Environmental and intracellular regulation of Francisella tularensis ripA

Background Francisella tularensis is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. RipA is a cytoplasmic membrane protein that is conserved among Francisella species and is required for intracellular growth. F. tularensis ripA deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm. Results Further analysis of ripA with respect to environmental effects on the growth of mutant strains and expression levels revealed that RipA is required for optimal growth at pH 7.5 but not pH 6.5. Using a combination of RT-PCR, ripA-lacZ transcriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that both ripA transcription and RipA protein levels were elevated in organisms grown at pH 7.5 as compared to organisms grown at pH 5.5. A number of genes, including iglA, that are required for intracellular growth are regulated by the transcriptional regulators MglA and SspA, and are induced upon infection of host cells. We quantified ripA and iglA expression at different stages of intracellular growth and found that the expression of each increased between 1 and 6 hours post infection. Given the similar intracellular expression patterns of ripA and iglA and that MglA and SspA are positive regulators of iglA we tested the impact of mglA and sspA deletions on ripA and iglA expression. In the deletion mutant strains iglA expression was reduced dramatically as expected, however ripA expression was increased over 2-fold. Conclusion Expression of ripA is required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern of ripA coincided with iglA, which is positively regulated by MglA and SspA. However, in contrast to their positive impact on iglA expression, MglA and SspA negatively impacted ripA expression in vitro

Variation in optineurin (OPTN) allele frequencies between and within populations

PURPOSE: To evaluate the extent to which mutations in the optineurin (OPTN) glaucoma gene play a role in glaucoma in different populations. METHODS: Case-controlled study of OPTN sequence variants in individuals with or without glaucoma in populations of different ancestral origins and evaluate previous OPTN reports. We analyzed 314 subjects with African, Asian, Caucasian and Hispanic ancestries included 229 cases of primary open-angle glaucoma, 51 cases of juvenile-onset open-angle glaucoma, 33 cases of normal tension glaucoma, and 371 controls. Polymerase chain reaction-amplified OPTN coding exons were resequenced and case frequencies were compared to frequencies in controls matched for ancestry. RESULTS: The E50K sequence variant was identified in one individual from Chile with normal tension glaucoma, and the 691_692insAG variant was found in one Ashkenazi Jewish individual from Russia. The R545Q variant was found in two Asian individuals with primary open-angle glaucoma; one of Filipino ancestry and one of Korean ancestry. In addition to presenting OPTN allele frequencies for Caucasian and Asian populations that have been the subject of previous reports, we also present information for populations of Hispanic and black African ancestries. CONCLUSIONS: Our study contributes additional evidence to support the previously reported association of the OPTN E50K mutation with glaucoma. After finding an additional 691_692insAG OPTN variant, we can still only conclude that this variant is rare. Combined analysis of our data with data from more than a dozen other studies indicates no association of R545Q with glaucoma in most populations. Those same studies disagree in their conclusions regarding the role of M98K in glaucoma. Our analysis of the combined data provides statistically significant evidence of association of M98K with normal tension glaucoma in Asian populations, but not in Caucasian populations; however, the validity of this conclusion is questionable because of large differences in allele frequencies between and within populations. It is currently not possible to tell how much of the underlying cause of the allele frequency difference is attributable to demographic, technical, or ascertainment differences among the studies

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

Pantothenate, commonly referred to as vitamin B5, is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG

Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca2+ entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3′s), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function

Deletion of ripA Alleviates Suppression of the Inflammasome and MAPK by Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis

STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs

STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca2+ entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER–plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites

