HPLC analysis scanning was performed using a diode array detector

HPLC analysis scanning was performed using a diode array detector model L-2450 (Hitachi, Japan) under the following conditions: ODS-80TM, i.d.=150 × 4.6 mm (Toso Co., Japan); MeCN, 0.06% TFA (30 : 70); flow rate, 0.8 mL min−1; and UV wavelength, 200–300 nm. LC-MS analysis was performed on LCMS2010 (Shimadzu) using reverse-phase INK 128 molecular weight HPLC [STR ODS-II, i.d.=150 × 2.0 mm; MeCN, 0.06% TFA (35 : 65); flow rate, 0.2 mL min−1; and UV wavelength, 220 nm]. Standard compounds of M-II, M-III, and M-VI were obtained from the fermentation broth of M. griseorubida A11725. The disruption cassette FRT-neo-oriT-FRT-attB was used to obtain the mycE disruption mutant of M. griseorubida. In previous

studies, the transconjugant of M. griseorubida has never been isolated with pSET152 as an intergeneric conjugation vector. Therefore, we estimated that M. griseorubida would not possess the bacteriophage φC31 attB site on the chromosome. The mycE-deleted plasmid pMG502, which had the mycinose biosynthetic gene cluster

region in which mycE was replaced with the disruption cassette, was generated with pSAN-lac as the suicide vector. pSAN-lac DAPT cell line was constructed with pUC18 and pIJ350 as an E. coli–Streptomyces shuttle vector, but the plasmid has never been amplified in M. griseorubida cells (data not shown). Plasmid pMG502 was transferred from E. coli to M. griseorubida A11725 by intergeneric conjugation, and some neomycin-resistant (neor) and thiostrepton-sensitive (thios) transconjugants were isolated. PCR was used to verify that the chromosomal copy of A11725 mycE was deleted by double cross-over. Using the primers mycEF and mycERBam annealing

outside the disruption cassette, the 1.4- and 1.2-kb amplified fragments were observed in TPMA0014 and the wild strain A11725, respectively (Fig. 2b). The size difference indicated that TPMA0014 was the mycE disruption mutant. M-VI was detected in the EtOAc extract from the FMM culture broth of TPMA0014 at 7.63 min (Fig. 3). PI-1840 However, the productivity of M-VI by TPMA0014 was very low (0.08 μg mL−1), and it was estimated that the direction of neo gene transcription had a negative effect on the productivity. We also isolated another mycE disruption mutant in which the direction of the neo gene was opposite to the mycinose biosynthesis gene cluster. The neor and thios transconjugant TPMA0003, which was isolated by the introduction of pMG503 into A11725, was confirmed to be a mycE disruption mutant by PCR (Fig. 2b); the M-VI productivity (13.8 μg mL−1) of TPMA0003 was higher than that of TPMA0014 (Fig. 3). Furthermore, three unknown peaks E-1, E-2, and E-3 were observed in the chromatogram of the extract of TPMA0003 at 5.62, 6.95, and 6.28 min, respectively. LC-MS was performed for the extract to measure the molecular weight of these metabolites (E-1; m/z 684, E-2; m/z 684, and E-3; m/z 698).

However, intracellular M bovis CFU decreases drastically after 2

However, intracellular M. bovis CFU decreases drastically after 24 h, which could be attributed to the massive cellular death observed. The CFU assessment shows no significant difference in the intracellular bacterial load of M. bovis between MDMs from tuberculosis and healthy control cattle. BTB is a chronic infectious disease caused by the pathogen M. bovis and continues to pose a threat to livestock

worldwide. Mycobacterium bovis is the causative agent of most cases of tuberculosis in cattle and M. bovis Beijing strains cause a substantial proportion of tuberculosis cases worldwide (Chen et al., 2009; Kremer et al., 2009). Understanding the specific immune response to BTB will aid in developing improved control and diagnostic strategies. Studies on tuberculosis in humans indicate that innate immunity, LDE225 TLR signaling and the Th1/Th2 bias of the immune response are essential for host defense against tuberculosis (Doherty & Arditi, 2004; Winek et al., 2009; Ahmad, 2011). However, these specific cell signal pathways and immune responses are poorly defined in cattle. Meade et CB-839 chemical structure al. (2006)

