Obtaining the Fluorescent Chitosan for Investigations in the Analytical Ultracentrifuge

1) In order to achieve the visibility of the chitosan macromolecule for the UV optical system of the analytical ultracentrifuge on investigation of the molecular characteristics and polymers interactions, the labeling of chitosan by a new fluorophore of fluorescein-5-isothiocyanat was carried out. 2) Samples of fluorescent chitosan with two different degrees of fluorophore substitution and various degrees of acetylation were obtained. 3) The labeled chitosans with the fluorescein-5-isothiocyanat allowed estimating the sedimentation coefficient and the molecular characteristic in the analytical ultracentrifuge. 4) The sensitivity of the UV-optical system of the analytical ultracentrifuge for the obtained fluorescent samples of chitosan relatively to the fixation of the meniscus and the influence of the wavelength and rotation speed were estimated.

Radioactivity of nuclei in a centrifugal force field

Radioactivity of nuclei in a centrifugal force field of an ultracentrifuge is considered for heavy radioactive nuclei, i.e., for the same nuclei, but with a significant virtual mass thousands of times larger than the actual mass and is characterized by an angular momentum. As the nucleus leaves the centrifugal force field, the virtual mass disappears, but the spin number appears and/or changes. The role of centrifugal and gravitational forces in radioactive decay of nuclei is studied. According to the terminology of western researchers, such a virtual mass state is called the dynamic gravitation which is more adequate. The oscillator and possible changes in the nucleus state are considered under conditions of dynamic gravitation and taking into account features of atomic nucleus physics. To a first approximation, the drop model of the nucleus was used, in which shape fluctuations have much in common with geophysical and astrophysical analogues. Shape fluctuations of analogues strongly depend on the gravitational force g defined by their mass (or nucleus mass). Experiments were performed by radiometric measurements of transbaikalian uranium ore (1.5 g) with known composition in a centrifuge at various rotation rates or gravitational forces g. The existence of characteristic times or the effect of rotation frequencies (i.e., g) on atomic nuclei, which, along with the nucleus type itself, controls the nucleus response to perturbation (stability increase or decay), is found statistically significant.

Optimization of crude enzyme preparation methods for analysis of glutamine synthetase activity in phytoplank-ton and field samples

Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation （HC, 〈10 000 g）, ultracentrifugation （UC, 70000 g）, and ultrafiltration （UF） with 100 kit molecular weight cutoff, were compared using two diatom species （Asterionellopsis glacialis and Thalassiosira weissflogii）, and two dinoflagellate species （Alexandriura catenella and Prorocentrum donghaiense） as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity （GSA） in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods （HC and UF） overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.

CHARACTERIZATION OF NANOSTRUCTURES:TOOLS AND CHALLENGES

Apart from long-known and applied nanostructures like carbon black for tyres or pigments for coatings nanotechnology has created highly sophisticated structures used for nano/molecular electronics,diagnostics,drug delivery, UV-absorbers etc.Often the main question to be solved analytically is the local determination of tiny amounts of chemicals resulting in an ever increasing need for highly sensitive as well as locally resolved techniques.Applications of techniques like mass spectroscopy,transmission elect...

Rapid Isolation of Large Amount of Plasma VLDL and LDL by a Two Step Ultracentrifugation

A rapid method to obtain large amount of VLDL and LDL by ultracentrifugation is described. The mixture of VLDL and LDL was isolated and concentrated from plasma by an ultracentrifugation at 265 000 g for 2h. VLDL and LDL were separated and purified by a further ultracentrifugation at 265 000g for 3h.This method combines the advantages of both sequential flotation ultracentri-fugation and density gradient ultracentrifugation. It can process a large volume of plasma in a short time. The purity of isolated VLDL and LDL was confirmed by the lipoprotein electrophoresis on agarose gel and PAGE and by the apolipoprotein electrophoresis on SDS-PAGE. This rapid.economical method is of great value in practical application.

Identification of a Morphogenic Intermediate of the Bacteriophage Mu Baseplate

Bacteriophage morphogenesis is a model system for investigating sequential molecular assembly. The Mu phage is one of the most classical Myoviridae. Although it is well known as a mobile genetic element, the details of its morphogenesis remain unclear. Analysis of conditional lethal mutants and genome analysis of the Mu phage have suggested that genes 42, 43, 44, 45, 46, 47, and 48 are essential for its baseplate assembly. Since we have already reported X-ray structures of the products of genes 44 (gp44) and 45 (gp45), we here tried to purify the remaining Mu phage baseplate subunits, gp42, gp43, gp46, gp47, and gp48, to investigate the baseplate assembly process. In the case of gp42 expression, the transformed E. coli cells showed growth inhibition after induction and no gp42 fractions were observed. However, gp43, gp46, gp47, and gp48 were successfully expressed and purified, although gp48 could not be applied to further analysis, because the amount of soluble fraction was very low. Based on analytical ultracentrifugation, we concluded that gp43 formed a monomer, gp46 was a monomer, and gp47 occurred as both a monomer and dimer in solution. Moreover, we found that gp43 and gp45 formed an intermediate complex in the baseplate assembly process.

