MiR-34b promotes oxidative stress and induces cellular senescence through TWIST1 in human cervical cancer.

PURPOSE: The aim of this research was to elucidate the role of miR-34b in cervical cancer progression and the underlying mechanism behind the miR-34b-mediated tumor suppression. The study revealed the role of miR-34b as a senescence inducer and serves as a potential therapeutic target in developing combination therapy with senotherapeutics. METHODS: MiR-34b was ectopically expressed in cervical cancer cell lines using a tetracycline inducible system and its effects on cell viability, apoptosis, senescence, DNA damage and oxidative stress were studied using MTT assay, acridine orange/ ethidium bromide staining, senescence associated ß-galactosidase assay, gamma H2AX foci staining assay, western blotting and specific dyes for the detection of total and individual ROS species. RESULTS: Ectopic expression of miR-34b promoted cellular senescence but no significant induction of apoptosis was observed in cervical cancer cell lines. MiR-34b promoted increase in oxidative stress through increase in total and individual ROS species and contributed to increase in cellular senescence. Mechanistically, miR-34b mediates its action by targeting TWIST1 as evidenced by the similar actions of TWIST1 shRNA in cervical cancer cell lines. Furthermore, our study revealed TWIST1 is one of the most significant targets of miR-34b targetome and identified RITA as a novel senolytic agent for use in combination therapy with miR-34b. CONCLUSION: MiR-34b promotes cellular senescence and oxidative stress by targeting TWIST1, a known oncogene and EMT regulator. This study delved into the mechanism of miR-34b-mediated tumor suppression and provided novel insights for development of miR-34b based therapeutics for cervical cancer.

Shear stress and microRNAs for better metastatic cancer management.

Metastasis is the process by which cancer cells move from the primary location to establish themselves in a new location in the human body. It is still a significant challenge in cancer management because it is responsible for 90% of cancer-related deaths. In this work, we present an idea to use shear stress encountered by all metastasizing cells as an elegant means to deactivate metastasizing cancer cells. Shear-induced ROS and cross-talk between ROS and miRNA play crucial roles in deactivating metastasizing cancer cells. In addition, there exists a vast therapeutic potential for miRNAs. Therefore, this study explores the effect of shear on miRNAs and reactive oxygen species (ROS), the two molecular mediators in the proposed {shear-stress}-{miRNA}-{metastasizing-cancer-cell-deactivation} approach. In this context, to understand the effect of defined shear on HCT116 colon cancer cells, they were cultivated in a defined shear environment provided by an appropriately designed and fabricated cone-and-plate device. Shear rate affected the culture growth characteristics and the specific intracellular reactive oxygen species level (si-ROS). HCT116 cell growth was observed at 0 and 0.63 s-1 but not at 1.57 s-1 or beyond. Shear rate induced upregulation of the hsa-miR-335-5p but induced downregulation of hsa-miR-34a-5p. Furthermore, the specific levels of hsa-miR-335-5p, hsa-miR-26b-5p, and hsa-miR-34a-5p negatively correlated with specific intracellular (si)-hydroxyl radical levels. In addition, some messenger RNAs (mRNAs) in HCT116 cells showed a differential expression under shear stress, notably the ROS-associated mRNA of PMAIP1. The above miRNAs (and possibly some mRNAs) could be targeted to manage colon cancer metastasis.

A reactive species reactions module for integration into genome-scale metabolic models for improved insights: Application to cancer.

Reactive species (RS) play significant roles in many disease contexts. Despite their crucial roles in diseases including cancer, the RS are not adequately modeled in the genome-scale metabolic (GSM) models, which are used to understand cell metabolism in disease contexts. We have developed a scalable RS reactions module that can be integrated with any Recon 3D-derived human metabolic model, or after fine-tuning, with any metabolic model. With RS-integration, the GSM models of three cancers (basal-like triple negative breast cancer (TNBC), high grade serous ovarian carcinoma (HGSOC) and colorectal cancer (CRC)) built from Recon 3D, precisely highlighted the increases/decreases in fluxes (dysregulation) occurring in important pathways of these cancers. These dysregulations were not prominent in the standard cancer models without the RS module. Further, the results from these RS-integrated cancer GSM models suggest the following decreasing order in the ease of ferroptosis-targeting to treat the cancers: TNBC > HGSOC > CRC.