examined the gene expression profiles of PBMCs from BTB-infected and healthy cattle and demonstrated the differential expression of innate immunity-related genes. In this study, gene expressions of MDMs cells from tuberculosis and healthy groups stimulated with M. bovis were detected. Seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in immune responses were examined. In MDMs, the expression of the seven examined genes was increased in both stimulated tuberculosis and stimulated healthy cattle. The expression of the proinflammatory cytokine TNF-α, IL1β and its receptor IL1R1 markedly increased, indicating that these genes may play a key role in the early interaction of host cells and M. bovis. The expression of these three genes, although elevated in response to M. bovis stimulation,

showed no significant difference between the two groups. This finding may indicate that the macrophages from tuberculosis cattle have a capability similar to healthy cattle in generating proinflammatory cytokine (IL1β and TNF-α) during early immune response to M. bovis stimulation. In agreement, Clomifene it is frequently reported that the tuberculosis infection could induce a burst of inflammatory cytokines IL1β and TNF-α in the infected location (Arcila et al., 2007; Qiu et al., 2008; Winek et al., 2009). Two Toll-like receptor genes (TLR2 and TLR4) were examined. The two genes have been studied widely, because they are very important in innate immunity and TLR signaling aids the activation of antigen-specific T cells (Cooper, 2009). Previous studies demonstrated that M. tuberculosis products can be recognized by TLR2 or TLR4 (Aliprantis et al., 1999; Underhill et al., 1999; Abel et al., 2002).

However, intracellular M bovis CFU decreases drastically after 2

However, intracellular M. bovis CFU decreases drastically after 24 h, which could be attributed to the massive cellular death observed. The CFU assessment shows no significant difference in the intracellular bacterial load of M. bovis between MDMs from tuberculosis and healthy control cattle. BTB is a chronic infectious disease caused by the pathogen M. bovis and continues to pose a threat to livestock

worldwide. Mycobacterium bovis is the causative agent of most cases of tuberculosis in cattle and M. bovis Beijing strains cause a substantial proportion of tuberculosis cases worldwide (Chen et al., 2009; Kremer et al., 2009). Understanding the specific immune response to BTB will aid in developing improved control and diagnostic strategies. Studies on tuberculosis in humans indicate that innate immunity, selleck inhibitor TLR signaling and the Th1/Th2 bias of the immune response are essential for host defense against tuberculosis (Doherty & Arditi, 2004; Winek et al., 2009; Ahmad, 2011). However, these specific cell signal pathways and immune responses are poorly defined in cattle. Meade et HIF-1 pathway al. (2006)

examined the gene expression profiles of PBMCs from BTB-infected and healthy cattle and demonstrated the differential expression of innate immunity-related genes. In this study, gene expressions of MDMs cells from tuberculosis and healthy groups stimulated with M. bovis were detected. Seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in immune responses were examined. In MDMs, the expression of the seven examined genes was increased in both stimulated tuberculosis and stimulated healthy cattle. The expression of the proinflammatory cytokine TNF-α, IL1β and its receptor IL1R1 markedly increased, indicating that these genes may play a key role in the early interaction of host cells and M. bovis. The expression of these three genes, although elevated in response to M. bovis stimulation,

showed no significant difference between the two groups. This finding may indicate that the macrophages from tuberculosis cattle have a capability similar to healthy cattle in generating proinflammatory cytokine (IL1β and TNF-α) during early immune response to M. bovis stimulation. In agreement, 17-DMAG (Alvespimycin) HCl it is frequently reported that the tuberculosis infection could induce a burst of inflammatory cytokines IL1β and TNF-α in the infected location (Arcila et al., 2007; Qiu et al., 2008; Winek et al., 2009). Two Toll-like receptor genes (TLR2 and TLR4) were examined. The two genes have been studied widely, because they are very important in innate immunity and TLR signaling aids the activation of antigen-specific T cells (Cooper, 2009). Previous studies demonstrated that M. tuberculosis products can be recognized by TLR2 or TLR4 (Aliprantis et al., 1999; Underhill et al., 1999; Abel et al., 2002).

