Negligible Cell Death Research Articles

Clonal dynamics of surface-driven growing tissues.

The self-organization of cells into complex tissues relies on a tight coordination of cell behavior. Identifying the cellular processes driving tissue growth is key to understanding the emergence of tissue forms and devising targeted therapies for aberrant growth, such as in cancer. Inferring the mode of tissue growth, whether it is driven by cells on the surface or by cells in the bulk, is possible in cell culture experiments but difficult in most tissues in living organisms (in vivo). Genetic tracing experiments, where a subset of cells is labeled with inheritable markers, have become important experimental tools to study cell fate in vivo. Here we show that the mode of tissue growth is reflected in the size distribution of the progeny of marked cells. To this end, we derive the clone size distributions using analytical calculations in the limit of negligible cell migration and cell death, and we test our predictions with an agent-based stochastic sampling technique. We show that for surface-driven growth the clone size distribution takes a characteristic power-law form with an exponent determined by fluctuations of the tissue surface. Our results propose a possible way of determining the mode of tissue growth from genetic tracing experiments.

The role of Type I interferons in the pathogenesis of foot-and-mouth disease virus in cattle: A mathematical modelling analysis

Type I interferons (IFN) are the first line of immune response against infection. In this study, we explore the interaction between Type I IFN and foot-and-mouth disease virus (FMDV), focusing on the effect of this interaction on epithelial cell death. While several mathematical models have explored the interaction between interferon and viruses at a systemic level, with most of the work undertaken on influenza and hepatitis C, these cannot investigate why a virus such as FMDV causes extensive cell death in some epithelial tissues leading to the development of lesions, while other infected epithelial tissues exhibit negligible cell death. Our study shows how a model that includes epithelial tissue structure can explain the development of lesions in some tissues and their absence in others. Furthermore, we show how the site of viral entry in an epithelial tissue, the viral replication rate, IFN production, suppression of viral replication by IFN and IFN release by live cells, all have a major impact on results.

Numbers Matter: The Role of Cell Dose in the Treatment of Osteosarcoma Using Mesenchymal Stromal Cells as Cellular Vehicles

AbstractA promising approach enhancing osteosarcoma (OS) prognosis involves the combination of various techniques, such as chemo‐ and photodynamic therapy, delivered through nanocarriers for synergistic cell death. Among the potential candidates for improving drug accumulation at the tumor site, mesenchymal stromal cells (MSCs) exhibit a significant advantage due to their tumor‐homing ability and intracellular drug retention. This study evaluates the efficacy of chemo‐releasing and photoactive bimodal nanoparticles, kPCe6 NPs, delivered via MSCs. In vitro analyses show that cells internalize and retain kPCe6 NPs in a dose‐dependent manner and that kPCe6‐loaded cells induce massive tumor cell death in a tridimensional tumor model. Results from an in vivo orthotopic OS murine model show negligible tumor cell death upon peritumoral administration of two doses containing 106 loaded cells. To gain insight into this observation, this work investigates the role of cell dose in treatment efficacy. The results indicate that achieving a tumor reduction higher than 90% requires a substantial number of loaded cells, approximately 35% of the entire tumor mass, highlighting the criticality of the cell dose for the success of this therapeutic approach and its potential impact on clinical translation in OS patients, particularly when the number of tumor cells is limited.

Nanocomposites of Nitrogen-Doped Graphene Oxide and Manganese Oxide for Photodynamic Therapy and Magnetic Resonance Imaging