An integrated approach to understand fluid shear stress-driven and reactive oxygen species-mediated metastasis of colon adenocarcinoma through mRNA-miRNA-lncRNA-circRNA networks.

Development of colon adenocarcinoma (COAD) metastasis involves several mediators including fluid shear stress (FSS), intracellular ROS levels, and non-coding RNAs. In our present study, we identified and investigated the role of regulatory non-coding RNA molecules specifically involved in COAD metastasis and their association with FSS and ROS. Interactions between the mRNAs associated with FSS and ROS, the corresponding microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in COAD metastasis were used to generate the mRNA-miRNA-lncRNA-circRNA network. Experimental validation of the identified RNA hubs using quantitative real-time PCR demonstrated a direct effect of the FSS on their expression levels in cancer cells. FSS resulted in the downregulation of HMGA1 and RAN, as well as the upregulation of HSP90AA1, PMAIP1 and BIRC5. Application of shear stress also led to downregulation of hsa-miR-26b-5p and hsa-miR-34a-5p levels in HCT116 cells. Further, functional enrichment and survival analysis of the significant miRNAs, as well as the OncoPrint and the survival analyses of the selected mRNAs were performed. Subsequently, their functional role was also corroborated with existing literature. Ten significant miRNA hubs were identified, out of which hsa-miR-17-5p and hsa-miR-20a-5p were found to interact with lncRNA (CCAT2) while hsa-miR-335 was found to interact with four circRNAs. Fifteen significant miRNAs were identified in 10 different modules suggesting their importance in FSS and ROS-mediated COAD metastasis. Finally, 10 miRNAs and 3 mRNAs associated with FSS and/or ROS were identified as significant overall survival markers; 33 mRNAs were also identified as metastasis-free survival markers whereas 15 mRNAs showed > 10% gene alterations in TCGA-COAD data and may serve as promising therapeutic biomarkers in the COAD metastasis.

Metabolic modeling of host-microbe interactions for therapeutics in colorectal cancer.

The onset of colorectal cancer (CRC) is often attributed to gut bacterial dysbiosis, and thus gut microbiota are highly relevant in devising treatment strategies. Certain gut microbes, like Enterococcus spp., exhibit remarkable anti-neoplastic and probiotic properties, which can aid in silver nanoparticle (AgNPs) induced reactive oxygen species (ROS)-based CRC treatment. However, the effects of AgNPs on gut microbial metabolism have not been reported thus far. In this study, a detailed systems-level understanding of ROS metabolism in Enterococcus durans (E. durans), a representative gut microbe, was gained using constraint-based modeling, wherein, the critical association between ROS and folate metabolism was established. Experimental studies involving low AgNP concentration treatment of E. durans cultures confirmed these modeling predictions (an increased extracellular folate concentration by 52%, at the 9th h of microbial growth, was observed). Besides, the computational studies established various metabolic pathways involving amino acids, energy metabolites, nucleotides, and SCFAs as the key players in elevating folate levels on ROS exposure. The anti-cancer potential of E. durans was also studied through MTT analysis of HCT 116 cells treated with microbial culture (AgNP treated) supernatant. A decrease in cell viability by 19% implicated the role of microbial metabolites (primarily folate) in causing cell death. The genome-scale modeling approach was then extended to extensively model CRC metabolism, as well as CRC-E. durans interactions in the context of CRC treatment, using tissue-specific metabolic models of CRC and healthy colon. These findings on further validation can facilitate the development of robust and effective cancer therapy.

