Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.     

In Vivo Indomethacin Treatment Causes Microgial Activation in Adult Mice

The current study was undertaken to study the role of prostaglandins in regulating microglial activation. Mice were treated with indomethacin (2 g/ml) in their drinking water to selectively inhibit cyclooxygenase activity. After 4–8 days, the effect of inhibiting prostaglandin synthesis on microglial activity was evaluated. This was accomplished by analyzing microglial expression of Mac-1 (C3 complement receptor) as an indicator of activation. Mac-1 expression was assessed by immunohistochemistry of fixed brain cryosections, and by flow cytometric analysis of immunostained single cell suspensions. Both methods demonstrated that compared to age-matched, untreated controls, brains of indomethacin-treated mice had increased levels of Mac-1 expression, suggesting an increase in the state of microglial activation. These results demonstrate the importance of prostaglandins in down regulating microglial activity, and that inhibition of prostaglandin synthesis with indomethacin may act to increase the reactivity of the brain's immune system.     

Comparison of Dn4- and Dn7-carrying spring wheat genotypes artificially infested with Russian wheat aphid (Homoptera: Aphididae) biotype 1

Genetic resistance is a useful control strategy for managing Russian wheat aphid, Diuraphis noxia (Mordvilko), in wheat, Triticum aestivum L. In 2003, a Russian wheat aphid population (denoted as biotype 2) identified in Colorado was virulent to genotypes carrying the Dn4 Russian wheat aphid resistance gene, necessitating the rapid identification and deployment of new sources of resistance. Although the Dn7 gene had shown excellent resistance to Russian wheat aphid biotypes 1 and 2 in evaluations in the greenhouse, no information is available on the amount of protection provided by Dn7 under field conditions. The objective of this study was to compare the reaction of Dn4- and Dn7-carrying spring wheat genotypes under artificial infestation by Russian wheat aphid biotype 1 in the field. Irrigated field experiments were conducted in 2003 and 2004 in a split-split plot arrangement with six replications. The whole plot treatment was infestation level (control, 1x, and 10x Russian wheat aphids), and the subplot treatment was resistance source (Dn4- and Dn7-carrying genotypes). The sub-subplot treatment consisted of side-by-side planting of resistant and susceptible genotypes. The Dn4 subplot was significantly more damaged than the Dn7 subplot in 2003, but not in 2004. Interaction effects observed in 2004 suggested an advantage of Dn7 relative to Dn4 in terms of reduced Russian wheat aphid abundance and plant damage. Deployment of the Dn7 Russian wheat aphid resistance gene should provide protection in the field comparable with that provided by the Dn4 resistance gene for management of Russian wheat aphid biotype 1.     

Dendritic-cell-peptide immunization provides immunoprotection against bcr-abl -positive leukemia in mice

 Chronic myelogenous leukemia (CML) is a clonal disorder characterized by proliferation of cells that possess the bcr-abl fusion gene resulting in the production of one of two possible chimeric 210-kDa tyrosine kinase proteins. Since these chimeric proteins are expressed only in leukemic cells they have the potential to serve as tumor-specific antigens for cytotoxic T lymphocytes (CTL). Using the 12B1 murine leukemia cell line, derived by retroviral transformation of BALB/c bone marrow cells with the bcr-abl (b₃a₂) fusion gene, we have demonstrated that intravenous inoculation of 12B1 cells into BALB/c mice results in a disseminated acute leukemia analogous to human CML in blast crisis. Histological sections of liver and spleen and polymerase chain reaction analysis of peripheral blood, bone marrow, liver, spleen and lymph nodes confirmed the presence of bcr-abl ⁺ leukemia cells in these murine tissues, while Western blot data demonstrated the expression of the fusion protein in 12B1 cells. Immunization of mice with dendritic cells (DC) loaded with the synthetic bcr-abl chimeric nonapeptide, GFKQSSKAL, led to a 150 times higher frequency of bcr-abl-specific CTL precursors in the spleen than in mice immunized with peptide alone. In vitro re-stimulation of DC-peptide-primed splenocytes resulted in substantial secretion of interferon γ and augmented cytolytic activity against 12B1 targets. Finally, vaccination with peptide-loaded DC significantly prolonged survival of BALB/c mice that were challenged with 12B1 leukemia. The capacity to generate bcr-abl-specific CTL in vivo by DC-based immunization may have clinical implications in the treatment of CML. Received: 14 July 2000 / Accepted: 18 October 2000     

Evaluation of sampling and screening techniques for tiered monitoring of toxic cyanobacteria in lakes

Exposure to cyanotoxins can pose serious human health consequences both through drinking water and recreational use of a contaminated waterway. We adapted a tiered framework proposed by the World Health Organization (WHO) for monitoring cyanobacteria populations and assessing public health risks and implemented the program in Lake Champlain. This study focused on evaluating the sampling protocols employed in this adapted monitoring program over two field seasons (2003 and 2004). Using a paired sampling design, we evaluated whether 63-μm Wisconsin net samples adequately represented whole-water conditions and whether chlorophyll a concentration could serve as a useful predictor of cyanobacteria density and microcystin concentration. We also evaluated the spatial and temporal dynamics of blooms and their implications for monitoring. Our results suggest that using threshold values of either potentially toxic cyanobacteria density counted using a rapid screening protocol or chlorophyll a concentration serve as initial indicators of potentially high levels of cyanotoxin; however, the utility of chlorophyll a is system-dependent. Whole-water samples provide more accurate estimates of population density and higher microcystin concentrations than net samples, offering a more cautionary approach to assessing risk to recreational lake users. Shoreline samples generally showed higher cyanotoxin concentrations than offshore, but restricting sampling to the shoreline may miss early warnings of bloom development.     