Cancer is a leading cause of death worldwide. Conventional methods of cancer treatment, including chemotherapy and radiotherapy, are associated with multiple side effects. Recently, photodynamic therapy (PDT) has emerged as an effective therapeutic modality for cancer treatment without adversely affecting normal tissue. In this study, we synthesized nitrogen doped graphene (NDG) and conjugated it with Mn3O4 nanoparticles to produce NDG-Mn3O4 nanocomposite with the aim of testing its bimodal performance including PDT and magnetic resonance imaging (MRI). We did not use any linker or binder for conjugation between NDG and Mn3O4, rather they were anchored by a milling process. The results of cell viability analysis showed that NDG-Mn3O4 nanocomposites caused significant cell death under laser irradiation, while control and Mn3O4 nanoparticles showed negligible cell death. We observed increased generation of singlet oxygen after exposure of NDG-Mn3O4 nanocomposites, which was directly proportional to the duration of laser irradiation. The results of MRI showed concentration dependent enhancement of signal intensity with an increasing concentration of NDG-Mn3O4 nanocomposites. In conclusion, NDG-Mn3O4 nanocomposites did not cause any cytotoxicity under physiological conditions. However, they produced significant and dose-dependent cytotoxicity in cancer cells after laser irradiation. NDG-Mn3O4 nanocomposites also exhibited concentration-dependent MRI contrast property, suggesting their possible application for cancer imaging. Further studies are warranted to test the theranostic potential of NDG-Mn3O4 nanocomposites using animal models of cancer.

Antimicrobial and Anticancer Activity of Corydalis solida

Aim: The present study, it was aimed to evaluate the bioactive properties of Corydalis solida.
 Material and Methods: In the study, the anticancer activity of ethanolic extracts prepared from C. solida was determined on HCT116 colon cancer, AGS gastric cancer and HepG2 hepatocellular carcinoma cell lines and HUVEC cells, healthy control cell line. Well diffusion method was used to determine the antimicrobial properties of solida. For this purpose, ethanolic extracts were used for antimicrobial activity against four bacterial isolates (Escherichia coli, Bacillus cereus, Staphylococcus aureus and Klebsiella oxytoca) and three yeast strains (Candida albicans, C. tropicalis and C .glabrata).
 Results: Corydalis solida plant extract produced significant antiproliferative effect in HCT116 (colon cancer), AGS (gastric cancer) and HepG2 (liver cancer) cell lines. This effect was more remarkable in the HepG2 cell line. In addition, negligible cell death in HUVEC cells indicated that the plant was not toxic to healthy cells. Plant extract application also caused significant Caspase-3, 8 and 9 activation in HepG2 and HCT116 cells, consistent with the antiproliferative effect. Antimicrobial studies have shown that the extract made inhibition zone on bacteria.
 Conclusion: In the study, it was determined that the ethanol extract of Corydalis solida had anticancer effect. In addition, the extract had inhibitory properties on bacteria. The data obtained from the study are qualified to support further pharmacological studies.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ Tcells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ Tcells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Polymer-Coated Nanoparticles for Therapeutic and Diagnostic Non-10B Enriched Polymer-Coated Boron Carbon Oxynitride (BCNO) Nanoparticles as Potent BNCT Drug.

Boron neutron capture therapy (BNCT) is a powerful and selective anti-cancer therapy utilizing 10B-enriched boron drugs. However, clinical advancement of BCNT is hampered by the insufficient loading of B-10 drugs throughout the solid tumor. Furthermore, the preparation of boron drugs for BNCT relies on the use of the costly B-10 enriched precursor. To overcome these challenges, polymer-coated boron carbon oxynitride (BCNO) nanoparticles, with ~30% of boron, were developed with enhanced biocompatibility, cell uptake, and tumoricidal effect via BNCT. Using the ALTS1C1 cancer cell line, the IC50 of the PEG@BCNO, bare, PEI@BCNO were determined to be 0.3 mg/mL, 0.1 mg/mL, and 0.05 mg/mL, respectively. As a proof-of-concept, the engineered non-10B enriched polymer-coated BCNO exhibited excellent anti-tumor effect via BNCT due to their high boron content per nanoparticle and due to the enhanced cellular internalization and retention compared to small molecular 10B-BPA drug. The astrocytoma ALTS1C1 cells treated with bare, polyethyleneimine-, and polyethylene glycol-coated BCNO exhibited an acute cell death of 24, 37, and 43%, respectively, upon 30 min of neutron irradiation compared to the negligible cell death in PBS-treated and non-irradiated cells. The radical approach proposed in this study addresses the expensive and complex issues of B-10 isotope enrichment process; thus, enabling the preparation of boron drugs at a significantly lower cost, which will facilitate the development of boron drugs for BNCT.