Polystyrene nanoplastics diminish the toxic effects of Nano-TiO2 in marine algae Chlorella sp.

Widespread usage of nano-TiO2 in various commercial products and their consequent release into the seawater pose a severe threat to marine biota. Nanoplastics, a secondary pollutant in the marine environment, could influence adverse effects of nano-TiO2. The main goal of the present study was to investigate the influence of the differently functionalized polystyrene nanoplastics (COOH-PSNPs, NH2-PSNPs, and Plain-PSNPs) on the acute toxic effects of P25 nano-TiO2 in marine algae Chlorella sp. Three different concentrations of nano-TiO2, 0.25, 0.5, and 1 mg/L, mixed with 5 mg/L of the PSNPs were employed in this study. A substantial increase was noted in mean hydrodynamic sizes of nano-TiO2 when they were mixed with the PSNPs. This hetero-aggregation would reduce the bioavailability of the particles to the algae. The presence of the PSNPs in the mixture reduced the toxicity of nano-TiO2 significantly. A signficant decline in the oxidative stress parameters like total ROS, superoxide (), and hydroxyl radical generation was noted for the mixture of nano-TiO2 with the PSNPs in comparison with the pristine counterparts. The lipid peroxidation, and the antioxidant enzyme activities in the cells correlated well with the reactive species generation results. The treatments with the mixture resulted in notable enhancement in the esterase activity in the cells. The Independent Action model suggested antagonistic interactions between PSNPs and nano-TiO2. The results from this study clearly demonstrate that nano-TiO2 in presence of the PSNPs exerted significantly reduced cytotoxic effects in Chlorella sp, in comparison with the pristine particles.

Influence of native ureolytic microbial community on biocementation potential of Sporosarcina pasteurii.

Microbially induced calcium carbonate precipitation (MICP)/Biocementation has emerged as a promising technique for soil engineering applications. There are chiefly two methods by which MICP is applied for field applications including biostimulation and bioaugmentation. Although bioaugmentation strategy using efficient ureolytic biocementing culture of Sporosarcina pasteurii is widely practiced, the impact of native ureolytic microbial communities (NUMC) on CaCO3 mineralisation via S. pasteurii has not been explored. In this paper, we investigated the effect of different concentrations of NUMC on MICP kinetics and biomineral properties in the presence and absence of S. pasteurii. Kinetic analysis showed that the biocementation potential of S. pasteurii is sixfold higher than NUMC and is not significantly impacted even when the concentration of the NUMC is eight times higher. Micrographic results revealed a quick rate of CaCO3 precipitation by S. pasteurii leading to generation of smaller CaCO3 crystals (5-40 µm), while slow rate of CaCO3 precipitation by NUMC led to creation of larger CaCO3 crystals (35-100 µm). Mineralogical results showed the predominance of calcite phase in both sets. The outcome of current study is crucial for tailor-made applications of MICP.

Insights into the influence of cell concentration in design and development of microbially induced calcium carbonate precipitation (MICP) process.

Microbially induced calcium carbonate precipitation (MICP) process utilising the biogeochemical reactions for low energy cementation has recently emerged as a potential technology for numerous engineering applications. The design and development of an efficient MICP process depends upon several physicochemical and biological variables; amongst which the initial bacterial cell concentration is a major factor. The goal of this study is to assess the impact of initial bacterial cell concentration on ureolysis and carbonate precipitation kinetics along with its influence on the calcium carbonate crystal properties; as all these factors determine the efficacy of this process for specific engineering applications. We have also investigated the role of subsequent cell recharge in calcium carbonate precipitation kinetics for the first time. Experimental results showed that the kinetics of ureolysis and calcium carbonate precipitation are well-fitted by an exponential logistic equation for cell concentrations between optical density range of 0.1 OD to 0.4 OD. This equation is highly applicable for designing the optimal processes for microbially cemented soil stabilization applications using native or augmented bacterial cultures. Multiple recharge kinetics study revealed that the addition of fresh bacterial cells is an essential step to keep the fast rate of precipitation, as desirable in certain applications. Our results of calcium carbonate crystal morphology and mineralogy via scanning electron micrography, energy dispersive X-ray spectroscopy and X-ray diffraction analysis exhibited a notable impact of cell number and extracellular urease concentration on the properties of carbonate crystals. Lower cell numbers led to formation of larger crystals compared to high cell numbers and these crystals transform from vaterite phase to the calcite phase over time. This study has demonstrated the significance of kinetic models for designing large-scale MICP applications.

