nonan-2-yl]prop-2-en-1-one (7b), a potent compound with high metabolic stabilities in human and mouse liver microsomes. Compound 7b showed a dose-dependent antitumor effect on subcutaneous administration in an NCI-H1373 xenograft mouse model.

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle.

BACKGROUND: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. METHODS: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. RESULTS: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. CONCLUSION: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Six New Phenylpropanoid Derivatives from Chemically Converted Extract of Alpinia galanga (L.) and Their Antiparasitic Activities.

Chemical conversion of the extract of natural resources is a very attractive way to expand the chemical space to discover bioactive compounds. In order to search for new medicines to treat parasitic diseases that cause high morbidity and mortality in affected countries in the world, the ethyl acetate extract from the rhizome of Alpinia galanga (L.) has been chemically converted by epoxidation using dioxirane generated in situ. The biological activity of chemically converted extract (CCE) of A. galanga (L.) significantly increased the activity against Leishmania major up to 82.6 ± 6.2 % at 25 µg/mL (whereas 2.7 ± 0.8% for the original extract). By bioassay-guided fractionation, new phenylpropanoids (1-6) and four known compounds, hydroquinone (7), 4-hydroxy(4-hydroxyphenyl)methoxy)benzaldehyde (8), isocoumarin cis 4-hydroxymelein (9), and (2S,3S,6R,7R,9S,10S)-humulene triepoxide (10) were isolated from CCE. The structures of isolated compounds were determined by spectroscopic analyses of 1D and 2D NMR, IR, and MS spectra. The most active compound was hydroquinone (7) with IC50 = 0.37 ± 1.37 µg/mL as a substantial active principle of CCE. In addition, the new phenylpropanoid 2 (IC50 = 27.8 ± 0.34 µg/mL) also showed significant activity against L. major compared to the positive control miltefosine (IC50 = 7.47 ± 0.3 µg/mL). The activities of the isolated compounds were also evaluated against Plasmodium falciparum, Trypanosoma brucei gambisense and Trypanosoma brucei rhodeisense. Interestingly, compound 2 was selectively active against trypanosomes with potent activity. To the best of our knowledge, this is the first report on the bioactive "unnatural" natural products from the crude extract of A. galanga (L.) by chemical conversion and on its activities against causal pathogens of leishmaniasis, trypanosomiasis, and malaria.

A covalent small molecule inhibitor of glutamate-oxaloacetate transaminase 1 impairs pancreatic cancer growth.

Metabolic programs are rewired in cancer cells to support survival and tumor growth. Among these, recent studies have demonstrated that glutamate-oxaloacetate transaminase 1 (GOT1) plays key roles in maintaining redox homeostasis and proliferation of pancreatic ductal adenocarcinomas (PDA). This suggests that small molecule inhibitors of GOT1 could have utility for the treatment of PDA. However, the development of GOT1 inhibitors has been challenging, and no compound has yet demonstrated selectivity for GOT1-dependent cell metabolism or selective growth inhibition of PDA cell lines. In contrast, potent inhibitors that covalently bind to the transaminase cofactor pyridoxal-5'-phosphate (PLP), within the active site of the enzyme, have been reported for kynurenine aminotransferase (KAT) and gamma-aminobutyric acid aminotransferase (GABA-AT). Given the drug discovery successes with these transaminases, we aimed to identify PLP-dependent suicide substrate-type GOT1 inhibitors. Here, we demonstrate that PF-04859989, a known KAT2 inhibitor, has PLP-dependent inhibitory activity against GOT1 and shows selective growth inhibition of PDA cell lines.

Anti-malarial activity of traditional Kampo medicine Coptis rhizome extract and its major active compounds.

