An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug – clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.     

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.     

Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r² = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.The accumulation of lipids in microalgae is currently a field of intense research: with their high photosynthetic efficiency and rapid growth rates, these organisms hold great potential both for sustainable production of biofuels (Chisti, 2007) and as a nutrition source (de Jesus Raposo et al., 2013). As in all living cells, lipids in microalgae are present in membranes, such as the plasma and organelle membranes. Some microalgae also accumulate lipids, mainly triacylglycerols, in intracellular droplets (De Martino et al., 2011; White et al., 2012). One such microalga is Phaeodactylum tricornutum, a unicellular photoautotrophic diatom and a well-studied model organism. It has a sequenced genome and is known to produce long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and small amounts of docosahexaenoic acid (DHA; Alonso et al., 1998). The long-chain n-3 PUFA-producing properties has made it particularly interesting within the areas of functional food and nutraceuticals. Under conditions of nitrogen starvation, P. tricornutum accumulates larger amounts of fatty acids, albeit of the more saturated nature (Yongmanitchai and Ward, 1991). However, in response to low irradiance, P. tricornutum has been reported to increase its content of PUFAs, especially EPA (Thompson et al., 1990). In order to optimize strain selection and algal cultivation conditions in relation to lipid accumulation/lipid profile, accurate tools to quantify total lipids as well as the degree of unsaturation are required.Solvent extractions followed by methylation, gas chromatography coupled to flame ionization detection, and gas chromatography-mass spectrometry (GC-MS) detection belong to the standard techniques used for algal lipid analysis. However, these are cumbersome and require relatively large amounts of solvents and sample. The resulting quantitative information on lipid amounts is related to total cell mass, which may introduce artifacts, as the cell mass is also influenced by other metabolic parameters. Furthermore, these bioanalytical techniques provide rough population averages without information on the intracellular location or distribution. As an alternative, microscopy techniques are increasingly being used, with the benefit that lipid droplets can be evaluated directly in single cells with high precision. Fluorescence microscopy is the most widespread technique, relying on lipid-specific fluorescent markers: Nile Red is commonly used, but its poor permeability through the cell walls causes difficulties, as does its nonspecific binding (Chen et al., 2009). Other fluorophores like BODIPY 505/515 also have been suggested for live-cell studies (Cooper et al., 2010; Wong and Franz, 2013). Still, invasive techniques are needed, requiring solvents to facilitate the transport of the labeling molecules into the cell, potentially inducing stress responses that may affect cell metabolism. Furthermore, there is little knowledge available on how the accumulation of lipophilic dyes in lipid droplets and the fluorescence emission are influenced by environmental conditions such as temperature, pH, and deposited light doses. It has also been shown that the fluorescence intensity emitted from the dyes cannot be related directly to the local lipid concentration, excluding quantitative measurements (De la Hoz Siegler et al., 2012). In algae/plant cell biology, the applicability of fluorescence microscopy is also limited due to the fact that algae/plant cells generate strong autofluorescence, potentially interfering with the lipid/fatty acid signals. In order to take algal lipid quantification one step further, microscopy techniques, not being dependent on exogenous fluorophores and allowing efficient separation of the lipid/fatty acid signal from the autofluorescence, are desirable.In label-free coherent anti-Stokes Raman scattering (CARS) microscopy, images are formed by probing intrinsic molecular vibrations through a nonlinear four-wave-mixing process (Cheng and Xie, 2004). Briefly, the frequency difference of two coherent near-infrared excitation beams (the pump beam at shorter wavelengths and the Stokes beam at longer wavelengths) are tuned to match the frequency of the target molecular vibration_. As a result, resonant oscillators are coherently driven in the sample focal volume and an enhanced blue-shifted CARS signal is generated. Due to the nonlinear nature of the CARS process, emission is generated only in the high-intensity region of focused laser beams, allowing optical sectioning of the specimen. By tuning the frequency difference of the excitation beams to match the resonance frequency of carbon-hydrogen (C-H) vibrations, especially abundant in lipids, three-dimensional images of lipids can be recorded without any staining (Enejder et al., 2010). As the CARS emission scales with the square of the concentration of C-H bonds, quantitative data on intracellular amounts of lipids can be extracted (Cheng and Xie, 2004). However, cells with chromophores, such as algae and plant cells, generate exceptionally strong two-photon fluorescence, the spectral tails of which tend to bleed through the most efficient optical filters typically used for separation of the CARS signal. As an alternative approach, we have incorporated a time-correlated single-photon counting system (Enejder et al., 2010), allowing us to distinguish the long-decay fluorescence component from the instant CARS signal by time gating.The capability of conventional CARS imaging for microalgae was recently demonstrated with proof-of-principle data, showing that individual, larger lipid droplets can be resolved visually, in contrast to conventional Raman microscopy (He et al., 2012). In this study, we introduce CARS microscopy with time-gated detection, also enabling the identification of subpicogram lipid-rich regions in the vicinity of strongly autofluorescent chloroplasts. This is particularly important because cellular storage lipids in algae are primarily synthesized in the chloroplasts and then budded off from the envelope membranes as nascent lipid droplets (Fan et al., 2011). Hence, time-gated CARS microscopy paves the way for high-precision quantification of the complete intracellular distribution of lipid stores, including the emerging droplets within and adjacent to chloroplasts. We show the feasibility of the approach for quantitative lipid analysis of large populations of living P. tricornutum cultivated under three different growth conditions: a control (Ctrl) group, a light-limited (LL) group to increase the EPA level, and a nitrogen-starved (NS) group to increase total lipid accumulation. We studied the lipid metabolism in approximately 100 cells per category over time and report detailed visual information on the dynamics of lipid droplet formation throughout a life cycle from budding droplets to, in some cases, giant lipid stores. We show that biologically relevant quantitative and qualitative data on intracellular lipid stores can be extracted at a precision of less than 1 pg cell⁻¹ despite adjacent chromophores. Data extracted from the CARS images are further compared with solvent extraction and quantification of fatty acids using GC-MS. We further illustrate the potential to assess whether the different growth conditions promote the synthesis of more PUFAs by detecting shifts in lipid fluidity and saturation per individual lipid droplet from CARS C-H vibration ratio images.     