Novel Hydroxytyrosol-Donepezil Hybrids as Potential Antioxidant and Neuroprotective Agents.

It is well-accepted that the endogenous antioxidant protection system progressively decays in elderly people, and that the oxidative stress contributes to different neurodegenerative disorders such as Alzheimer’s Diseases (AD). The lower incidence of AD in countries which feature the Mediterranean Diet was associated to the high consumption of extra virgin olive oil and its polyphenolic fraction, in particular hydroxytyrosol. The protective role of these bio-phenols against oxidative stress, suggested that we combine their antioxidant/free radical scavenging activity with donepezil, an active ingredient which has just been approved for the treatment of AD. Different synthetic strategies were tested to conjugate the two different synthons in good yields. Additionally, a nitro-hydroxytyrosol derivative was synthesized to extend the application to other neurodegeneration inflammatory models. Then, their bioactivity was measured in different chemical and biological tests on a human neuroblastoma cell line (SHSY-5Y). Remarkable results on cell viability and the regulation of the redox state of cells were obtained. All hybrids showed negligible cell death under 1 μM and are stable and non toxic. Reactive oxygen species (ROS) measurements showed that the nitro-hybrid was the more effective one at reducing the ROS amount to physiological values. Then, in light of the bio-metal hypothesis of diverse neurodegenerative disorders, we tested these new compounds on the chelation properties of redox-active metals. The nitro-hybrid was able to chelate all of the tested metal cations, suggesting that we propose it as potential lead compound for a new class of neuroprotective antioxidant agents.

Intensification of pyrene degradation by native Candida tropicalis MTCC 184 with sonication: Kinetic and mechanistic investigation

Candida tropicalis is a well–known yeast strain for degradation of polycyclic aromatic hydrocarbons. In this paper, we have reported intensification of biodegradation of pyrene with sonication. This study has also attempted to deduce the mechanistic features of intensification induced by sonication. The initial statistical optimization of biodegradation parameters revealed following results: pH = 6, temperature = 33ºC, agitation speed = 164 rpm and % inoculum = 8.07% v/v and pyrene degradation of ~53%. Investigations of the yeast growth pattern on a wide range of salt concentrations revealed that the strain could withstand upto 20% NaCl concentration. The kinetics of pyrene removal followed the 1st order kinetics with rate constant of 0.0667 day−1 and half–life of 10.39 days. Optimum sonication duty cycle using flow cytometry was 10% with negligible cell death. Exposure of reaction mixture to 33 kHz sonication in log phase resulted in 30% augmentation in pyrene elimination. The expression of overall proteins present in the samples were examined by SDS–PAGE. This analysis revealed overexpression of catechol 2,3–dioxygenase (–meta route metabolism) in the ultrasound–treated samples. FE–SEM micrographs did not show any cell rupture or damage during sonication.

Exploring therapeutic potential of azelaic acid loaded NLCs for the treatment of acne vulgaris

Azelaic acid (AZA), a naturally occurring dicarboxylic acid is reported to be effective in management of mild to moderate acne vulgaris. However, incidence of dose-related side effect limits its therapeutic applicability. The study is an endeavour to establish the anti-acne potential of pre-formulated and optimized Azelaic acid (AZA) loaded nano-structured lipid carriers (NLCs). The current investigation comprise of characterization of optimized nano preparation (NLCs suspension) or nano formulation (aloe-vera based nanogel) for its skin retentability, skin compatibility, anti-inflammatory, cytotoxicity and anti-microbial potential. Confocal laser scanning microscopic (CLSM) studies of skin revealed an intense fluorescence concentrated over the deeper layers of the skin, in comparison to superficial fluorescence of marketed preparation, indicating the superior carrier abilities of NLCs to the target site (epidermal-dermal junction). No signs of redness, dryness/oedema was observed for AZA NLCs in Draize skin patch test and the results were comparable to that of control. Cytotoxicity studies conducted employing Saccharomyces cerevisiae model also demonstrated a negligible cell death (<10%) upon application of nano preparation, confirming their biocompatible nature. The topical application of nano-preparation on P.acnes infected mice ear, suggested its anti-inflammatory and antimicrobial properties. Thus, above studies highlighted NLCs gel to be a promising vehicle for enhancing the tissue targetability and efficacy of AZA without compromising its safety and therapeutic profile.