Eco-corona formation lessens the toxic effects of polystyrene nanoplastics towards marine microalgae Chlorella sp.

Unabated use of nanoplastics (<1 µm) in the consumer products and their consequent release to the marine environment poses a substantial threat to the marine ecosystem. The toxic impact of the nanoplastics on marine microalgae is yet to be explored in detail, and the role of reactive oxygen species generation remains largely unclear. The algal exudates constitute a significant part of the natural organics present in the marine system that may readily adsorb over the nanoplastics to form eco-corona. In the current work a marine alga, Chlorella sp., was considered a bioindicator organism and the effects of eco-corona formation in lessening the toxic impact of the nanoplastics was analyzed. Three differently functionalized polystyrene nanoplastics (PS NPs): Aminated (NH2-PS NPs), Carboxylated (COOH-PS NPs) and Plain nanoplastics were aged (12, 24, and 48 h) in the EPS containing medium to facilitate eco-corona formation. Decline in cell viability, membrane integrity, and photosynthetic yield were considered to be principle toxicity indicators. The role of oxidative stress as key mode of action (MOA) was studied considering generation of overall reactive oxygen species, and specific radicals (hydroxyl and superoxide) as relevant markers. The changes in antioxidant enzyme activities (superoxide dismutase, and catalase) were also measured. The results clearly indicate a significant decline in the oxidative stress and corresponding lessening of the toxic effects due to eco-corona formation on the PS NPs. The response varied with surface charge on the NPs and ageing duration. Considering the increasing importance of the nanoplastics as one of the major emerging pollutants in marine ecosystem, this study strongly suggests that the EPS mediated eco-corona formation may substantially lessen their toxic burden.

Mechanism of the oxidative stress-mediated increase in lipid accumulation by the bacterium, R. opacus PD630: Experimental analysis and genome-scale metabolic modeling.

Appropriate species of oleaginous bacteria, with their high growth rates and lipid accumulation capabilities, can be good contenders for industrial triacylglycerol (TAG) production, compared to microalgae. Further, oxidative stress (OS) can be used to significantly increase TAG yields in oleaginous microbes, but the mechanism is unexplored. In a first, this study explored the mechanism behind OS-mediated increase in TAG accumulation by the bacterium, Rhodococccus opacus PD630, through experimental analysis and metabolic modelling. Two mechanisms that could increase acetyl-CoA (TAG-precursor) levels were hypothesized based on literature information. One was OS-mediated inactivation of the aconitase (TCA cycle), and another was the inactivation of the triosephosphate isomerase (TPI; glycolysis). The results negated the involvement of aconitase in increased acetyl-CoA levels. Analysis of the metabolic model showed that inactivation of TPI, re-routed the flux through the pentose phosphate pathway (PPP), supplying both NADPH and acetyl-CoA for TAG synthesis. Additionally, inactivation of TPI increased TAG flux by 143%, whereas, inactivating both TPI and aconitase, increased it by 152%. We present experimental evidence for OS-mediated decrease in TPI activity and increase in activity of glucose-6-phosphate dehydrogenase (PPP enzyme). The findings indicate that increased flux through PPP can be explored to improve TAG accumulation on a large-scale.

Improved redox anti-cancer treatment efficacy through reactive species rhythm manipulation.