Effects of Storage Time and Hydrogen Peroxide on the Formation of Soy Globulin 15S in 11S Dilute Solutions Investigated by Analytical Ultracentrifugation

Two soy protein 11S fractions with different surface sulfhydryl contents were prepared.Utilizing analytical ultracentrifugation,the effects of storage time and hydrogen peroxide at different concentrations(0.5-100 mmol/L)on the two 11S fractions were investigated.Results show that after removing 2-mercaptoethanol(2-ME)by size exclusion chromatography,the 11S fraction with high surface sulfhydryl content(2.0 mol sulfhydryl/mol 11S)progressively formed 15S and 21S in dilute solutions during storage at 4℃ for 82 days.While,the 11s fraction with low surface sulfhydryl content(0.2 mol sulfhydryl/mol 11S)was stable under the same condition.Moreover,after treating the 11s with high surface sulfhydryl content with 1 mmol/L H_(2)O_(2),the weight percentage of 15S reached the maximum value of 20%.The 15S induced by air and H_(2)O_(2)could be totally converted to 11S with the addition of 10 mmol/L 2-ME,which could be attributed to that the disulfide bond linking two 11S molecules is on the surface of the 15S and easily accessible to the reducing agent 2-ME.This study helps us to deeply understand the formation mechanism of 15S and the stability of 11S.

Wastewater-based epidemiological investigation of SARS-CoV-2 in Porto Alegre,Southern Brazil

Wastewater-based epidemiology(WBE)may be successfully used to comprehensively monitor and determine the scale and dynamics of some infections in the community.We monitored severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)RNA in raw wastewater samples from Porto Alegre,Southern Brazil.The samples were collected and analyzed every week between May 2020 to May 2021.Meanwhile,different social restrictions were applied according to the number of hospitalized patients in the region.Weekly samples were obtained from two wastewater treatment plants(WWTP),named Navegantes and Serraria.To determine the SARS-CoV-2 RNA titers in wastewater,we performed RT-qPCR analysis targeting the N gene(N1).The highest titer of SARS-CoV-2 RNA was observed between epidemiological weeks(EWs)33-37(August),42-43(October),45-46(November),49-51(December)in 2020,and 1-3(January),7-13(February to March)in 2021,with viral loads ranging from 1-106-3-106 genomic copies/Liter.An increase in positive confirmed cases followed such high viral loads.Depending on the sampling method used,positive cases increased in 6-7 days and 15 days after the rise of viral RNA titers in wastewater,with composite sampling methods showing a lower time lag and a higher resolution on the analyses.The results showed a direct relation between strict social restrictions and the loads of detected RNA reduction in wastewater,corroborating the number of confirmed cases.Differences in viral loads between different sampling points and methods were observed,as composite samples showed more stable results during the analyzed period.Besides,viral loads obtained from samples collected at Serraria WWTP were consistently higher than the ones obtained at Navegantes WWTP,indicating differences in local dynamics of SARS-CoV-2 spread in different regions of Porto Alegre.In conclusion,wastewater sampling to monitor SARS-CoV-2 is a robust tool to evaluate the viral loads contributing to hospitalized patients’data and confirmed cases.In addition,SARS-CoV-2 detection in sewage may inform and alert the government when there are asymptomatic or nontested patients.

Tunable Separation of Single-Walled Carbon Nanotubes by Dual-Surfactant Density Gradient Ultracentrifugation

We present a systematic study of the effects of surfactants in the separation of single-walled carbon nanotubes （SWNTs） by density gradient ultracentrifugation （DGU）. Through analysis of the buoyant densities, layer positions, and optical absorbance spectra of SWNT separations using the bile salt sodium deoxycholate （DOC） and the anionic salt sodium dodecyl sulfate （SDS）, we clarify the roles and interactions of these two surfactants in yielding different DGU outcomes. The separation mechanism described here can also help in designing new DGU experiments by qualitatively predicting outcomes of different starting recipes, improving the efficacy of DGU and simplifying post-DGU fractionation.

Density gradient ultracentrifugation for colloidal nanostructures separation and investigation

In this article,we review the advancement in nanoseparation and concomitant purification of nanoparticles(NPs) by using density gradient ultracentrifugation technique(DGUC) and demonstrated by taking several typical examples.Study emphasizes the conceptual advances in classification,mechanism of DGUC and synthesis-structure-property relationships of NPs to provide the significant clue for the further synthesis optimization.Separation,concentration,and purification of NPs by DGUC can be achieved at the same time by introducing the water/oil interfaces into the separation chamber.We can develop an efficient method ‘‘lab in a tube" by introducing a reaction zone or an assembly zone in the gradient to find the surface reaction and assembly mechanism of NPs since the reaction time can be precisely controlled and the chemical environment change can be extremely fast.Finally,to achieve the best separation parameters for the colloidal systems,we gave the mathematical descriptions and computational optimized models as a new direction for making practicable and predictable DGUC separation method.Thus,it can be helpful for an efficient separation as well as for the synthesis optimization,assembly and surface reactions as a potential cornerstone for the future development in the nanotechnology and this review can be served as a plethora of advanced notes on the DGUC separation method.

Preparation of plasma membrane of sperm cells of Zea mays and preliminary analysis of membrane protein

Just like in animals, the essence of fertilization process in plants is the fusion of male andfemale gametes. Although it is already possible, with the aid of cytological manipulation, toachieve in vitro fusion of isolated egg and sperm cell to form zygote, it remains to be solvedwhether the fusion of male and female gametes in nature is at random or there exists some kindof 'recognition'.

Methods for extracellular vesicle isolation from cancer cells

Cells are known to release different types of vesicles such as small extracellular vesicles(sEVs)and large extracellular vesicles(LEVs).sEVs and LEVs play important roles in intercellular communication,pre-metastatic niche formation,and disease progression;both can be detected cell culture media and biological fluids.sEVs and LEVs contain a variety of protein and RNA cargo,and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins.It has recently been established that standard isolation techniques,such as differential ultracentrifugation,yield a mixed population of EVs.However,density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris.Here,we describe the most common methods used to isolate sEVs from cell culture medium,mouse and human plasma,and a new technique for isolating sEVs from tissues as well.This article also provides detailed procedures to isolate LEVs.