Mr Arnaud Cannet, entomologist (University Hospital of Nice, Fran

Mr Arnaud Cannet, entomologist (University Hospital of Nice, France), Dr Véronique Blanc, biologist (Hospital of Antibes–Juan-les-Pins, France), Professor Pierre Marty (Laboratoire de Parasitologie–Mycologie, Centre Hospitalier Universitaire de Nice, and Inserm U895/Université

de Nice-Sophia Antipolis, Nice, France), Dr Cameron Webb (Department of Medical Entomology University of Sydney, Australia), and Janet Jacobson for editorial assistance. This research has been funded by the French Ministry of Health, Projet Hospitalier de Recherche Clinique 2009 (P. D., PHRC 2010 09-API-01). This review is part of a research program entitled “Cimex lectularius find more or Bedbugs: Vector of Infectious Agents and Pathogenic Role. The Infectiopole Sud Scientific Cooperation Foundation provided funds for the camera and microscope. The author states that he has no conflicts of interest to declare. “
“Background. Rifaximin has been shown to be effective in treating and preventing travelers’ diarrhea (TD) during the summer season. Methods. The goal of this double-blinded multicenter trial was to assess the efficacy and safety of rifaximin 550 mg administered once daily for 14 days compared with placebo in the prevention of TD during the dry season in Mexico. Results. There were 101 participants randomized. Overall, 25 participants developed TD during the 3 weeks of the study: 22% from the

rifaximin group and 29% from the placebo group (p = 0.4). Mild diarrhea (defined as only one or two unformed stools during a 24-h period plus at least one abdominal PR171 symptoms) developed in only 3 (6%) participants taking rifaximin compared with 10 (21%) taking placebo during the first week of study (p = 0.03). No clinically significant or serious adverse events were reported. Conclusions. Antibiotic prophylaxis of TD in Mexico during the dry season needs to be further studied and its benefits weighed against the benefits of self-treatment. Travelers’ diarrhea (TD), which occurs in approximately 40% of international travelers visiting high-risk areas,1 is caused by bacteria in approximately 80% of cases.2 A variety of drugs with antimicrobial effects have been used in Palbociclib mw the prevention of TD during periods

of risk of no greater than 2 weeks, including doxycycline,3 bismuth subsalicylate,4 trimethoprim-sulfamethoxazole,5 and fluoroquinolones.6 Prophylaxis with antibacterial drugs is not generally recommended because of adverse effects of systemically absorbed drugs and risk of antimicrobial resistance for drugs that have important uses outside the gut. Rifaximin is a nonsystemic, gut-selective antibiotic that has activity against enteric bacterial pathogens causing TD in multiple areas of the world,7 and has been shown to be effective in treating TD in studies carried out in Mexico.8 Previous clinical trials have been carried out during summer months in Mexico showing that a once daily dose of rifaximin (one, two, or three 200 mg tablets) was effective in preventing TD.

Consequently, the cytosolic N-terminal domain becomes accessible

Consequently, the cytosolic N-terminal domain becomes accessible and stabilizes the interaction between phospho-KdpE and the DNA. In parallel, KdpD transfers the phosphoryl group to the response regulator KdpE that dimerizes, and binds with increased affinity to the KdpE-binding site upstream the kdp-promoter/operator region (Fig. ICG-001 in vitro 2a). Other proteins modulate the signaling cascade. Under conditions of hyperosmolarity, the

production of the universal stress protein UspC is enhanced. UspC interacts with the Usp domain within KdpD, and scaffolds the KdpD/phospho-KdpE/DNA complex at a high intracellular K+ concentration (Fig. 2b). Nonphosphorylated IIANtr of the Ntr-PTS interacts with KdpD and shifts

KdpD into the ‘ON’ state when E. coli needs more K+ due to increased metabolic requirements. selleck chemicals llc The phosphorylation state of IIANtr is influenced by the transport-PTS, and therefore, cells are able to adjust K+ uptake according to the available C source (Fig. 2c). The link between Ntr-PTS and the Kdp system ensures K+ homeostasis according to the metabolic state of E. coli. During the past 15 years of research on the molecular mechanism of stimulus perception and signaling by the KdpD/KdpE histidine kinase/response regulator system, we have been realizing that a ‘simple’ two-component system is more complex than thought before. We have learnt that KdpD has the capability to integrate diverse stimuli to allow best adaptation of E. coli Parvulin in different environments. Moreover, the link between Kdp and Ntr-PTS demonstrates an elegant mechanism to connect gene regulation with metabolic requirements to ensure K+ homeostasis under various cellular conditions. It is quite possible that the currently known collection of accessory proteins for histidine kinase/response

regulator systems reflects only a small portion of the complex regulatory interaction network within a prokaryotic cell. Continuous progress of bacterial genome projects has resulted in the availability of several hundred bacterial genome sequences to date. These analyses elucidated that KdpD/KdpE is one of the most widespread histidine kinase/response regulator systems among bacteria and archaea, indicating the importance of this ‘simple’ two-component system for various bacterial lifestyles. This work was financially supported by the Deutsche Forschungsgemeinschaft (Exc114-1) and the BMBF (SysMO, project KOSMOBAC). We are grateful to Dr Boris Görke for critically reading the manuscript. “
“Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms naturally present in many foods and those have proved to be effective mucosal delivery vectors. Moreover, some specific strains of LAB exert beneficial properties (known as probiotic effect) on both human and animal health.