Rhythms in the pseudo-steady state (PSS) levels of reactive species (RS), particularly superoxide and hydroxyl radicals, exist in cancer cells. The RS rhythm characteristics, particularly frequency and amplitude, are entrained (reset) by the anticancer compounds/drugs. In this work, we show for the first time that the phase of the RS rhythm at which the drug is added is significantly important in determining the cytotoxicity of anticancer compounds/drugs such as menadione and curcumin, in two different cancer cell lines. Curcumin, the more effective of the two drugs (IC50 = 15 µM, SiHa; 6 µM, HCT116) induced reset of superoxide and hydroxyl rhythms from 15.4 h to 9 h, and 25 h to 11 h respectively, as well as caused increases in these radical levels. However, menadione (IC50 = 20 µM, SiHa; 17 µM, HCT116) affected only the superoxide levels. Drug treatment at different time points/phase of the RS rhythm resulted in a maximum of 27% increase in cytotoxicity, which is significant. Further, we report for the first time, an unexpected absence of a correlation between the intracellular PSS RS and antioxidant levels; thus, the practice of using antioxidant enzyme levels as surrogate markers of intracellular oxidative stress levels may need a re-consideration. Therefore, the RS rhythm could be a fundamental/generic target to manipulate for improved cancer therapy.

Entrainment of superoxide rhythm by menadione in HCT116 colon cancer cells.

Reactive oxygen species (ROS) are primary effectors of cytotoxicity induced by many anti-cancer drugs. Rhythms in the pseudo-steady-state (PSS) levels of particular intracellular ROS in cancer cells and their relevance to drug effectiveness are unknown thus far. We report that the PSS levels of intracellular superoxide (SOX), an important ROS, exhibit an inherent rhythm in HCT116 colon cancer cells, which is entrained (reset) by the SOX inducer, menadione (MD). This reset was dependent on the expression of p53, and it doubled the sensitivity of the cells to MD. The period of oscillation was found to have a linear correlation with MD concentration, given by the equation, T, in h = 23.52 - 1.05 [MD concentration in µM]. Further, we developed a mathematical model to better understand the molecular mechanisms involved in rhythm reset. Biologically meaningful parameters were obtained through parameter estimation techniques; the model can predict experimental profiles of SOX, establish qualitative relations between interacting species in the system and serves as an important tool to understand the profiles of various species. The model was also able to successfully predict the rhythm reset in MD treated hepatoma cell line, HepG2.

Oxidative stress decreases the redox ratio and folate content in the gut microbe, Enterococcus durans (MTCC 3031).

Gut microbiome plays an important role in determining the effectiveness of cancer therapy. The composition of the microbiome is crucial to maintain good digestive health in the host, and to prevent and treat colorectal cancers. Most cancer therapies employ oxidative stress, which disturbs the redox status of the cell, and consequently affect growth, reductive biosynthesis and cell death. Therefore, oxidative stress can undesirably affect the gut microbiome. Hence, it is important to understand the impact of oxidative stress on gut bacteria to devise effective treatment strategies. The current study induces oxidative stress in the model gut bacterium Enterococcus durans (MTCC 3031) with menadione and H2O2. Oxidative stress considerably decreased the redox ratio (NADPH/NADP), an indicator of the redox status, by 55% (menadione) and 28% (H2O2). In addition, an oxidative stress induced decrease in redox ratio decreased folate synthesis by the bacteria, which is an undesirable consequence for the host, since folate deficiency can induce colorectal cancer. Further, oxidative stress considerably decreased growth and the biomass density by 61% (menadione) and 21% (H2O2). Thus, maintenance of the cellular redox status and management of oxidative stress in the gut microbiome may be crucial to the effectiveness of cancer treatment strategies.

Effect of light on the kinetics and equilibrium of the textile dye (Reactive Red 120) adsorption by Helianthus annuus hairy roots.

