Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

Reduced major minerals and increased minor nutrients improve micropropagation in three apple cultivars

Mineral nutrient medium requirements for propagation of in vitro shoots of apple (Malus domestica Borkh) ‘Golden Delicious’, ‘Maksat’, and ‘Voskhod’ were studied using response surface methodology (RSM). The mineral nutritional factors evaluated were based on Murashige and Skoog (MS) mineral nutrients (NH₄NO₃, KNO₃, CaCl₂, KH₂PO₄, MgSO₄, and minor nutrients), with concentrations ranging from 0.5 to 3.0× the MS concentrations. Nine plant growth qualities were evaluated. The most significant factors were NH₄NO₃ at 0.5 to 1.0× MS, and minor nutrients at 2.0× MS. Most of the other factors were optimal at 0.5×. The quality rating was highest when minor nutrients were 2.0× MS, and most other nutrients were standard concentrations or lower. Increased KH₂PO₄ and minor nutrients were the most significant for improved multiplication, and higher KNO₃ for shoot length. Optimized media were developed for each cultivar based on these models. The cultivars were grown on the three individual optimized media, a general medium based on the three optimizations, and MS. The optimized medium for each cultivar was significantly better for shoot quality and shoot length of each cultivar than MS, but the generalized medium of minors at 2.0× and NH₄NO₃, CaCl₂, and MgSO₄ at 0.5× MS, was significantly better for two of the three cultivars and not significantly different for the third. The next step to develop a final optimized medium will require the evaluation of the minor nutrients, determination of optimal concentrations of each, and screening a wide range of Malus germplasm on the finalized medium.

     

Early functional muscle regeneration after myotoxic injury in mice is unaffected by nNOS absence

Nitric oxide (NO) is an important signaling molecule produced in skeletal muscle primarily via the neuronal subtype of NO synthase (NOS1, or nNOS). While many studies have reported NO production to be important in muscle regeneration, none have examined the contribution of nNOS-derived NO to functional muscle regeneration (i.e., restoration of the muscle's ability to produce force) after acute myotoxic injury. In the present study, we tested the hypothesis that genetic deletion of nNOS would impair functional muscle regeneration after myotoxic injury in nNOS(-/-) mice. We found that nNOS(-/-) mice had lower body mass, lower muscle mass, and smaller myofiber cross-sectional area and that their tibialis anterior (TA) muscles produced lower absolute tetanic forces than those of wild-type littermate controls but that normalized or specific force was identical between the strains. In addition, muscles from nNOS(-/-) mice were more resistant to fatigue than those of wild-type littermates (P < 0.05). To determine whether deletion of nNOS affected muscle regeneration, TA muscles from nNOS(-/-) mice and wild-type littermates were injected with the myotoxin notexin to cause complete fiber degeneration, and muscle structure and function were assessed at 7 and 10 days postinjury. Myofiber cross-sectional area was lower in regenerating nNOS(-/-) mice than wild-type controls at 7 and 10 days postinjury; however, contrary to our original hypothesis, no difference in force-producing capacity of the TA muscle was evident between the two groups at either time point. Our findings reveal that nNOS is not essential for functional muscle regeneration after acute myotoxic damage.     

Anastomosis Group Determination of Rhizoctonia solani Kühn (Telemorph: Thanatephorus cucumeris) Isolates from Tomatoes Grown in Aydin,Turkey and their Disease Reaction on Various Tomato Cultivars

In the Province of Aydin‐Turkey most frequently Fusarium spp. and secondly Rhizoctonia solani Kühn were isolated from the roots and crown of tomato plants. Based on affinities for hyphal fusion with test isolates, all R. solani isolates were identified as AG‐4. The tomato cultivars which were grown in soil infested with R. solani AG‐4 exhibited different reactions. From the point of symptom expression and the rate of seedling emergence Sunny 6066 F1 was found to be the most resistant cultivar, whereas Rio Grande, Rio Fuego, NDM 725, Interpeel and Konia were the most susceptible cultivars.     

Complex dynamics of osteoclast formation and death in long-term cultures

Background

Osteoclasts, cells responsible for bone resorption, contribute to the development of degenerative, metabolic and neoplastic bone diseases, which are often characterized by persistent changes in bone microenvironment. We aimed to investigate the dynamics of osteoclast formation and death in cultures that considerably exceeded the length of standard protocol and to design a mathematical model describing osteoclastogenesis.