Progesterone and free-cholesterol (FC) obstructed each other’s ef

Progesterone and free-cholesterol (FC) obstructed each other’s effects against the H. pylori cell. Taken in sum, these results suggest that progesterone and FC may bind to the identical region on the H. pylori cell surface. We expect these findings to contribute to the development of a novel anti-H.

pylori steroidal agent. Helicobacter pylori colonizes the human gastric epithelium and causes chronic gastritis and peptic ulcers (Marshall & Warren, 1983; Wyatt & Dixon, 1988; Graham, 1991). Over longer periods, it also contributes to the development of gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (Wotherspoon et al., 1991; Forman, the Eurogast Study Group, 1993). This bacterium possesses the unique biological feature GPCR & G Protein inhibitor of steroid assimilation. A recent study by our group demonstrated that H. pylori selectively absorbs 3β-OH and 3-OH steroids,

glucosylates only the former, and uses both steroids, with or without glucosylation, as membrane lipid components (Hosoda et al., 2009). A number of investigations, including our own, have revealed the physiological significance of steroid assimilation in H. pylori. Wunder et al. (2006) demonstrated that H. pylori evades the host immune KU 57788 systems by glucosylating the absorbed free-cholesterol (FC). Our own study found that H. pylori retains the steroid (FC or estrone) in order to reinforce the membrane lipid barrier and thereby resists the bacteriolytic action of the phosphatidylcholines (Shimomura et al., 2009). This confirms that certain steroids are beneficial to the survival of H. pylori. Conversely, other steroids have been found to impair the viability of H. pylori. After examining Astemizole the anabolic use of 10 steroid hormones in H. pylori, our

group proposed that three hormones, namely, estradiol, androstenedione, and progesterone, may have the potential to inhibit the growth of H. pylori (Hosoda et al., 2009). These findings led to our interest in the development of antibacterial steroidal agents for H. pylori. To explore the potential for this, we must first precisely clarify the inhibitory effects of those steroids on the growth of H. pylori. In this study, we do so by analyzing the anti-H. pylori actions of the steroid hormones. Four strains of H. pylori were investigated: NCTC 11638, ATCC 43504, A-13, and A-19. The A-13 and A-19 strains were clinical isolates from a patient with a gastric ulcer and a patient with a duodenal ulcer, respectively. The cultures were all grown in an atmosphere of 5% O2, 10% CO2, and 85% N2 at 37 °C (Concept Plus: Ruskinn Technology, Leeds, UK).

[14] Recommendations for serologic testing of immunity to hepatit

[14] Recommendations for serologic testing of immunity to hepatitis B vaccination vary

between countries. In Australia, serological testing is not performed after routine vaccination of adults (including travelers). However, anti-HBs antibody levels should be performed 1 to 2 months after vaccination in health-care workers, patients on hemodialysis, and individuals at risk of recurrent exposure to HBV.[14] There is no universal agreement on how to manage nonresponders to HBV vaccination. However, the Australian Immunization Guidelines suggest offering nonresponders either a fourth double dose or another three-dose vaccine series. Persistent nonresponders should be counseled to minimize exposure and offered immunoglobulin within 72 hours if significant Maraviroc research buy HBV exposure occurs.[14] Anti-HBs antibody levels decrease over time following a primary immunization course; however, the need for HBV boosting is controversial. The duration of protection www.selleckchem.com/products/Fulvestrant.html has been estimated to be at least 15 years[46-48] and even if titers of anti-HBs fall to <10 mIU/mL, a booster dose is likely to be unnecessary because of an effective amnesic response.[49] In the United States, HBV boosting is not recommended

for otherwise healthy individuals,[4] whereas some European countries (including the UK) recommend it.[50] The European Consensus Group on hepatitis B immunity and a recent review by Van Damme and Van Herck concluded that there was no evidence to recommend HBV boosting in healthy individuals including travelers.[50, 51] This issue will have increasing practical relevance as cohorts immunized as