BACKGROUND: Herbal medicine has been a rich source of new drugs exemplified by quinine and artemisinin. In this study, a variety of Japanese traditional herbal medicine ('Kampo') were examined for their potential anti-malarial activities. METHODS: A comprehensive screening methods were designed to identify novel anti-malarial drugs from a library of Kampo herbal extracts (n = 120) and related compounds (n = 96). The anti-malarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) strains of Plasmodium falciparum. The cytotoxicity was also evaluated using primary adult mouse brain cells. After being selected through the first in vitro assay, positive extracts and compounds were examined for possible in vivo anti-malarial activity. RESULTS: Out of 120 herbal extracts, Coptis rhizome showed the highest anti-malarial activity (IC50 1.9 µg/mL of 3D7 and 4.85 µg/mL of Dd2) with a high selectivity index (SI) > 263 (3D7) and > 103 (Dd2). Three major chlorinated compounds (coptisine, berberine, and palmatine) related to Coptis rhizome also showed anti-malarial activities with IC50 1.1, 2.6, and 6.0 µM (against 3D7) and 3.1, 6.3, and 11.8 µM (against Dd2), respectively. Among them, coptisine chloride exhibited the highest anti-malarial activity (IC50 1.1 µM against 3D7 and 3.1 µM against Dd2) with SI of 37.8 and 13.2, respectively. Finally, the herbal extract of Coptis rhizome and its major active compound coptisine chloride exhibited significant anti-malarial activity in mice infected with Plasmodium yoelii 17X strain with respect to its activity on parasite suppression consistently from day 3 to day 7 post-challenge. The effect ranged from 50.38 to 72.13% (P < 0.05) for Coptis rhizome and from 81 to 89% (P < 0.01) for coptisine chloride. CONCLUSION: Coptis rhizome and its major active compound coptisine chloride showed promising anti-malarial activity against chloroquine-sensitive (3D7) and -resistant (Dd2) strains in vitro as well as in vivo mouse malaria model. Thus, Kampo herbal medicine is a potential natural resource for novel anti-malarial agents.

Rapid identification of genes controlling virulence and immunity in malaria parasites.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

Prediction Model for Antimalarial Activities of Hemozoin Inhibitors by Using Physicochemical Properties.

The rapid spread of strains of malaria parasites that are resistant to several drugs has threatened global malaria control. Hence, the aim of this study was to predict the antimalarial activity of chemical compounds that possess anti-hemozoin-formation activity as a new means of antimalarial drug discovery. After the initial in vitro anti-hemozoin-formation high-throughput screening (HTS) of 9,600 compounds, a total of 224 hit compounds were identified as hemozoin inhibitors. These 224 compounds were tested for in vitro erythrocytic antimalarial activity at 10 µM by using chloroquine-mefloquine-sensitive Plasmodium falciparum strain 3D7A. Two independent experiments were conducted. The physicochemical properties of the active compounds were extracted from the ChemSpider and SciFinder databases. We analyzed the extracted data by using Bayesian model averaging (BMA). Our findings revealed that lower numbers of S atoms; lower distribution coefficient (log D) values at pH 3, 4, and 5; and higher predicted distribution coefficient (ACD log D) values at pH 7.4 had significant associations with antimalarial activity among compounds that possess anti-hemozoin-formation activity. The BMA model revealed an accuracy of 91.23%. We report new prediction models containing physicochemical properties that shed light on effective chemical groups for synthetic antimalarial compounds and help with in silico screening for novel antimalarial drugs.

Tryptophan-rich domains of Plasmodium falciparum SURFIN4.2 and Plasmodium vivax PvSTP2 interact with membrane skeleton of red blood cell.