Synthesis and anticholinesterase activity of novel non-hepatotoxic naphthyridine-11-amine derivatives.

In the present study, 14 novel naphthyridine-11-amine derivatives were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. 12-(4-Fluorophenyl)-1,2,3,4,7,8,9,10-octahydrodibenzo[b,g][1, 8]naphthyridin-11-amine (4a) was found to be the most potent AChE inhibitor with IC50 value of 0.091µM, and 12-(2,3-dimethoxyphenyl)-1,2,3,4,7,8,9,10-octahydrodibenzo[b,g][1,8]naphthyridin-11-amine (4h) exhibited the strongest inhibition against BuChE with IC50 value of 0.182µM. Additionally, hepatocellular carcinoma (HepG2) cell cytotoxicity assay for the synthesized compounds was investigated and the results showed negligible cell death. Log P values of the synthesized compounds were also calculated using ChemSketch program. Moreover, the blood-brain barrier (BBB) permeability of the potent AChE inhibitor (4a) was assessed by the widely used parallel artificial membrane permeability assay (PAMPA-BBB). The results showed that 4a is capable of crossing the BBB.

Green Reduced Graphene Oxide Toughened Semi-IPN Monolith Hydrogel as Dual Responsive Drug Release System: Rheological, Physicomechanical, and Electrical Evaluations.

Macroporous hydrogel monoliths having tailor-made features, conductivity, superstretchability, excellent biocompatibility, and biodegradability, have become the most nurtured field of interest in soft biomaterials. Green method assisted reduced graphene oxide has been inserted by in situ free radical gelation into semi-IPN hydrogel matrix to fabricate conducting hydrogel. Mechanical toughness has been implemented for the graphene-polymer physisorption interactions with graphene basal planes. Moreover, the as-prepared 3D scaffold type monolith hydrogel has been rheologically superior regarding their high elastic modulus and delayed gel rupturing. κ-Carragenaan, one of the components of the hydrogel, has biodegradable nature. The most significant outcome is their low electrical percolation threshold and reversibly ductile nature. Reversible ductility provides them with rubber-like consistency in flow conditions. Surprising, the hydrogels showed dual stimuli-responsiveness, that is, environmental pH and external electrical stimulation. Electro-stimulation has been adopted here for the first time in semi-IPN systems, which could be an ideal alternative for iontopheretic devices and pulsatile drug release through skin. Regarding this, the hydrogel also has been passed to biocompatibility assay; they are noncytotoxic and show cell proliferation without negligible cell death in live-dead assay. The porosity of the nanocomposite scaffold-like gels was also analyzed by microcomputed tomography (μ-CT), which exhibited their connectivity in cell/voids inside the matrix. Thus, the experimentations are on the support of biocompatible soft material for dual-responsive tunable drug delivery.

Vulnerability of corneal endothelial cells to mechanical trauma from indentation forces assessed using contact mechanics and fluorescence microscopy