infants become adult travelers. Plasma-derived and recombinant forms of HBV vaccine are comparable in terms of efficacy and durability. Plasma-derived vaccines are prepared by concentrating and purifying Tryptophan synthase plasma from HBsAg carriers and are used in developing countries. Concerns regarding the potential of plasma-derived products to transmit infections have led to the widespread use of recombinant HBV vaccines in Europe, the United States, and Australia.[4] Recombinant HBsAg is produced by cloning the HBV S gene in either yeast or mammalian cells. In the United States, two thimerosal free vaccines that express HBsAg [Engerix-B (GlaxoSmithKline, Brentford, UK) and Recombivax-HB (Merck, Rixensart, Belgium)] have been licensed. Engerix-B contains 20 µg of recombinant HBsAg adsorbed onto 0.5 mg of aluminum hydroxide. Recombivax-HB contains 10 µg of recombinant HBsAg protein adsorbed onto 0.5 mg of aluminum hydroxyphosphate sulfate. Recombivax-HB is available in Europe as HBVAXPRO.[52] In Europe, a recombinant HBsAg vaccine adjuvanted with ASO4 [Fendrix (GlaxoSmithKline)] is licensed for use in adolescents and adults with renal insufficiency. ASO4 is a novel adjuvant that contains aluminum hydroxide and monophosphoryl lipid A. The primary immunization schedule of recombinant HBsAg vaccine adjuvanted with ASO4 is four doses given at 0, 1, 2, and 6 months.

Migration of cerebellar granule cells (CGCs) requires multiple fa

Migration of cerebellar granule cells (CGCs) requires multiple factors. Mature brain-derived neurotrophic factor (BDNF) positively regulates the proliferation, migration, survival and differentiation of CGCs in rodents. However, the role of the BDNF precursor, proBDNF, in neuronal development remains unknown. In this study, we investigated the effect of proBDNF in vivo and in vitro on migration of CGCs. We demonstrate that proBDNF and its receptors p75 neurotrophin receptor (p75NTR) and sortilin are highly expressed in the cerebella

as determined by immunohistochemistry and Western blot. ProBDNF is released from cultured cerebellar neurons, and this release is increased by high potassium stimulation. ProBDNF inhibits migration of CGCs in vitro, and the neutralizing antibodies to proBDNF enhance such migration as assayed by transwell culture. In addition, proBDNF incorporated into an agarose plug reduces granule cell migration from such CP-673451 cost plugs, whereas the neutralizing antibodies attract these cells towards the plug. The application of proBDNF into the lateral ventricle significantly inhibits migration of CGCs out of the proliferative zone into the internal granular cell layer, whereas the neutralizing

see more antibodies enhance this migration. Furthermore, the effects of proBDNF on cell migration are lost in p75NTR−/− mice. Our data suggest that proBDNF negatively regulates migration of CGCs and this effect is mediated by p75NTR. We conclude that proBDNF has an opposing role in migration of CGCs to that of mature BDNF. “
“Pathology department, University of California, San Diego, La Jolla, CA, USA The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster,

mutations C59 research buy in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. In this study, using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, we demonstrate that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). We observed a dual regulatory role of FSK on GABAergic transmission, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, we show that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA.

The presence of CYN was confirmed in 16 samples The homology sea

The presence of CYN was confirmed in 16 samples. The homology searches revealed that amplified sequences of four water samples, which were selected from among all the samples, displayed a strong 99% homology to cyrJ gene of Aphanizomenon sp. 10E6. The culture of C. raciborskii did not contain the cyrJ gene nor the CYN. The specificity of C. raciborskii

was confirmed by application of a fragment of the rpoC1. These first genetic analyses have shown that Aphanizomenon seems to be the main cyanobacterial genus responsible for the production of CYN in the Polish lakes. The lack of toxigenicity of the isolated C. raciborskii suggests that it is possible that this invasive species does not demonstrate toxigenic activity in Polish LY2157299 ic50 water bodies. Climate change increases water temperatures and nutrient concentration and hence the intensity of eutrophication. In consequence, global warming causes massive cyanobacteria bloom in many water bodies

