Nitric Oxide Production: A Mechanism for Inhibition of Chlamydia Trachomatis Replication

Chlamydia trachomatis (CT) replicates in macrophages, but is inhibited by IFN-$\gamma$ or LPS. IFN-$\gamma$ and/or LPS induced nitrite production in mouse peritoneal macrophages, macrophage cell lines (RAW264.7 and J774A.1) and McCoy cells. Kinetic studies indicated that peak production occurred 48 hours post-treatment. CT infection itself was insufficient to induce nitrite production, but resulted in enhancement of nitrite production in IFN-$\gamma$-treated cells. Treatment with IFN-$\gamma$ or LPS resulted in significant inhibition of CT replication in these cells. Strong correlation between nitrite production and inhibition of CT replication was observed in RAW264.7 and J774A.1 cells (correlation coefficients: $-$0.93 and $-$0.94, p $\u3c$ 0.001). N\sp{\rm g}- monomethyl-L-arginine (L-NMMA) specifically inhibited nitrite production and partially reversed inhibition of CT replication in macrophage cell lines. NOS mRNA was measured in RAW264.7 cells by Northern blot and Dot blot hybridization. Strong correlation between NOS mRNA expression and inhibition of CT replication (correlation coefficient: $-$0.97, p $\u3c$ 0.05) was observed. Anti-TNF-$\alpha$ antibody completely neutralized the biological activity of TNF-$\alpha$ secreted by LPS-treated RAW264.7 cells, yet the antibody neither reduced nitrite production nor restored CT replication. Combination of the antibody and L-NMMA significantly enhanced restoration of CT replication. In peritoneal macrophages, inhibition of CT replication induced by IFN-$\gamma$ was partially restored by L-NMMA or anti-TNF-$\alpha$ antibody. In McCoy cells, inhibition of CT replication induced by IFN-$\gamma$ and LPS was not significantly restored by L-NMMA. Great restoration of CT replication by 1 mM L-NMMA was observed in LPS-treated J774A.1 cells (31%), but not in IFN-$\gamma$-treated cells (5%). Our data indicate that (1) NO production is one of the mechanisms for inhibition of CT replication in IFN-$\gamma$-activated peritoneal macrophages and RAW264.7 cells; (2) NO plays a significant role in CT inhibition in LPS-treated macrophage cell lines, but not peritoneal macrophages; (3) TNF-$\alpha$ may be associated with inhibition, but the mechanism(s) may not involve NO production; (4) NO production may not be the mechanism for CT inhibition in McCoy cells treated with IFN-$\gamma$ and LPS

Neuroprotection by Chitosan and Chitosan Nanoparticles

In the U.S., about 200,000 people are currently living with spinal cord injury (SCI). An estimated of 50%-70% of all SCI cases occurs in the range of ages between 15-35 years old. The destructive neurotrauma results in the majority of adult disability, even after patients suffering with SCI survived from the acute death. There are two stages involved in the progression of SCI, the primary stage and the secondary stage. The primary stage is mainly the mechanical damage to the central nervous system. The rapid collapse of the integrity of cell membrane and tissue is often one of the initial onsets. Centered by the cascade of biochemical disruption, such as aldehyde toxins, the secondary injury is responsible for the major clinical deficits in sensory and motor functions. Available pharmacological treatment for SCI includes high doses of steroids. However, the side effects of steroid therapy leave patients more susceptible for complications, such as infections, chronic pain and blood clots. The absence of standard of care have triggered waves of intense research leading to finding a cure for SCI . Based on our previous successful explorations of the neuroprotection by chitosan and chitosan nanoparticles (Chi-NPs) in SCI related cell and tissue studies, we further investigated the neuroprotective effects based on two major characteristics of chitosan: (1) molecular weight (MW) and (2) degree of acetylation (DA). Our results demonstrated that chitosan polymer blocked the random exchange of a probe, tetramethyl-rodamine (TMR) and an endogenous protein, lactate dehydrogenase (LDH), across mechanically compromised cell membrane, while a significant difference of the membrane sealing effect was not suggested among different MWs and DAs of chitosan polymer. A similar affinity of FITC-chitosan polymer at intact and injured spinal tissues was also suggested. To push the use of chitosan a step towards clinical tests, we incorporated the advantage of nanomedicine with our promising chitosan material . Different factors were investigated during the formation and the storage of Chi F-NPs. Two types of Chi-NPs (chitosan-triphosphate, Chi-TPPNPs and chitosan-dextran sulfate, Chi-DSNPs) were synthesized, with a range of size at 100-300nm and zeta-potentials of 30.65mV and -47.4mV, based on an ionic gelation method. Chi-DSNPs were shown to rescue necrotic BV-2 cells induced by a short incubation of hydrogen peroxide at 5.5mM. In addition, the conduction of somatosensory evoked potentials (SSEPs) through the lesion produced by the compression injury was partially restored after 1 week of the subcutaneous administration of Chi-DSNPs. We also found that polyethylene glycol (PEG)-coated silica NPs were significantly accumulated at the compression injured spinal tissues. The affinity of NPs at severed cell membranes was guided by PEG. Our experimental findings suggested that chitosan and Chi-NPs provided neuroprotective effects using both in vitro and in vivo models

