Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.     

Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection

The extent of lung regeneration following catastrophic damage and the potential role of adult stem cells in such a process remains obscure. Sublethal infection of mice with an H1N1 influenza virus related to that of the 1918 pandemic triggers massive airway damage followed by apparent regeneration. We show here that p63-expressing stem cells in the bronchiolar epithelium undergo rapid proliferation after infection and radiate to interbronchiolar regions of alveolar ablation. Once there, these cells assemble into discrete, Krt5+ pods and initiate expression of markers typical of alveoli. Gene expression profiles of these pods suggest that they are intermediates in the reconstitution of the alveolar-capillary network eradicated by viral infection. The dynamics of this p63-expressing stem cell in lung regeneration mirrors our parallel finding that defined pedigrees of human distal airway stem cells assemble alveoli-like structures in vitro and suggests new therapeutic avenues to acute and chronic airway disease.     

Phosphate removal from the returned liquor of municipal wastewater treatment plant using iron-reducing bacteria

AIM: The application of iron-reducing bacteria (IRB) to phosphate removal from returned liquor (liquid fraction after activated sludge digestion and anaerobic sludge dewatering) of municipal wastewater treatment plant (WWTP) was studied. METHODS AND RESULTS: An enrichment culture and two pure cultures of IRB, Stenotrophomonas maltophilia BK and Brachymonas denitrificans MK identified by 16S rRNA gene sequencing, were produced using returned liquor from a municipal WWTP as carbon and energy source, and iron hydroxide as oxidant. The final concentration of phosphate increased from 70 to 90 mg l(-1) in the control and decreased from 70 to 1 mg l(-1) in the experiment. The mass ratio of removed P to produced Fe(II) was 0.17 g P g(-1) Fe(II). The strain S. maltophilia BK showed the ability to reduce Fe(III) using such xenobiotics as diphenylamine, m-cresol, 2,4-dichlorphenol and p-phenylphenol as sole sources of carbon under anaerobic conditions. CONCLUSIONS: Bacterial reduction of ferric hydroxide enhanced the phosphate removal from the returned liquor. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the facultative anaerobes S. maltophilia BK and B. denitrificans MK to reduce Fe(III) was shown. These micro-organisms can be used for anaerobic removal of phosphate and xenobiotics by bacterial reduction of ferric ions.     

Neural development in human embryonic stem cells-applications of lentiviral vectors

The derivation of neural lineages from human embryonic stem cells (hESCs) in vitro is based largely on exposure of hESCs to exogenous signals and substrates, designed to mimic conditions in the developing embryo. However, selection of specific lineages and the discovery of gene function in human neural development may be enhanced by the ability to intrinsically regulate gene expression. Recombinant lentiviral vectors provide an efficient method to stably introduce genes into hESC and their differentiating derivatives. Here we review the methods used to derive neural cells from hESCs, transduction of these cells with lentiviral vectors, and improvements that have been made to the vectors to enhance viral integration and transgene expression. Finally, we explore prospects for future uses of lentiviral vectors in hESC research, including their applications in library screening for drug development, zinc finger nucleases for gene editing and optogenetics to interrogate cellular pathways and function.     

A unified theory for upscaling aerobic granular sludge sequencing batch reactors

Aerobic granular sludge sequencing batch reactors (SBR) are a promising technology for treating wastewater. Increasing evidence suggests that aerobic granulation in SBRs is driven by selection pressures exerted on microorganisms. Three major selection pressures have been identified as follows: settling time, volume exchange ratio and discharge time. This review demonstrates that these three major selection pressures can all be unified to one, the minimal settling velocity of bio-particles, that determines aerobic granulation in SBRs. The unified selection pressure theory is a useful guide for manipulating and optimizing the formation and characteristics of aerobic granules in SBRs. Furthermore, the unified theory provides a single engineering basis for scale up of aerobic granular sludge SBRs.     

The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.     

The Impact of Prolonged Storage of Red Blood Cells on Cancer Survival

Background

The duration of storage of transfused red blood cells (RBC) has been associated with poor clinical outcomes in some studies. We sought to establish whether prolonged storage of transfused RBC in cancer patients influences overall survival (OS) or cancer recurrence.

Methods and Findings

Patients diagnosed with cancer at The Ottawa Regional Cancer Centre between January 01, 2000 and December 31, 2005 were included (n = 27,591) where 1,929 (7.0%) received RBC transfusions within one year from diagnosis. Transfused RBC units were categorized as “new” if stored for less than 14 days, “intermediate” if stored between 14 and 28 days and “old” if stored for more than 28 days. Baseline characteristics between the comparative groups were compared by ANOVA test. Categorical variables and continuous variables were compared using Chi-squared and Wilcoxan rank-sum tests respectively. Overall survival was not associated with duration of storage of transfused RBC with a median survival of 1.2, 1.7, 1.1 years for only new, intermediate and old RBC units respectively (p = 0.36). Cancer recurrence was significantly higher in patients who received a RBC transfusion than those who did not (56.3% vs 33.0% respectively; p<0.0001) but was not affected by the duration of storage of transfused RBC (p = 0.06). In multivariate analysis, lung cancer, advanced stage, chemotherapy, radiation, cancer-related surgery and cancer recurrence were associated with inferior OS (p<0.05), while age, advanced stage, lung cancer, and more than 6 units of blood transfused were associated with cancer recurrence (p<0.05). The duration of storage of RBC before transfusion was not associated with OS or cancer recurrence in multivariate analysis.

Conclusion

In patients diagnosed with cancer, the duration of storage of transfused RBC had no impact on OS or cancer recurrence. This suggests that our current RBC storage policy of providing RBC of variable duration of storage for patients with malignancy is safe.     

Hydraulic selection pressure-induced nitrifying granulation in sequencing batch reactors

The effect of hydraulic selection pressure on the development of nitrifying granules was investigated in four column-type sequencing batch reactors (SBR). The nature of SBR is cycle operation, thus SBR cycle time can serve as a main hydraulic selection pressure imposed on the microbial community in the system. No nitrifying granulation was observed in the SBR operated at the longest cycle time of 24 h, due to a very weak hydraulic selection pressure, while the washout of nitrifying sludge was found in the SBR run at the shortest cycle time of 3 h, and led to a failure of nitrifying granulation. Excellent nitrifying granules with a mean diameter of 0.25 mm and specific gravity of 1.014 were developed in a SBR operated at cycle times of 6 h and 12 h, respectively. The results further showed that a short cycle time would stimulate microbial activity, production of cell polysaccharides and also improve the cell hydrophobicity. These hydraulic selection pressure-induced microbial changes favour the formation of nitrifying granules. This work, probably for the first time, shows that nitrifying granules can be developed at a proper hydraulic selection pressure in terms of SBR cycle time. Nitrifying granulation is a novel biotechnology which has a great potential for wastewater nitrification.     

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.