Using an online phycocyanin fluorescence probe for rapid monitoring of cyanobacteria in Macau freshwater reservoir

Monitoring of cyanobacteria and their toxins are traditionally conducted by cell counting, chlorophyll-a (chl-a) determination and cyanotoxin measurements, respectively. These methods are tedious, costly, time consuming, and insensitive to rapid changes in water quality and cyanobacterial abundance. We have applied and tested an online phycocyanin (PC) fluorescence probe for rapid monitoring of cyanobacteria in the Macau Storage Reservoir (MSR) that is experiencing cyanobacterial blooms. The relationships among cyanobacterial abundance, biovolume, cylindrospermopsin concentration, and PC fluorescence were analyzed using both laboratory and in-the-field studies. The performance of the probe was compared with traditional methods, and its advantages and limitations were assessed in pure and mixed cyanobacterial cultures in the laboratory. The proposed techniques successfully estimated the species including Microcystis and Cylindrospermopsis, two toxic species recently observed in the MSR. During February–November, 2010, the PC probe detected high correlations between PC and cell numbers (R ² = 0.71). Unlike the chl-a content, which indicates only the total algal biomass, the PC pigment specifically indicates cyanobacteria. These results support the PC parameter as a reliable estimate of cyanobacterial cell number, especially in freshwater bodies where the phytoplankton community and structure are stable. Thus, the PC probe is potentially applicable to online monitoring of cyanobacteria.     

Somatic embryogenesis and shoot organogenesis from interspecific hybrid embryos of Vigna glabrescens and V. radiata

Somatic embryogenesis occurred on cotyledons of morphologically abnormal embryos derived from Vigna glabrescens x V. radiata crosses and cultured on Murashige and Skoog (MS) medium without growth regulators. The frequency of 15–17 day old embryos that gave rise to somatic embryos increased from 8% to 29% by application a mixture of 100 mg/l gibberelllc acid, 25 mg/l -naphthaleneacetic acid (NAA) and 5 mg/l kinetin daily to the pedicels of the developing pods. However, only callus formed on immature hybrid embryos of the reciprocal cross. These callus tissues occasionally gave rise to shoots via organogenesis when transferred to MS medium with 2 mg/l N⁶-benzyladenine and 0.05 mg/l NAA. Treatment of pods with growth regulators did not influence the frequency of organogenic callus. Selfed embryos of the parents did not form somatic embryos in culture, nor did callus derived from the selfed embryos produce shoots. Thus, the ability to redifferentiate appears to be associated with interspecific hybridity.     

Fertilization and early embryo development in reciprocal interspecific crosses of Phaseolus

Summary Fertilization and early embryo and endosperm development were examined in Phaseolus vulgaris x P. acutifolius, P. vulgaris x P. lunatus crosses and their reciprocals. The number and length of pollen tubes were not different between selfings and interspecific crosses. Fertilization was completed in all matings and the time of fertilization was maternally dependent which may reflect the degree of maturation of embryo sacs at pollination. A large difference between reciprocal crosses was found in the time of endosperm and embryo division in relation to the time of fertilization. When P. vulgaris was the female parent and P. acutifolius the male parent, endosperm division occurred at the same time as in P. vulgaris upon selfing, while in P. vulgaris x P. lunatus crosses the time of endosperm division was intermediate as compared with the two parents. The time lapse between fertilization and endosperm and embryo division in P. acutifolius x P. vulgaris crosses was longer than in either parent upon selfing. In P. lunatus x P. vulgaris crosses, endosperm division occurred in only 7–12% of the ovules at 72 hours after pollination. Embryo development in these ovules was limited to the four cell stage although the endosperm was at the free nuclei stage. The severe delay in embryo and endosperm divisions may be the major cause of early pod abscission in P. lunatus x P. vulgaris crosses.Technical paper No. 4929 of the Oregon Agricultural Experiment Station. Research was supported by the Oregon Agricultural Experiment Station, the Research Council of Oregon State University (NIH Biomedical Research Support Grand RR07079) and the Processor Research Council of Oregon. A.R. is supported by an African Graduate Fellowship from the African-American Institute.     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

Human mesenchymal stromal cells could deliver erythropoietin and migrate to the basal layer of hair shaft when subcutaneously implanted in a murine model

Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. MIDGE (an acronym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. The objective of this study was to test the ability of the human MSC (hMSC) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. hMSC nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hMSC resulted in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hMSC harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration.     

The NS1 Protein of Influenza A Virus Interacts with Cellular Processing Bodies and Stress Granules through RNA-Associated Protein 55 (RAP55) during Virus Infection

The nonstructural protein (NS1) of influenza A virus performs multiple functions in the virus life cycle. Proteomic screening for cellular proteins which interact with NS1 identified the cellular protein RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1, RAP55, and other P-body-associated proteins (Ago1, Ago2, and DCP1a) were confirmed using coimmunoprecipitation and cellular colocalization assays. Overexpression of RAP55 induced RAP55-associated stress granule formation and suppressed virus replication. Knockdown of RAP55 with small interfering RNA (siRNA) or expression of a dominant-negative mutant RAP55 protein with defective interaction with P-bodies blocked NS1 colocalization to P-bodies in cells. Expression of NS1 inhibited RAP55 expression and formation of RAP55-associated P-bodies/stress granules. The viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was coexpressed. Restriction of virus replication via P-bodies occurred in the early phases of infection, as the number of RAP55-associated P-bodies in cells diminished over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules was associated with RNA binding and mediated via a protein kinase R (PKR)-interacting viral element. Mutations introduced into either RNA binding sites (R38 and K41) or PKR interaction sites (I123, M124, K126, and N127) caused NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules while NS1 counteracts this host restriction mechanism.     

Use-dependent inhibition of Na+ currents by benzocaine homologs.

Most local anesthetics (LAs) elicit use-dependent inhibition of Na+ currents when excitable membranes are stimulated repetitively. One exception to this rule is benzocaine, a neutral LA that fails to produce appreciable use-dependent inhibition. In this study, we have examined the use-dependent phenomenon of three benzocaine homologs: ethyl 4-diethylaminobenzoate, ethyl 4-ethoxybenzoate, and ethyl 4-hydroxybenzoate. Ethyl 4-hydroxybenzoate at 1 mM, like benzocaine, elicited little use-dependent inhibition of Na+ currents, whereas ethyl 4-diethylaminobenzoate at 0.15 mM and ethyl 4-ethoxybenzoate at 0.5 mM elicited substantial use-dependent inhibition--up to 55% of peak Na+ currents were inhibited by repetitive depolarizations at 5 Hz. Each of these compounds produced significant tonic block of Na+ currents at rest and shifted the steady-state inactivation curve (h infinity) toward the hyperpolarizing direction. Kinetic analyses showed that the decaying phase of Na+ currents during a depolarizing pulse was significantly accelerated by all drugs, thus suggesting that these drugs also block the activated channel. The recovery time course for the use-dependent inhibition of Na+ currents was relatively slow, with time constants of 6.8 and 4.4 s for ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate, respectively. We conclude that benzocaine and 4-hydroxybenzoate interact with the open and inactivated channels during repetitive pulses, but during the interpulse the complex dissociates too fast to accumulate sufficient use-dependent block of Na+ currents. In contrast, ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate dissociate slowly from their binding site and consequently elicit significant use-dependent block. A common LA binding site suffices to explain the presence and absence of use-dependent block by benzocaine homologs during repetitive pulses.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.