BACKGROUND: Plasmodium falciparum dramatically alters the morphology and properties of the infected red blood cells (iRBCs). A large group of exported proteins participate in these parasite-host interactions occurring at the iRBC membrane skeleton. SURFIN4.2 is one of iRBC surface protein that belongs to surface-associated interspersed protein (SURFIN) family. Although the intracellular tryptophan-rich domain (WRD) was proposed to be important for the translocation of SURFINs from Maurer's clefts to iRBC surface, the molecular basis of this observation has yet to be defined. The WRDs of P. falciparum SURFIN proteins and their orthologous Plasmodium vivax subtelomeric transmembrane proteins (PvSTPs) show homology to the intracellular regions of PfEMP1 and Pf332, both of which are involved in RBC membrane skeleton interactions, and contribute to malaria pathology. METHODS: Two transfected lines expressing recombinant SURFINs (NTC-GFP and NTC-4.2WRD2-GFP) of the 3D7 sequence were generated by transfection in P. falciparum. In vitro binding assays were performed by using recombinant WRDs of SURFIN4.2/PvSTP2 and inside-out vesicles (IOVs). The interactions between the recombinant WRDs of SURFIN4.2/PvSTP2 with actin and spectrin were evaluated by the actin spin down assay and an enzyme-linked immunosorbent assay based binding assays, respectively. RESULTS: The recombinant SURFINs (NTC-4.2WRD2-GFP), in which the second WRD from SURFIN4.2 was added back to NTC-GFP, show diffused pattern of fluorescence in the iRBC cytosol. Furthermore, WRDs of SURFIN4.2/PvSTP2 were found to directly interact with the IOVs of RBC, with binding affinities ranging from 0.26 to 0.68 µM, values that are comparable to other reported parasite proteins that bind to the RBC membrane skeleton. Further experiments revealed that the second WRD of SURFIN4.2 bound to F-actin (K d = 5.16 µM) and spectrin (K d = 0.51 µM). CONCLUSIONS: Because PfEMP1 and Pf332 also bind to actin and/or spectrin, the authors propose that the interaction between WRD and RBC membrane skeleton might be a common feature of WRD-containing proteins and may be important for the translocation of these proteins from Maurer's clefts to the iRBC surface. The findings suggest a conserved mechanism of host-parasite interactions and targeting this interaction may disrupt the iRBC surface exposure of Plasmodium virulence-related proteins.

Selections, frameshift mutations, and copy number variation detected on the surf 4.1 gene in the western Kenyan Plasmodium falciparum population.

BACKGROUND: Plasmodium falciparum SURFIN4.1 is a putative ligand expressed on the merozoite and likely on the infected red blood cell, whose gene was suggested to be under directional selection in the eastern Kenyan population, but under balancing selection in the Thai population. To understand this difference, surf 4.1 sequences of western Kenyan P. falciparum isolates were analysed. Frameshift mutations and copy number variation (CNV) were also examined for the parasites from western Kenya and Thailand. RESULTS: Positively significant departures from neutral expectations were detected on the surf 4.1 region encoding C-terminus of the variable region 2 (Var2) by 3 population-based tests in the western Kenyan population as similar in the Thai population, which was not covered by the previous analysis for eastern Kenyan population. Significant excess of non-synonymous substitutions per nonsynonymous site over synonymous substitutions per synonymous site was also detected in the Var2 region. Negatively significant departures from neutral expectations was detected on the region encoding Var1 C-terminus consistent to the previous observation in the eastern Kenyan population. Parasites possessing a frameshift mutation resulting a product without intracellular Trp-rich (WR) domains were 22/23 in western Kenya and 22/36 in Thailand. More than one copy of surf 4.1 gene was detected in western Kenya (4/24), but no CNV was found in Thailand (0/36). CONCLUSIONS: The authors infer that the high polymorphism of SURFIN4.1 Var2 C-terminus in both Kenyan and Thai populations were shaped-up by diversifying selection and maintained by balancing selection. These phenomena were most likely driven by immunological pressure. Whereas the SURFIN4.1 Var1 C-terminus is suggested to be under directional selection consistent to the previous report for the eastern Kenyan population. Most western Kenyan isolates possess a frameshift mutation that would limit the expression of SURFIN4.1 on the merozoite, but only 60% of Thai isolates possess this frameshift, which would affect the level and type of the selection pressure against this protein as seen in the two extremities of Tajima's D values for Var1 C-terminus between Kenyan and Thai populations. CNV observed in Kenyan isolates may be a consequence of this frameshift mutation to increase benefits on the merozoite surface.

The first nationwide surveillance of antibacterial susceptibility patterns of pathogens isolated from skin and soft-tissue infections in dermatology departments in Japan.