The study demonstrates for the first time that light influences the adsorption equilibrium and kinetics of a dye by root culture system. The azo dye (Reactive Red 120) adsorption by the hairy roots of H. annuus followed a pseudo first-order kinetic model and the adsorption equilibrium parameters were best estimated using Langmuir isotherm. The maximum dye adsorption capacity of the roots increased 6-fold, from 0.26 mg g-1 under complete dark conditions to 1.51 mg g-1 under 16/8 h light/dark photoperiod. Similarly, adsorption rate of the dye and removal (%) also increased in the presence of light, irrespective of the initial concentration of the dye (20-110 mg L-1). The degradation of the azo dye upon adsorption by the hairy roots of H. annuus was also confirmed. In addition, a strategy for simultaneous dye removal and increased alpha-tocopherol (industrially relevant) production by H. annuus hairy root cultures has been proposed and demonstrated.

Chitosan-agarose scaffolds supports chondrogenesis of Human Wharton's Jelly mesenchymal stem cells.

Tissue engineering strategies for cartilage aim to restore the complex biomechanical and biochemical properties of the native cartilage. To mimic the in vivo microenvironment, we developed a novel scaffold based on chitosan-agarose (CHAG scaffold) resembling the properties of native cartilage extracellular matrix (ECM) that aids in vitro cartilage formation. The CHAG scaffolds had pore size ranging from 75 to 300 µm and the degradation of 18% over 6 months in PBS. L929 cells and Human Wharton's Jelly-Mesenchymal Stem Cells (HWJ-MSCs) attached well and grew in the CHAG scaffolds. HWJ-MSCs seeded on CHAG scaffolds and cultured in chondrogenic medium were able to differentiate into chondrogenic lineage. Simultaneous supplementation of growth factors (BMP-2, TGF-ß3) significantly enhanced chondrogenesis and neo ECM synthesis. CHAG scaffolds seeded with HWJ-MSCs cultured in chondrogenic media supplemented with both BMP-2 and TGF-ß3 produced 12.71 ± 1.0 µg GAG/µg DNA compared to the one which received no or either of the growth factors. Our findings suggest that CHAG scaffolds could be used as a biomaterial scaffold for cell mediated repair approaches based on HWJ-MSCs for articular cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1845-1855, 2017.

UVA-induced reset of hydroxyl radical ultradian rhythm improves temporal lipid production in Chlorella vulgaris.

We report for the first time that the endogenous, pseudo-steady-state, specific intracellular levels of the hydroxyl radical (si-OH) oscillate in an ultradian fashion (model system: the microalga, Chlorella vulgaris), and also characterize the various rhythm parameters. The ultradian rhythm in the endogenous levels of the si-OH occurred with an approximately 6 h period in the daily cycle of light and darkness. Further, we expected that the rhythm reset to a shorter period could rapidly switch the cellular redox states that could favor lipid accumulation. We reset the endogenous rhythm through entrainment with UVA radiation, and generated two new ultradian rhythms with periods of approximately 2.97 h and 3.8 h in the light phase and dark phase, respectively. The reset increased the window of maximum lipid accumulation from 6 h to 12 h concomitant with the onset of the ultradian rhythms. Further, the saturated fatty acid content increased approximately to 80% of total lipid content, corresponding to the peak maxima of the hydroxyl radical levels in the reset rhythm.

Simultaneous increases in specific growth rate and specific lipid content of Chlorella vulgaris through UV-induced reactive species.

A challenge in algae-based bio-oil production is to simultaneously enhance specific growth rates and specific lipid content. We have demonstrated simultaneous increases in both the above in Chlorella vulgaris through reactive species (RS) induced under ultraviolet (UV) A and UVB light treatments. We postulated that the changes in photosystem (PS) stoichiometry and antenna size were responsible for the increases in specific growth rate. UVB treatment excited PSII, which resulted in a twofold to sevenfold increase in PSII/PSI ratio compared to control. An excited PSII caused a 2.7-fold increase in the specific levels of superoxide and a twofold increase in the specific levels of hydroxyl radicals. We have established that the increased specific intracellular RS (si-RS) levels increased the PSII antenna size by a significant 10-fold as compared to control. In addition, the 8.2-fold increase in specific lipid content was directly related to the si-RS levels. We have also demonstrated that the RS induced under UVA treatment led to a 3.2-fold increase in the saturated to unsaturated fatty acid ratio. Based on the findings, we have proposed and demonstrated a UV-based strategy, which achieved an 8.8-fold increase in volumetric lipid productivity.