Corneal endothelial cell (CEC) loss occurs from tissue manipulation during anterior segment surgery and corneal transplantation as well as from contact with synthetic materials like intraocular lenses and tube shunts. While several studies have quantified CEC loss for specific surgical steps, the vulnerability of CECs to isolated, controllable and measurable mechanical forces has not been assessed previously. The purpose of this study was to develop an experimental testing platform where the susceptibility of CECs to controlled mechanical trauma could be measured. The corneal endothelial surfaces of freshly dissected porcine corneas were subjected to a range of indentation forces via a spherical stainless steel bead. A cell viability assay in combination with high-resolution fluorescence microscopy was used to visualize and quantify injured/dead CEC densities before and after mechanical loading. In specimens subjected to an indentation force of 9 mN, the mean ± SD peak contact pressure P0 was 18.64 ± 3.59 kPa (139.81 ± 26.93 mmHg) in the center of indentation and decreased radially outward. Injured/dead CEC densities were significantly greater (p ≤ 0.001) after mechanical indentation of 9 mN (167 ± 97 cells/mm2) compared to before indentation (39 ± 52 cells/mm2) and compared to the sham group (34 ± 31 cells/mm2). In specimens subjected to “contact only” – defined as an applied indentation force of 0.65 mN – the peak contact pressure P0 was 7.31 ± 1.5 kPa (54.83 ± 11.25 mmHg). In regions where the contact pressures was below 78% of P0 (<5.7 kPa or 42.75 mmHg), injured/dead CEC densities were within the range of CEC loss observed in the sham group, suggesting negligible cell death. These findings indicate that CECs are highly susceptible to mechanical trauma via indentation, supporting the established “no-touch” policy for ophthalmological procedures. While CECs can potentially remain viable below contact pressures of 5.7 kPa (42.75 mmHg), this low threshold suggests that prevention of indentation-associated CEC loss may be challenging.

Fluorescent Carbon Dots Derived from Maillard Reaction Products: Their Properties, Biodistribution, Cytotoxicity, and Antioxidant Activity.

Food-borne nanoparticles have received great attention because of their unique physicochemical properties and potential health risk. In this study, carbon dots (CDs) formed during one of the most important chemical reactions in the food processing field, the Maillard reaction from the model system including glucose and lysine, were investigated. The CDs purified from Maillard reaction products emitted a strong blue fluorescence under ultraviolet light with a fluorescent quantum yield of 16.30%. In addition, they were roughly spherical, with sizes of around 4.3 nm, and mainly composed of carbon, oxygen, hydrogen, and nitrogen. Their surface groups such as hydroxyl, amino, and carboxyl groups were found to possibly enable CDs to scavenge DPPH and hydroxyl radicals. Furthermore, the cytotoxicity assessment of CDs showed that they could readily enter HepG2 cells while causing negligible cell death at low concentration. However, high CDs concentrations were highly cytotoxic and led to cell death via interference of the glycolytic pathway.

Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population.

SummaryTaxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer.

Reduction of pulmonary toxicity of metal oxide nanoparticles by phosphonate-based surface passivation

BackgroundThe wide application of engineered nanoparticles has induced increasing exposure to humans and environment, which led to substantial concerns on their biosafety. Some metal oxides (MOx) have shown severe toxicity in cells and animals, thus safe designs of MOx with reduced hazard potential are desired. Currently, there is a lack of a simple yet effective safe design approach for the toxic MOx. In this study, we determined the key physicochemical properties of MOx that lead to cytotoxicity and explored a safe design approach for toxic MOx by modifying their hazard properties.ResultsTHP-1 and BEAS-2B cells were exposed to 0–200 μg/mL MOx for 24 h, we found some toxic MOx including CoO, CuO, Ni2O3 and Co3O4, could induce reactive oxygen species (ROS) generation and cell death due to the toxic ion shedding and/or oxidative stress generation from the active surface of MOx internalized into lysosomes. We thus hypothesized that surface passivation could reduce or eliminate the toxicity of MOx. We experimented with a series of surface coating molecules and discovered that ethylenediamine tetra (methylene phosphonic acid) (EDTMP) could form stable hexadentate coordination with MOx. The coating layer can effectively reduce the surface activity of MOx with 85-99% decrease of oxidative potential, and 65-98% decrease of ion shedding. The EDTMP coated MOx show negligible ROS generation and cell death in THP-1 and BEAS-2B cells. The protective effect of EDTMP coating was further validated in mouse lungs exposed to 2 mg/kg MOx by oropharyngeal aspiration. After 40 h exposure, EDTMP coated MOx show significant decreases of neutrophil counts, lactate dehydrogenase (LDH) release, MCP-1, LIX and IL-6 in bronchoalveolar lavage fluid (BALF), compared to uncoated particles. The haematoxylin and eosin (H&E) staining results of lung tissue also show EDTMP coating could significantly reduce the pulmonary inflammation of MOx.ConclusionsThe surface reactivity of MOx including ion shedding and oxidative potential is the dominated physicochemical property that is responsible for the cytotoxicity induced by MOx. EDTMP coating could passivate the surface of MOx, reduce their cytotoxicity and pulmonary hazard effects. This coating would be an effective safe design approach for a broad spectrum of toxic MOx, which will facilitate the safe use of MOx in commercial nanoproducts.