CHOLESTEROL REGULATION OF PULMONARY ENDOTHELIAL CALCIUM ENTRY FOLLOWING CHRONIC HYPOXIA

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) is associated with diminished ATP-induced endothelial Ca2+ entry as well as membrane cholesterol in pulmonary arteries. Store-operated Ca2+ entry (SOCE) and depolarization-induced Ca2+ entry are major components of the response to ATP and are similarly decreased after CH. Because endothelium-dependent vasodilation is closely associated with pulmonary endothelial [Ca2+]i, the blunted agonist-induced Ca2+ influx in pulmonary artery endothelial cells (PAEC) may contribute to the development of CH-induced PH. Interestingly, impaired agonist-induced Ca2+ influx in PAEC following CH can be restored by membrane cholesterol supplementation. In the current studies, we hypothesized that impaired Ca2+ entry in PAEC following CH is due to decreased membrane cholesterol. We demonstrated that substitution of cholesterol with its functionally inactive epimer epicholesterol, greatly attenuated ATP-induced Ca2+ influx in PAEC from control rats. Whereas epicholesterol similarly blunted endothelial SOCE in PAEC from both groups, cholesterol supplementation restored diminished SOCE in PAEC from CH rats while having no effect in controls. Similar effects of cholesterol manipulation on T-type Ca2+ vii channel-mediated Ca2+ influx were observed in PAEC. Additionally, the role of cholesterol in SOCE mediated by Orai1, a Ca2+ selective ion channel, was examined in PAEC. Whereas cholesterol restored endothelial SOCE in CH rats, both epicholesterol and the Orai1 inhibitor, AnCoA4, attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with epicholesterol. In cultured pulmonary endothelial cells, using pharmacological inhibition and siRNA knockdown of Orai1, we found that epicholesterol, AnCoA4 and Orai1 siRNA each inhibited SOCE compared to their respective controls. Epicholesterol had no additional effect following knockdown of Orai1. Finally, we found that endothelial stromal interaction molecule 1 (STIM1)-Orai1 interaction, which is essential for SOCE, requires membrane cholesterol. Our studies support a novel regulatory role for membrane cholesterol in agonist-induced Ca2+ entry and its components. Our observation also demonstrated that altered membrane cholesterol homeostasis may contribute to impaired endothelial Ca2+ influx pathways following CH

Left orderability and Anosov flows

Let $M$ be a connected, closed, orientable, irreducible $3$-manifold. We show that: if $M$ admits a co-orientable taut foliation which is the stable or unstable foliation of an Anosov flow, then $\pi_1(M)$ is left orderable. In addition, if $M$ admits an Anosov flow, then either $\pi_1(M)$ is left orderable or $\pi_1(M)$ has an index $2$ left orderable subgroup. Combined with a theorem of Calegari, if $M$ is hyperbolic and admits a co-orientable taut foliation $\mathcal{F}$, then either $\pi_1(M)$ is left orderable, or there are a pair of very full genuine laminations transverse to $\mathcal{F}$.Comment: 14 pages, 13 figures. Comments are welcome

Modelling Emissions Reduction Strategies for Passenger Air Transport

This dissertation aims to evaluate the potential for and the effectiveness of two strategies that could reduce carbon dioxide (CO2) emissions from passenger aviation. The two strategies consist of market-based measures (MBMs) and the substitution of high-speed rail (HSR) for air transport. To assess the first mitigation strategy an econometric, itinerary-based airfare model, which explicitly captures airline operating costs, is developed and estimated for different world regions. Based on the estimated cost pass-through elasticities, the impact of a carbon tax is tested for the European and Asia-Pacific markets. Because of the higher cost pass-through elasticity in the Asia-Pacific market, a carbon tax would lead to higher airfares, lower demand, and thus greater emissions reductions in the Asia-Pacific compared to the European market. For the second mitigation strategy, i.e. the HSR substitution for air transport, this dissertation takes China’s transportation network as a case study. In a first step, an empirical study explores how airline supply has already been affected by the introduction of HSR since 2008. The results show that the HSR substitution has led to operational CO2 emissions savings from aviation in the order of 6.52-7.44 million tonnes over the period 2009-2015, depending on assumptions on the electricity intensity of Chinese HSR trains. In a second step, the dissertation explores how the enhanced introduction of HSR may affect future aviation CO2 emissions. To accomplish this objective, the future demand for inter-city high-speed transportation between 2016 and 2050 and the mode shares of HSR and air travel are estimated with an econometric model. The projected aviation demand under the planned 2025 HSR network is then compared against the demand under the 2015 HSR network. The marginal net savings of lifecycle CO2 emissions resulting from the HSR substitution are calculated from the “avoided” emissions in aviation and the additional emissions generated from transporting the diverted demand by HSR. The results show that, if China continues decarbonizing its power generation sector and achieves zero-carbon power generation in 2050, the cumulative marginal net savings of CO2 emissions could be at 736-960 million tonnes, depending on assumptions on China’s future population, GDP per capita, and jet fuel prices. The annual average of this amount between 2016 and 2050 are equivalent to 39-50% of the 53.8 million tonnes CO2 emissions from domestic aviation in 2015