(Delpla et al., 2009; Nõges et al., 2011). Ultimately, cyanobacterial blooms and their toxins pose a serious threat to public health through water supply systems, recreation or agriculture, and to the natural environment. The problem of cyanobacteria responsible for the production of microcystins (MCs) belonging to the cyanobacterial hepatotoxins is common. In Poland, regular blooms with domination of microcystin-producing cyanobacteria Planktothrix agardhii or Microcystis aeruginosa find more and MCs concentration reaching 212.7 μg L−1 have been documented well (Pawlik-Skowrońska et al., 2008; Mankiewicz-Boczek et al., 2006; Mazur-Marzec et al., 2010). Recently, the occurrence of other cyanotoxin (representing the group of cytotoxins), cylindrospermopsin (CYN), with maximum 1.8 μg L−1, has been reported in the Western Poland (Kokociński et al., 2009). CYN is a stable alkaloid, which is able to inhibit synthesis of proteins. Liver is the main target of the CYN activity; however, other organs, such as kidneys, lungs, thymus, spleen, adrenal glands, intestinal tract, Baricitinib immune system and heart, might

also be affected (Falconer, 1999; Carmichael, 2001; van Apeldoorn et al., 2007; Žegura et al., 2011). Moreover, CYN is genotoxic and probably more hazardous to human and animal health than MCs (Žegura et al., 2011). Therefore, it seems to be important not only to estimate the concentration of CYN in the water but also to determine the source of CYN to identify early warning signals and better prevention against the CYN-producing cyanobacteria. In 1992, the strain of Cylindrospermopsis raciborskii from Australia was characterized as potent producer of CYN (Ohtani et al., 1992). So far the CYN-producing C. raciborskii strains have been isolated from Australian and Asian water bodies (Carmichael, 2001; Schembri et al., 2001; Fergusson & Saint, 2003; Mihali et al., 2008; Stüken & Jakobsen, 2010).

, 1994; Ritchie & Waldor, 2005; Mann et al, 2007) Also present

, 1994; Ritchie & Waldor, 2005; Mann et al., 2007). Also present on the surface of Y. pestis is the highly immunogenic F1 capsular antigen which composes a proteinaceous capsule (Meyer et al., 1974a, b; Friedlander et al., 1995). The expression of the F1 antigen is find more temperature regulated and encoded by the

caf operon on the pFra plasmid (Chen & Elberg, 1977; Galyov et al., 1990). The capsule is synthesized in large quantities (Davis et al., 1996) and allows Y. pestis to be antiphagocytic and prevents adhesion to epithelial cells (Williams et al., 1972; Liu et al., 2006). Currently, there is no approved plague vaccine for human use in the United States. The killed whole cell-based vaccine (Plague vaccine, USP) was discontinued in 1999 because it does not protect against pneumonic plague (Heath et al., 1998), the

most likely learn more disease route for use of Y. pestis as a bioweapon. The recombinant F1-LcrV fusion protein was demonstrated to be protective in an animal model of pneumonic plague (Powell et al., 2005). However, adding to the difficulties of developing a successful vaccine, the LcrV antigen is very heterogeneous across Yersinia species (Anisimov et al., 2007). Live vaccines offer exposure to the full antigenic spectrum from a pathogen and would not be subject to the limitations encountered with vaccine development based on a limited set of recombinant proteins. This strategy has been used in preventing infectious diseases by many pathogens (Agin et al., 2005; Feunou et al., 2008; Pasquali et al., 2008), but the only human-approved, live bacterial vaccine currently available for research

purposes in the U.S. is the attenuated LVS strain of Francisella tularensis (Isherwood et al., 2005). Based on an attenuated Pgm− strain, the live EV76 vaccine against Y. pestis is protective against pneumonic plague and induces a high antibody titer (Byvalov et al., 1984), but its use has been discontinued due to chronic infections and adverse reactions (Meyer et al., 1974a, b; Welkos et al., 2002). The use of genetically engineered attenuated pathogens as vaccines, on the other hand, offers the potential to circumvent such deleterious side effects. In the current Tenofovir work, we show that a ΔyscN Y. pestis mutant is highly attenuated in mice but also protects them against lethal doses of the fully virulent CO92 strain in a subcutaneous (s.c.) model of plague. The fully virulent CO92 parent strain (Doll et al., 1994), ΔyscN mutant (Swietnicki et al., 2011), and CO92 pLcr− (USAMRIID collection) strains of Y. pestis were maintained on sheep blood agar plates or in heart infusion (HI) broth. When growth occurred at 37 °C, HI broth was supplemented with either 2.5 mM CaCl2 or 20 mM MgCl2 and 20 mM sodium oxalate (MOX), as indicated.