Design, synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC50 value of 43 nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.

Synthesis, anticholinesterase activity and molecular modeling study of novel carbamate-substituted thymol/carvacrol derivatives

New thymol and carvacrol derivatives with the carbamate moiety were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. 5-isopropyl-2-methylphenyl(3-fluorophenyl)carbamate (29) was found to be the most potent AChE inhibitor with IC50 values of 2.22μM, and 5-isopropyl-2-methylphenyl (4-fluorophenyl)carbamate (30) exhibited the strongest inhibition against BuChE with IC50 value of 0.02μM. Additionally, the result of H4IIE hepatoma cell toxicity assay for compounds 18, 20, 29, 30 and 35 showed negligible cell death at 0.07–10μM. Moreover in order to better understand the inhibitory profiles of these molecules, molecular modeling studies were applied. Binding poses of studied compounds at the binding pockets of AChE and BuChE targets were determined. Predicted binding energies of these compounds as well as structural and dynamical profiles of molecules at the target sites were estimated using induced fit docking (IFD) algorithms and post-processing molecular dynamics (MD) simulations methods (i.e., Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) approaches).

Endogenous metabolism of anaerobic ammonium oxidizing bacteria in response to short-term anaerobic and anoxic starvation stress

Under starvation conditions, bacteria could consume intracellular macromolecules, which deteriorates the performance of the bioreactor. However, the fundamental endogenous metabolism of anaerobic ammonium oxidation (anammox) bacteria is poorly understood. Here, we describe the endogenous metabolism of anammox bacteria biomass under short-term anaerobic and anoxic starvation stress (4.5d and 40h, respectively) by protein, mRNA and biochemical analyses. The live/dead staining results showed negligible cell death rates. The activity decay rates (decrease in specific activity of active bacteria) were 0.128d−1 and 0.629d−1 under anaerobic and anoxic starvation conditions, respectively. The degradation rates of extracellular polymeric substances (EPS) and hydrazine dehydrogenase protein (HDH) were approximately 4- and 5-fold higher under anoxic starvation than under anaerobic starvation tests, suggesting the electron-donor roles for EPS and HDH in denitrification. The greater consumption of intracellular macromolecules during anoxic starvation corresponded with the greater decrease in anammox activities under this condition. Under both starvation conditions, the mRNA decay rates of nirS, hzsA, and hdh mRNA (representing metabolic stability) of anammox bacteria decreased from their normal levels. Such control of specific mRNAs is important for economizing protein synthesis and aids the cells’ recovery after starvation stress. These identified endogenous characteristics of anammox bacteria provide new insights into starvation stress responses at the transcriptional and translational levels, and will assist the optimization of anammox processes in bioreactors, which frequently experience starvation conditions.

Locally-concentrated sonicated microbubbles: Potential as an antibiotic

While the antimicrobial effects of ultrasound have been well established, application as a bactericide in medicine has been hindered in part by significant variation in reported efficacy. Non-thermal mechanisms of cell death are generally attributed to cavitation, prompting us to investigate the potential for locally applied microbubbles to serve as a catalyst for reliably increasing bacterial death, while sparing surrounding cells. This preliminary study was limited to gram-negative bacteria (E-coli) suspended in buffered saline. Preliminary work at 40 kHz continuous-wave sonication established a threshold time for our setup of 15 minutes, below which negligible cell death was observed relative to control samples. Experiments were then performed with and without the introduction of octofluropropane-filled lipid shells microbubbles, sonicated over the 15 minute period. Thermometry and passive cavitation monitoring (PCD) were both conducted. Analysis by dilution and plating indicate a 65-80% reduction in b...