Stress induced lipid production in Chlorella vulgaris: relationship with specific intracellular reactive species levels.

Microalgae have significant potential to be an important alternative energy source, but the challenges to the commercialization of bio-oil from microalgae need to be overcome for the potential to be realized. The application of stress can be used to improve bio-oil yields from algae. Nevertheless, the understanding of stress effects is fragmented due to the lack of a suitable, direct quantitative marker for stress. The lack of understanding seems to have limited the development of stress based strategies to improve bio-oil yields, and hence the commercialization of microalgae-based bio-oil. In this study, we have proposed and used the specific intracellular reactive species levels (siROS) particularly hydroxyl and superoxide radical levels, separately, as direct, quantitative, markers for stress, irrespective of the type of stress induced. Although ROS reactions are extremely rapid, the siROS level can be assumed to be at pseudo-steady state compared to the time scales of metabolism, growth and production, and hence they can be effective stress markers at particular time points. Also, the specific intracellular (si-) hydroxyl and superoxide radical levels are easy to measure through fluorimetry. Interestingly, irrespective of the conditions employed in this study, that is, nutrient excess/limitation or different light wavelengths, the cell concentrations are correlated to the siROS levels in an inverse power law fashion. The composite plots of cell concentration (y) and siROS (x) yielded the correlations of y = k1 · x(-0.7) and y = k2 · x(-0.79) , for si-hydroxyl and si-superoxide radical levels, respectively. The specific intracellular (si-) neutral lipid levels, which determine the bio-oil productivity, are related in a direct power law fashion to the specific hydroxyl radical levels. The composite plot of si-neutral lipid levels (z) and si-hydroxyl radical level (x) yielded a correlation of z = k3 · x(0.65) . More interestingly, a nutrient shift caused a significant change in the sensitivity of neutral lipid accumulation to the si-hydroxyl radical levels.

Influence of viscoelasticity of protein on the toughness of bone.

Bone is an ultrafine composite of protein (collagen) and mineral (hydroxyapatite). An analysis to determine the influence of the viscoelasticity of protein on the toughness of bone at the ultrafine scale is conducted by developing a discrete lattice model appropriate for the ultrafine scale called the incremental continuous damage random fuse model (ICDRFM). Collagen viscoelasticity at ultrafine scale is shown to contribute significantly to the toughness of bone. The results obtained are important in the design of biomimetic ultratough artificial composites.

High hydrogen peroxide concentration in the feed-zone affects bioreactor cell productivity with liquid phase oxygen supply strategy.

Liquid phase oxygen supply strategy (LPOS), in which hydrogen peroxide (H(2)O(2)) is used to supply oxygen to the bioreactor, leads to low cell productivity despite high specific productivities of relevant metabolites. We hypothesized that high H(2)O(2) concentrations in the feed-zone led to local cell death, which in turn, lead to lower cell productivity. To test the hypothesis, a mathematical model was developed. Bacillus subtilis 168 was used as the model system in this study. The model simulations of cell concentrations in the bioreactor-zone were verified with the experimental results. The feed-zone H(2)O(2) concentrations remained 12-14 times higher than bulk bioreactor concentrations. The high local concentrations are expected to cause local cell killing, which explains the decrease in overall cell production by 50% at 300 rpm compared to conventional cultivation. Further, among the four different feed strategies studied using the model, dissolved oxygen (DO) controlled H(2)O(2) feed strategy caused least local cell killing and improved overall cell production by 34%.