Biologically active Phytophthora mating hormone prepared by catalytic asymmetric total synthesis

A Phytophthora mating hormone with an array of 1,5-stereogenic centers has been synthesized by using our recently developed methodology of catalytic enantioselective conjugate addition of Grignard reagents. We applied this methodology in a diastereo- and enantioselective iterative route and obtained two of the 16 possible stereoisomers of Phytophthora hormone α1. These synthetic stereoisomers induced the formation of sexual spores (oospores) in A2 mating type strains of three heterothallic Phytophthora species, P. infestans, P. capsici, and P. nicotianae but not in A1 mating type strains. The response was concentration-dependent, and the oospores were viable. These results demonstrate that the biological activity of the synthetic hormone resembles that of the natural hormone α1. Mating hormones are essential components in the sexual life cycle of a variety of organisms. For plant pathogens like Phytophthora, sexual reproduction is important as a source of genetic variation. Moreover, the thick-walled oospores are the most durable propagules that can survive harsh environmental conditions. Sexual reproduction can thus greatly affect disease epidemics. The availability of synthetic compounds mimicking the activity of Phytophthora mating hormone will be instrumental for further unravelling sexual reproduction in this important group of plant pathogens.

Expression profile of CREB knockdown in myeloid leukemia cells.

BackgroundThe cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.MethodsTo understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA.ResultsBy combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA.ConclusionWe have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

Analisis Tingkat Kepuasan Pelanggan Terhadap Pelayanan Apotek Kimia Farma Jakarta Menggunakan Model Servqual (Studi Kasus Pada Tiga Apotek)

Since policy sector pharmaceutical affairs 1993 by government about deregulations sector community pharmacy, competition business community pharmacy in Jakarta was very strict. For win something this competitions, Kimia Farma communitypharmacy as the biggest community pharmacy network in Indonesia have propagan-dized vision be the foremost business networking community pharmacy in Indonesiaand mission first-rate services for customers with customer satisfaction. The objec-tive of this research is detect satisfaction level of service quality of Kimia Farma community pharmacy Jakarta, in particular Kimia Farma community pharmacy -1Kemayoran, Kimia Farma community pharmacy-48 Matraman, Kimia Farma com-munity pharmacy-147 Duren Sawit. Beside This research also objective for knowed vision, mission of Kimia Farma community pharmacy was reached. This researchusing survey method with quisionaire Single Cross Sectional Study approaches.Research using Servqual (Service Quality) model was  discovered by Parasuraman, Zeithaml and Berry use five dimensions of service quality, that was tangible, reli-ability, responsiveness, assurance, and emphaty. Satisfaction level measured with Gap analysis that was difference expectation before customer receiving the service and perception after that; and Cartesian Diagram analysis, that was mapping atributservice quality on Cartesian Diagram. Analysis difference on customer satisfaction within community pharmacy used analysis varian (Anova). Result: dimension that highest satisfaction level was emphaty (gap -0.37 or satisfactions levels 91.88 %);attribute that highest satisfaction level was well designed interior/exterior building (gap -0.14 or satisfaction levels 96.4 %). Mapping attribute on Cartesian Diagram majority on quadrat B, means service quality Kimia Farma community pharmacy was sufficient. Based on hypothesis test, was not difference significant satisfactions level Kimia Farma community pharmacy was research (alpha > 0.05). Conclusions:generally satisfactions level Kimia Farma community pharmacy that researched nearly satisfy, with satisfaction levels within community pharmacy was not significant difference

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors

CREB is a critical regulator of normal hematopoiesis and leukemogenesis

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation

Short-term heat acclimation is effective and may be enhanced rather than impaired by dehydration