Modelling of organizational system of deliveries within transportation company

Globalny transport samochodowy wykorzystywany w wielu gałęziach gospodarki zapewnia dostawy do klientów. Fragmentaryzacja działań firm transportowych komplikuje wykonywanie zadań i nie pozwala na optymalizację dostaw. Powoduje to wydłużenie czasu realizacji, podnosi koszty i nie jest elastyczne na potrzeby klienta. Konsekwencją tego jest malejące zadowolenie klienta z logistycznej obsługi, co w efekcie prowadzi do ich utraty, a co za tym idzie spadku zysków. Przedsiębiorstwa transportowe powinny ukierunkować się na zamiany w podejściu procesowym, bowiem transport odgrywa znaczącą rolę w procesach logistycznych. W rezultacie jego optymalizacja zmierza do usprawnienia procesów bezpośrednio wpływających na wzrost wartości przedsiębiorstwa.Global trucking used in various branch of industry enables deliveries to respective customers. Fragmentation of activities of transport companies makes complex providing some tasks and not allows for optimization of deliveries. It causes delays of realization of deliveries, rises costs and is not flexible towards customer needs. As a consequence, customer satisfaction from logistic service is going down and it leads to losing of customers and decreasing of profit. Transportation companies should aim in changing into processes approach, respecting the significant role of transportation in logistic processes. In result optimization of transportation process aims in rationalization of global processes affecting the growth of the company

Effect of qualitative determinants of dairy products on choosing discount stores as place of purchasing them by young buyers

Przemiany zachodzące w sektorze handlowym powodują, że współczesny rynek produktów żywno- ściowych charakteryzuje się dużą zmiennością i nieprzewidywalnością. Znajomość i zrozumienie czynni- ków kształtujących wybór produktów i miejsc ich zakupu przez nabywców jest ważnym elementem kreo- wania strategii konkurencyjnej podmiotów działających na danym rynku W Polsce obserwowany jest rozwój sieci dyskontowych. Do niedawna polskich nabywców zachęcały do sklepów dyskontowych prze- de wszystkim niskie ceny, jednak z czasem rosnące wymagania klientów tych sklepów stały się głównym czynnikiem rozwoju nowych modeli sprzedaży w dyskontach. Rozpoczęły one proces zmian koncepcji swoich formatów poprzez ulepszenie oferty towarów pod względem jakości. Celem pracy była ocena wpływu wybranych cech jakościowych produktów żywnościowych na decy- zje nabywców związane z wyborem sklepów dyskontowych jako miejsca zakupu. Część badawczą opra- cowano na podstawie badań sondażowych, które zostały przeprowadzone w 2016 roku na grupie 358 młodych nabywców produktów mleczarskich, przy wykorzystaniu metody doboru celowego. W zbieraniu danych wykorzystano metodę CAWI wspomaganą metodą PAPI. W analizach statystycznych opisujących zachowania badanych nabywców zastosowano analizę czynnikową, a do modelowania decyzji konsumen- tów w zakresie wyboru miejsca zakupu (tj. sklepu dyskontowego) wykorzystano model probitowy. W przeprowadzonych badaniach potwierdzono kluczowe znaczenie cech jakościowych przy podejmowa- niu decyzji zakupowych młodych nabywców produktów mleczarskich. Po przeanalizowaniu wpływu czynników jakościowych na wybór sklepu dyskontowego jako miejsca zakupu produktów mleczarskich stwierdzono, że najsilniejszy wpływ na decyzje młodych konsumentów miały cechy sensoryczne oraz zdrowotność produktów

Efficiency of dairy companies in the Lublin province

Przedstawiono wyniki badań efektywności technicznej przedsiębiorstw mleczarskich zlokalizowanych na Lubelszczyźnie. Badania przeprowadzono w latach 2010-2012 we wszystkich lubelskich mleczarniach, które miały obowiązek przekazywania swoich sprawozdań finansowych do Krajowego Rejestru Sądowego (12 spośród 14 podmiotów). Oceny dokonano w oparciu o metodę DEA oraz wybrane wskaźniki finansowe. Przeprowadzone badania wykazały różnice w efektywności technicznej i efektywności skali pomiędzy badanymi mleczarniami. Jedynie trzy spośród dwunastu analizowanych podmiotów były w pełni efektywne w całym badanym okresie.The article presents the results of the study on technical efficiency of dairy enterprises located in the Lublin region. The research was conducted in the years 2010-2012 in all dairies from the Lublin region which are obligated to submit their financial reports to the National Court Register. In order to assess the dairies performance the DEA method and selected financial ratios were applied. The study showed differences in technical efficiency and scale efficiency between dairy companies. Only three of twelve dairies were fully effective in the years 2010-2012