Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Representing an ensemble of NMR-derived protein structures by a single structure.

The usefulness of representing an ensemble of NMR-derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of phi psi torsion angles and the distribution of chi 1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the "average" of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the "best" rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue.     

Homeostasis of cell composition during prolonged darkness

The chemical composition of organisms in relation to their environmental resource availability is an area of intense research activity. We studied the changes in cell composition of the cyanobacterium Phormidium autumnale in response to prolonged darkness. Cells allocated their internal resources in a homeostatic manner, oxidizing all the three major cellular constituents in a proportional way. This resulted in constant C/N and carbohydrates, lipids and proteins ratios that remained unaltered throughout the whole incubation period. We propose the maintenance of balanced cell composition (homeostasis) as an evolutionary strategy related to environmental transitory changes.     

Effect of blending lignin biopolymer on the biodegradability of polyolefin plastics

The ability of the lignin-degrading microorganism Phanerochaete chrysosporium to attack polyethylene and polypropylene was investigated using a series of polymer blends containing 10, 20 and 30% lignin obtained from the waste product of pulp and paper industry. In the cultivation medium, lignin peroxidase and Mn(II)peroxidase activities were detected. Degradation was verified by quantitative u.v. spectrophotometric analysis of the cultivation medium and by liberation of CO₂ from the blends. Measurement of the tensile strength after 30-days cultivation showed that the mechanical properties of the polymer blends were decreased during the biodegradation process. The isolation of oligomer fractions by tetrahydrofuran (THF) extraction of biodegraded polymers and their characterization by gel permeation chromatography (GPC), u.v. and Fourier transmission infrared (FTIR) spectroscopy indicates that biotransformation of the lignin component during the cultivation process initiates partial biodegradation of the synthetic polymer matrix.     

蝉虫草样品的分级萃取与化学成分分析

利用索氏萃取技术,依次采用石油醚、乙酸乙酯、丙酮、无水乙醇和甲醇等5种溶剂对蝉虫草纯粉进行分级萃取,运用傅里叶变换红外光谱分析法和气相色谱-质谱联用技术对5级萃取物进行分析鉴定。傅里叶变换红外光谱分析显示萃取物中含有与烯烃类、羧酸类、酯类、醇类和酮类等化合物相关的C-H、C=O、C-O和C=C等官能团。气相色谱-质谱联用技术共鉴定出有机小分子化合物34种,以酯类和脂肪酸类为主,多为碳链长度为15-20的长链脂肪酸及对应的酯,其中十八碳不饱和脂肪酸相对含量高达28.95%;分别存在于两种或以上萃取物中的有机化合物共有11种;仅存于石油醚萃取物中的化合物6种,乙酸乙酯萃取物中3种,丙酮萃取物中2种,无水乙醇萃取物中6种,甲醇萃取物中6种。在一定极性范围内,利用溶剂的极性梯度变化,可实现蝉虫草中活性物质的按极性梯度分离;采用分级萃取技术可有效分离蝉虫草中部分化学成分。鉴定结果充实了蝉虫草中化合物的种类资源,为蝉虫草中活性物质谱图库的完善、构效关系的建立及蝉虫草产品的利用开发提供支撑。     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.     

Highly Solvating Electrolytes for Lithium–Sulfur Batteries

There is a critical need to evaluate lithium–sulfur (Li–S) batteries with practically relevant high sulfur loadings and minimal electrolyte. Under such conditions, the concentration of soluble polysulfide intermediates in the electrolyte drastically increases, which can alter the fundamental nature of the solution‐mediated discharge and thereby the total sulfur utilization. In this work, an investigation into various high donor number (DN) electrolytes that allow for increased polysulfide dissolution is presented, and the way in which this property may in fact be necessary for increasing sulfur utilization at low electrolyte and high loading conditions is demonstrated. The solvents dimethylacetamide, dimethyl sulfoxide, and 1‐methylimidazole are holistically evaluated against dimethoxyethane as electrolyte co‐solvents in Li–S cells, and they are used to investigate chemical and electrochemical properties of polysulfide species at both dilute and practically relevant conditions. The nature of speciation exhibited by lithium polysulfides is found to vary significantly between these concentrations, particularly with regard to the S₃^?? species. Furthermore, the extent of the instability in conventional electrolyte solvents and high DN solvents with both lithium metal and polysulfides is thoroughly investigated. These studies establish a basis for future efforts into rationally designing an optimal electrolyte for a lean electrolyte, high energy density Li–S battery.     

Fragments and dust after Holmium laser lithotripsy with or without “Moses technology”: How are they different?

Urinary stones can be readily disintegrated by Holmium:YAG laser (Holmium laser lithotripsy), resulting in a mixture of small stone dust particles, which will spontaneously evacuate with urine and larger residual fragments (RF) requiring mechanical retrieval. Differences between fragments and dust have not been well characterized. Also, it remains unknown how the recently introduced “Moses technology” may alter stone disintegration products. Three complementary analytical techniques have been used in this study to offer an in‐depth characterization of disintegration products after in vitro Holmium laser lithotripsy: stereoscopic microscopy, scanning electron microscopy and Fourier‐transform infrared spectroscopy. Dust was separated from fragments based on its floating ability in saline irrigation. Depending on initial crystalline constituents, stone dust either conserved attributes found in larger RFs or showed changes in crystalline organization. These included conversion of calcium oxalate dihydrate towards calcium oxalate monohydrate, changes in carbapatite spectra towards an amorphous phase, changes of magnesium ammonium phosphate towards a differing amorphous and crystalline phase and the appearance of hydroxyapatite on brushite fragments. Comparatively, “Moses technology” produced more pronounced changes. These findings provide new insights suggesting a photothermal effect occurring in Holmium laser lithotripsy. Figure: Appearance of hydroxyapatite hexagons on stone dust collected after Holmium laser lithotripsy of a brushite stone using “Moses technology.”      

Physiological model using diffuse reflectance spectroscopy for nonmelanoma skin cancer diagnosis

Diffuse reflectance spectroscopy (DRS) is a noninvasive, fast, and low‐cost technology with potential to assist cancer diagnosis. The goal of this study was to test the capability of our physiological model, a computational Monte Carlo lookup table inverse model, for nonmelanoma skin cancer diagnosis. We applied this model on a clinical DRS dataset to extract scattering parameters, blood volume fraction, oxygen saturation and vessel radius. We found that the model was able to capture physiological information relevant to skin cancer. We used the extracted parameters to classify (basal cell carcinoma [BCC], squamous cell carcinoma [SCC]) vs actinic keratosis (AK) and (BCC, SCC, AK) vs normal. The area under the receiver operating characteristic curve achieved by the classifiers trained on the parameters extracted using the physiological model is comparable to that of classifiers trained on features extracted via Principal Component Analysis. Our findings suggest that DRS can reveal physiologic characteristics of skin and this physiologic model offers greater flexibility for diagnosing skin cancer than a pure statistical analysis. Physiological parameters extracted from diffuse reflectance spectra data for nonmelanoma skin cancer diagnosis.     

Studies on fermentative production of squalene

Fermentative production of squalene under anaerobic conditions using commercially available compressed baker's yeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yield of squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 g g^–1 respectively, dry weight of yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain were achieved chromatographically. The purified squalene was characterized spectroscopically against an authentic standard.