Most heat acclimation data are from regimes longer than 1 week, and acclimation advice is to prevent dehydration. Objectives: We hypothesized that (i) short-term (5-day) heat acclimation would substantially improve physiological strain and exercise tolerance under heat stress, and (ii) dehydration would provide a thermally independent stimulus for adaptation. Methods: Nine aerobically fit males heat acclimated using controlled-hyperthermia (rectal temperature 38.5°C) for 90 min on 5 days; once euhydrated (EUH) and once dehydrated (DEH) during acclimation bouts. Exercising heat stress tests (HSTs) were completed before and after acclimations (90-min cycling in T a 35°C, 60% RH). Results: During acclimation bouts, [aldosterone] plasma rose more across DEH than EUH (95%CI for difference between regimes: 40-411 pg ml -1 ; P=0.03; n=5) and was positively related to plasma volume expansion (r=0.65; P=0.05), which tended to be larger in DEH (CI: -1 to 10%; P=0.06; n=9). In HSTs, resting forearm perfusion increased more in DEH (by 5.9 ml 100 tissue ml -1 min -1 : -11.5 to -1.0; P=0.04) and end-exercise cardiac frequency fell to a greater extent (by 11 b min -1 : -1 to 22; P=0.05). Hydration-related effects on other endocrine, cardiovascular, and psychophysical responses to HSTs were unclear. Rectal temperature was unchanged at rest but was 0.3°C lower at end exercise (P < 0.01; interaction: P=0.52). Conclusions: Short-term (5-day) heat acclimation induced effective adaptations, some of which were more pronounced after fluid-regulatory strain from permissive dehydration, and not attributable to dehydration effects on body temperature. Am. J. Hum. Biol. 26:311-320, 2014. © 2014 Wiley Periodicals, Inc

Effect of permissive dehydration on induction and decay of heat acclimation, and temperate exercise performance

Purpose: It has been suggested that dehydration is an independent stimulus for heat 32 acclimation (HA), possibly through influencing fluid-regulation mechanisms and increasing 33 plasma volume (PV) expansion. There is also some evidence that HA may be ergogenic in 34 temperate conditions and that this may be linked to PV expansion. We investigated: i) the 35 influence of dehydration on the time-course of acquisition and decay of HA; ii) whether 36 dehydration augmented any ergogenic benefits in temperate conditions, particularly those related to PV expansion. Methods: Eight males (VO2max: 56.9(7.2) mL·kg-1 ·min-1 37 ) undertook 38 two HA programmes (balanced cross-over design), once drinking to maintain euhydration 39 (HAEu) and once with restricted fluid-intake (HADe). Days 1, 6, 11 and 18 were 60 min exercise- 40 heat stress tests (HST [40°C; 50%RH]), days 2-5 and 7-10 were 90 min, isothermal-strain 41 (Tre~38.5°C), exercise-heat sessions. Performance parameters (VO2max, lactate threshold, 42 efficiency, peak power output [PPO]) were determined pre and post HA by graded exercise test 43 (22°C; 55 %RH). Results: During isothermal-strain sessions hypohydration was achieved in 44 HADe and euhydration maintained in HAEu (average body mass loss -2.71(0.82)% vs. - 45 0.56(0.73)%, P<0.001), but aldosterone concentration, power output and cardiovascular strain 46 were unaffected by dehydration. HA was evident on day 6 (reduced end-exercise Tre [- 0.30°C(0.27)] and exercise heart rate [-12(15) beats.min-1 47 ], increased PV [+7.2(6.4)%] and sweat-loss [+0.25(0.22) L.hr-1 48 ], P<0.05) with some further adaptations on day 11 (further reduced end-exercise Tre [-0.25(0.19)°C] and exercise heart rate [-3(9) beats.min-1 49 ], P<0.05). 50 These adaptations were not notably affected by dehydration and were generally maintained 7- 51 days post HA. Performance parameters were unchanged, apart from increased PPO (+16(20) 52 W, irrespective of condition). Conclusions: When thermal-strain is matched, permissive 53 dehydration which induces a mild, transient, hypohydration does not affect the acquisition and 54 decay of HA, or endurance performance parameters. Irrespective of hydration, trained 55 individuals require >5 days to optimise HA

Delivery of cytoplasmic and apoplastic effectors from <i>Phytophthora infestans </i>haustoria by distinct secretion pathways

• The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralise host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease.• We used transient Agrobacterium-mediated in planta expression, transformation of P. infestans with fluorescent protein-fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection.• The cytoplasmic effector Pi04314, expressed as an mRFP-fusion protein with a signal peptide to secrete it from plant cells, does not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans is translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites-of-action in the host nucleus. The well-characterised apoplastic effector EPIC1 was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. In contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells.• P. infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways