Do-It-Yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 °C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues (1,2). The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn' (2), which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces (2). In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks (1). These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials (3). However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use. In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.     

Assignment of MS-based metabolomic datasets via compound interaction pair mapping

Assignment of physical meaning to mass spectrometry (MS) data peaks is an important scientific challenge for metabolomics investigators. Improvements in instrumental mass accuracy reduce the number of spurious database matches, however, this alone is insufficient for accurate, unique high-throughput assignment. We present a method for clustering MS instrumental artifacts and a stochastic local search algorithm for the automated assignment of large, complex MS-based metabolomic datasets. Artifact peaks and their associated source peaks are grouped into “instrumental clusters.” Instrumental clusters, peaks grouped together by shared peak shape in the temporal domain, serve as a guide for the number of assignments necessary to completely explain a given dataset. We refine mass only assignments through the intersection of peak correlation pairs with a database of biochemically relevant interaction pairs. Further refinement is achieved through a stochastic local search optimization algorithm that selects individual assignments for each instrumental cluster. The algorithm works by choosing the peak assignment that maximally explains the connectivity of a given cluster. We demonstrate that this methodology provides a significant advantage over standard methods for the assignment of metabolites in a UPLC-MS diabetes dataset. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chemical and principal-component analyses of the essential oils of Apioideae taxa (Apiaceae) from central Balkan

The volatile constituents of the essential oils of 23 taxa belonging to the Apioideae subfamily were studied in detail. The investigated taxa were Pimpinella serbica (Vis.) Bentham & Hooker, Libanotis montana Cr., Cnidium silaifolium (Jacq.) Simk. ssp. orientale (Boiss.) Tutin, Bupleurum praealtum L., B. sibthorpianum S. S. var. diversifolium (Roch.) Hay, Aegopodium podagraria L., Torilis anthriscus (L.) Gmel., Orlaya grandiflora (L.) Hoffm., Laserpitium siler L., Laser trilobum (L.) Brokh., Chaerophyllum aureum L., C. hirsutum L., C. temulum L., Pastinaca sativa L., P. hirsuta Pancic., Tordylium maximum L., Physospermum cornubiense (L.) DC., Peucedanum alsaticum L., P. oreoselinum (L.) Moench, P. cervaria (L.) Cuss., P. austriacum (Jacq.) Koch, P. longifolium W. et K., and P. officinale L. All of these species grow wild in the central part of the Balkan Peninsula. The essential oils were found to be complex mixtures of various compounds, more than 100 constituents being in each taxon, with contributions of main products never exceeding 25% of the total content. Sesquiterpene hydrocarbons were found to be the main group of constituents of all taxa, except for Peucedanum species, where monoterpene hydrocarbons were identified as the main components. The chemotaxonomic value of the essential-oil composition is discussed according to results of principal-component analysis (PCA). The essential-oil composition mainly reflects current taxonomic relationships between the investigated taxa.     

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.     

Property control of sophorolipids: influence of fatty acid substrate and blending

Sophorolipids (SLs) were synthesized by fed-batch fermentation of Candida bombicola on glucose and either palmitic acid (SL-p), stearic acid (SL-s), oleic acid (SL-o) or linoleic acid (SL-l) and the structural distribution accurately determined by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The surfactant properties, including critical micelle concentration (CMC), minimum surface tension (min.ST) and oil-water interfacial tension (IFT) were measured by tensiometry. Minimum STs of 35–36 mN/m were obtained regardless of the substrate while IFTs ranged from 3–5 mN/m with the exception of SL-l, which had an IFT of 7 mN/m. The largest disparity occurred in the CMC values, which ranged from 35 ppm for SL-s to 250 ppm for SL-l. By manually mixing these four SLs in different ratios, it was possible to better control the CMC values without affecting the min.ST or IFT, which will prove beneficial as new applications for SLs are established. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and Dinophysis acuminata from New Zealand

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Interplant communication: airborne methyl jasmonate is essentially converted into JA and JA-Ile activating jasmonate signaling pathway and VOCs emission

Methyl jasmonate (MeJA) was identified as an airborne signal involved in mediating interplant defense response communications over a decade ago. However, how MeJA activates plant defense systems and what becomes of the compound after it has done so has, thus far, remained unknown. To investigate this, Achyranthes bidentata plants were exposed to deuterated methyl jasmonate (d₂MeJA) followed by absolute quantification of metabolic products of d₂MeJA, and emissions of volatile organic compound (VOC) as defensive markers. We found that d₂MeJA was metabolized mainly into deuterated jasmonic acid (d₂JA) and jasmonoyl isoleucine (d₂JA-Ile), and to a much lesser extent, deuterated jasmonoyl leucine (d₂JA-Leu). Increases in d₂JA-Ile/Leu and also endogenous JA-Ile/Leu were tightly co-related with, and significantly influenced the pattern and amount of, VOC emissions. The amount of accumulated d₂JA-IIe was 13.1-fold higher than d₂JA-Leu, whereas the amounts of JA-IIe and JA-Leu accumulated were almost identical. This study demonstrates that exogenous MeJA activates defensive systems (such as VOC emissions) in receiver plants by essentially converting itself into JA and JA-IIe and initiating a signal transduction leading to VOC emissions and induction of endogenous JA-IIe and JA-Leu, which in turn cause further amplification of VOC emissions.     

Outer arm fucosylation of N-glycans increases in sera of hepatocellular carcinoma patients

Serum glycans are promising markers for early-stage cancer detection, but the research remains challenging because low concentrations of serum glycoproteins are secreted from early-stage tumors. We have established an N-glycan profiling method using liquid chromatography electrospray ionization-mass spectrometry with high sensitive derivative, trimethyl(4-aminophenyl)ammonium chloride (TMAPA). The mass sensitivity of TMAPA-labeled oligosaccharides was enhanced more than 50 times compared with 2-aminopyridine (PA) labeled oligosaccharides, and the analytical period was significantly shortened compared with traditional HPLC 2D-mapping. Using this method, we found about 28 major N-linked oligosaccharides in human sera, and we investigated their alterations in patients who developed hepatocellular carcinoma (HCC). We found that outer arm fucosylation (attached GlcNAc via an alpha 1-3/4 linkage) in highly branched oligosaccharides increased significantly in sera of HCC patients. Normalizing the level of outer arm fucosylation by taking into account platelet concentration allowed us to distinguish more clearly between HCC and LC patients.     

聚氨酯／液晶（PU／EBBA）复合膜的血液相容性研究

以聚氨酯弹性体为基质材料,与液晶化合物EBBA共混后,由溶剂蒸发法浇铸成膜。偏光显微镜观察证实了复合膜中液晶相的存在。用动态凝血实验、血小板粘附实验和扫描电镜观察的方法研究了复合膜中液晶含量对材料抗凝血性能的影响。结果表明,只有当液晶含量达到30%（wt）时,复合膜的血液相容性随着液晶含量的增加有明显改善,同时发现复合膜表面吸附的血小板随着液晶含量的增加而明显减少。