Genome-Wide Search for Host Association Factors during Ovine Progressive Pneumonia Virus Infection

Ovine progressive pneumonia virus (OPPV) is an important virus that causes serious diseases in sheep and goats with a prevalence of 36% in the USA. Although OPPV was discovered more than half of a century ago, little is known about the infection and pathogenesis of this virus. In this report, we used RNA-seq technology to conduct a genome-wide probe for cellular factors that are associated with OPPV infection. A total of approximately 22,000 goat host genes were detected of which 657 were found to have been significantly up-regulated and 889 down-regulated at 12 hours post-infection. In addition to previously known restriction factors from other viral infections, a number of factors which may be specific for OPPV infection were uncovered. The data from this RNA-seq study will be helpful in our understanding of OPPV infection, and also for further study in the prevention and intervention of this viral disease.     

Motor Subtype as a Predictor of Future Working Memory Performance in Idiopathic Parkinson's Disease

Parkinson’s disease is a progressive neurodegenerative disorder associated with reduced spatial and verbal working memory ability. There are two established motor subtypes of PD, tremor dominant (TD) and postural instability and gait difficulty (PIGD). This study used structural equation modelling to explore the longitudinal relationship between the two subtypes and working memory assessed at a 2-year follow-up. The study comprised 84 males and 30 females (N = 114), aged between 39 and 85 (M = 64.82, SD = 9.23) with confirmed PD. There was no significant relationship between motor subtype at Time 1 and working memory at Time 2. Postural symptom severity at Time 1 predicted Time 2 spatial working memory for the PIGD subtype (p = .011) but not the TD subtype. Tremor symptoms were not associated with Time 2 working memory in either subtype. Predictive significance of Time 1 postural symptoms only in the PIGD subtype suggests an interaction between symptom dominance (subtype) and symptom severity that future subtyping should consider. This study demonstrates a predictive relationship between postural difficulties and working memory performance assessed at a 2-year follow-up. Establishing physical symptoms as predictors of cognitive change could have significant clinical importance.     

Minimal genomes,maximal productivity: comparative genomics of the photosystem and light-harvesting complexes in the marine cyanobacterium, <Emphasis Type="Italic">Prochlorococcus</Emphasis>

Although Prochlorococcus isolates possess the smallest genomes of any extant photosynthetic organism, this genus numerically dominates vast regions of the world’s subtropical and tropical open oceans and has evolved to become an important contributor to global biogeochemical cycles. The sequencing of 12 Prochlorococcus genomes provides a glimpse of the extensive genetic heterogeneity and, thus, physiological potential of the lineage. In this study, we present an up-to-date comparative analysis of major proteins of the photosynthetic apparatus in 12 Prochlorococcus genomes. Our analyses reveal a striking diversity within the Prochlorococcus lineage in the major protein complexes of the photosynthetic apparatus. The heterogeneity that has evolved in the photosynthetic apparatus suggests versatility in strategies for optimizing photosynthesis under conditions of environmental variability and stress. This diversity could be particularly important in ensuring the survival of a lineage whose individuals have evolved minimal genomes and, thus, relatively limited repertoires for responding to environmental challenges. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Abbreviated Exposure to Hypoxia Is Sufficient to Induce CNS Dysmyelination,Modulate Spinal Motor Neuron Composition,and Impair Motor Development in Neonatal Mice

Neonatal white matter injury (nWMI) is an increasingly common cause of cerebral palsy that results predominantly from hypoxic injury to progenitor cells including those of the oligodendrocyte lineage. Existing mouse models of nWMI utilize prolonged periods of hypoxia during the neonatal period, require complex cross-fostering and exhibit poor growth and high mortality rates. Abnormal CNS myelin composition serves as the major explanation for persistent neuro-motor deficits. Here we developed a simplified model of nWMI with low mortality rates and improved growth without cross-fostering. Neonatal mice are exposed to low oxygen from postnatal day (P) 3 to P7, which roughly corresponds to the period of human brain development between gestational weeks 32 and 36. CNS hypomyelination is detectable for 2–3 weeks post injury and strongly correlates with levels of body and brain weight loss. Immediately following hypoxia treatment, cell death was evident in multiple brain regions, most notably in superficial and deep cortical layers as well as the subventricular zone progenitor compartment. PDGFαR, Nkx2.2, and Olig2 positive oligodendrocyte progenitor cell were significantly reduced until postnatal day 27. In addition to CNS dysmyelination we identified a novel pathological marker for adult hypoxic animals that strongly correlates with life-long neuro-motor deficits. Mice reared under hypoxia reveal an abnormal spinal neuron composition with increased small and medium diameter axons and decreased large diameter axons in thoracic lateral and anterior funiculi. Differences were particularly pronounced in white matter motor tracts left and right of the anterior median fissure. Our findings suggest that 4 days of exposure to hypoxia are sufficient to induce experimental nWMI in CD1 mice, thus providing a model to test new therapeutics. Pathological hallmarks of this model include early cell death, decreased OPCs and hypomyelination in early postnatal life, followed by dysmyelination, abnormal spinal neuron composition, and neuro-motor deficits in adulthood.