To investigate the trends of antimicrobial resistance in pathogens isolated from skin and soft-tissue infections (SSTI) at dermatology departments in Japan, a Japanese surveillance committee conducted the first nationwide survey in 2013. Three main organisms were collected from SSTI at 30 dermatology departments in medical centers and 10 dermatology clinics. A total of 860 strains - 579 of Staphylococcus aureus, 240 of coagulase-negative Staphylococci, and 41 of Streptococcus pyogenes - were collected and shipped to a central laboratory for antimicrobial susceptibility testing. The patient profiles were also studied. Among all 579 strains of S. aureus, 141 (24.4%) were methicillin-resistant (MRSA). Among 97 Staphylococcus epidermidis strains, 54 (55.7%) were methicillin-resistant (MRSE). MRSA and MRSE were more frequently isolated from inpatients than from outpatients. Furthermore, these methicillin-resistant strains were also isolated more frequently from patients with histories of taking antibiotics within 4 weeks and hospitalization within 1 year compared to those without. However, there were no significant differences in MIC values and susceptibility patterns of the MRSA strains between patients with a history of hospitalization within 1 year and those without. Therefore, most of the isolated MRSA cases at dermatology departments are not healthcare-acquired, but community-acquired MRSA. S. pyogenes strains were susceptible to most antibiotics except macrolides. The information in this study is not only important in terms of local public health but will also contribute to an understanding of epidemic clones of pathogens from SSTI.

Three-dimensional analysis of morphological changes in the malaria parasite infected red blood cell by serial block-face scanning electron microscopy.

The human malaria parasite, Plasmodium falciparum, exhibits morphological changes during the blood stage cycle in vertebrate hosts. Here, we used serial block-face scanning electron microscopy (SBF-SEM) to visualize the entire structures of P. falciparum-infected red blood cells (iRBCs) and to examine their morphological and volumetric changes at different stages. During developmental stages, the parasite forms Maurer's clefts and vesicles in the iRBC cytoplasm and knobs on the iRBC surface, and extensively remodels the iRBC structure for proliferation of the parasite. In our observations, the Maurer's clefts and vesicles in the P. falciparum-iRBCs, resembling the so-called tubovesicular network (TVN), were not connected to each other, and continuous membrane networks were not observed between the parasitophorous vacuole membrane (PVM) and the iRBC cytoplasmic membrane. In the volumetric analysis, the iRBC volume initially increased and then decreased to the end of the blood stage cycle. This suggests that it is necessary to absorb a substantial amount of nutrients from outside the iRBC during the initial stage, but to release waste materials from inside the iRBC at the multinucleate stage. Transportation of the materials may be through the iRBC membrane, rather than a special structure formed by the parasite, because there is no direct connection between the iRBC membrane and the parasite. These results provide new insights as to how the malaria parasite grows in the iRBC and remodels iRBC structure during developmental stages; these observation can serve as a baseline for further experiments on the effects of therapeutic agents on malaria.

Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2.

BACKGROUND: Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown. METHODS: A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy. RESULTS: PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b. CONCLUSION: N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.

The rediscovery of malaria parasites of ungulates.

Over a hundred years since their first description in 1913, the sparsely described malaria parasites (genus Plasmodium) of ungulates have been rediscovered using molecular typing techniques. In the span of weeks, three studies have appeared describing the genetic characterization and phylogenetic analyses of malaria parasites from African antelope (Cephalophus spp.) and goat (Capra aegagrus hircus), Asian water buffalo (Bubalus bubalis), and North American white-tailed deer (Odocoileus virginianus). Here we unify the contributions from those studies with the literature on pre-molecular characterizations of ungulate malaria parasites, which are largely based on surveys of Giemsa-reagent stained blood smears. We present a phylogenetic tree generated from all available ungulate malaria parasite sequence data, and show that parasites from African duiker antelope and goat, Asian water buffalo and New World white-tailed deer group together in a clade, which branches early in Plasmodium evolution. Anopheline mosquitoes appear to be the dominant, if not sole vectors for parasite transmission. We pose questions for future phylogenetic studies, and discuss topics that we hope will spur further molecular and cellular studies of ungulate malaria parasites.