Methodology/Principal Findings

RAW 264.7 monocytic cells fuse to form multinucleated osteoclasts upon treatment with pro-resorptive cytokine RANKL. We have found that in long-term experiments (15–26 days), the dynamics of changes in osteoclast numbers was remarkably complex and qualitatively variable in different experiments. Whereas 19 of 46 experiments exhibited single peak of osteoclast formation, in 27 experiments we observed development of successive waves of osteoclast formation and death. Periodic changes in osteoclast numbers were confirmed in long-term cultures of mouse bone marrow cells treated with M-CSF and RANKL. Because the dynamics of changes in osteoclast numbers was found to be largely independent of monocytes, a two-species model of ordinary differential equations describing the changes in osteoclasts and monocytes was ineffective in recapitulating the oscillations in osteoclast numbers. Following experimental observation that medium collected from mature osteoclasts inhibited osteoclastogenesis in fresh cultures, we introduced a third variable, factor f, to describe osteoclast-derived inhibitor. This model allowed us to simulate the oscillatory changes in osteoclasts, which were coupled to oscillatory changes in the factor f, whereas monocytes changed exponentially. Importantly, to achieve the experimentally observed oscillations with increasing amplitude, we also had to assume that osteoclast presence stimulates osteoclast formation.

Conclusions/Significance

This study identifies the critical role for osteoclast autocrine regulation in controlling long-term dynamic of osteoclast formation and death and describes the complementary roles for negative and positive feedback mediators in determining the sharp dynamics of activation and inactivation of osteoclasts.     

Practicability of the Index of Trophic Completeness for running waters

Effects of stress caused by anthropogenic activities in rivers negatively act on the intricate system of trophic links within invertebrate communities and other components of the aquatic ecosystem. These effects can be made visible with the Index of Trophic Completeness (ITC), which was developed as an indicator for the functioning of the river ecosystem, based on the trophic classification of benthic macroinvertebrates. We tested the index using data collected from rivers exposed to different degree of anthropogenic pressure. In undisturbed rivers, all trophic guilds distinguished are present irrespective the part of the river studied and its geographical region. No significant seasonal effect on the outcomes was observed. Disturbances cause the extinction of specific trophic guilds, however due to overlap of effects, the result of an ITC outcome does not indicate the type of anthropogenic pressure. The ITC can be applied to the results of each combination of biotopes sampled, although one has to consider a varying biotope-density relation for species in the trophic guilds. Although the outcomes are projections of trophic guilds present, they can be arranged into quality classes.     

Macrophages survive hyperoxia via prolonged ERK activation due to phosphatase down-regulation

Macrophages exposed to hyperoxia in the lung continue to survive for prolonged periods. We previously reported (Nyunoya, T., Powers, L. S., Yarovinsky, T. O., Butler, N. S., Monick, M. M., and Hunninghake, G. W. (2003) J. Biol. Chem. 278, 36099-36106) that hyperoxia induces cell cycle arrest and sustained extracellular signal-related kinase (ERK) activity in macrophages. In this study, we determined the mechanisms of hyperoxia-induced ERK activation and how ERK activity plays a pro-survival role in hyperoxia-exposed cells. Inhibition of ERK activity decreased survival of hyperoxia-exposed macrophages. This was due, at least in part, to down-regulation of the pro-apoptotic Bcl-2 family member, BimEL. In determining the mechanism of ERK activation by hyperoxia, we found that ERK activation was not associated with hyperoxia-induced activation of the upstream ERK kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2. When we examined the ability of whole cell lysates from hyperoxia-exposed cells to dephosphorylate purified phosphorylated ERK, we found decreased ERK-directed phosphatase activity. Two particular ERK-directed phosphatases (protein phosphatase 2A and MAPK phosphatase-3) demonstrated decreased activity in hyperoxia-exposed cells. Moreover, whole cell lysates from normoxia-exposed cells depleted of PP2A or MAPK phosphatase-3 were also less able to dephosphorylate ERK. These data demonstrate that, in hyperoxia-exposed macrophages, sustained activation of ERK due to phosphatase down-regulation permits macrophage survival via effects on the balance between pro- and anti-apoptotic Bcl-2 family proteins.     

Molecular colony diagnostics: detection and quantitation of viral nucleic acids by in-gel PCR

When PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. Furthermore, because of the spatial separation of the products in the gel, no interference is observedfrom another simultaneously amplified target even if it is present at a 106 higher amount orfrom human nucleic acids that outweigh the target by up to a factor of 1,012, which is often true of clinical samples. All these features provide for an accurate and reliable assay of viruses even at very low amounts, that is, in cases most important to diagnostics.