Diversity patterns of the terrestrial snail fauna of Nyungwe Forest National Park (Rwanda), a Pleistocene refugium in the heart of Africa

We investigated the land snail fauna of Nyungwe Forest National Park in south‐western Rwanda. Fifty plots at altitudes between 1718 and 2573 m were studied. In total, 3461 specimens were collected and were assigned to 102 land snail species. With respect to land snail species, Nyungwe Forest is the richest forest known in Africa. A comparison with other forests in the northern Albertine Rift indicates that land snail species richness in this region is significantly correlated with distance from Pleistocene forest refugia. The high beta diversity in Nyungwe is the result of a high species turnover between sites, which has biogeographical and ecological origins. Nyungwe Forest is situated on the Congo–Nile divide where species of different geographical origin may meet. Moreover, Nyungwe Forest offers a high diversity of habitats because it extends across a wide range of altitudinal zones. Species richness decreased with increasing altitude. It was also correlated with the presence of bare rocks that offer additional microhabitats and shelter. Although the occurrences of different land snail species in Nyungwe Forest were significantly clustered, only a minority of the species could be assigned to a group of species with similar occurrences. The majority of the species respond individualistically to environmental variables. The significant nestedness of the occurrences of the land snail species in Nyungwe was mainly correlated with altitude. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 363–375.     

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes

The x₂ glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P^k-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x₂. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P₁^k phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x₂ and sialylated forms of x₂, whereas x₂ is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x₂. Knockdown experiments with siRNA against B3GALNT1 diminished x₂ levels. We conclude that x₂ fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x₂ joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P₁^k or P₂^k erythrocyte units are preferentially selected for transfusion to P^k patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels.     

Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge

The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge.     

Vitamin D,Vitamin D Binding Protein,and Longitudinal Outcomes in COPD

Background

Associations between Vitamin D₃ [25(OH)D], vitamin D binding protein (VDBP) and chronic obstructive pulmonary disease (COPD) are previously reported. We aimed to further investigate these associations on longitudinal outcomes.

Methods

426 COPD patients from western Norway, GOLD stage II-IV, aged 40–76, were followed every six-month from 2006 through 2009 with spirometry, bioelectrical impedance measurements and registration of exacerbation frequency. Serum 25(OH)D and VDBP levels were determined at study-entry by high-performance liquid chromatography coupled with mass spectrometry and enzyme immunoassays respectively. Yearly change in lung function and body composition was assessed by generalized estimating equations (GEE), yearly exacerbation rate by negative binomial regression models, and 5 years all-cause mortality by Cox proportional-hazard regression.

Results

1/3 of the patients had vitamin D deficiency (<20ng/mL) and a greater decline in both FEV1 and FVC, compared to patients with normal levels; for FEV1 this difference only reached statistical significance in the 28 patients with the lowest levels (<10ng/mL, p = 0.01). Neither 25(OH)D nor VDBP levels predicted exacerbation rate, change in fat free mass index or risk of death.

Conclusion

Severe vitamin D deficiency may affect decline in lung function parameters in COPD. Neither 25(OH)D nor VDBP levels did otherwise predict markers of disease progression.     

Seasonal effects on egg production and level of paternity in a natural population of a simultaneous hermaphrodite snail

In a seasonal environment, the suitable time window for females to reproduce is restricted by both environmental conditions and the availability of males. In simultaneous hermaphrodites, which are female and male at the same time, selection on a trait that is solely beneficial for one sexual function cannot occur independently. Therefore, it is assumed that the optimal time window for reproduction is a compromise between the two sexual functions in simultaneous hermaphrodites, mediated by environmental conditions. We examined seasonal patterns of reproduction and the resulting paternity in a natural population of the simultaneously hermaphroditic land snail Arianta arbustorum. Adult and premature individuals (snails in a short protandric phase) were collected on four occasions over the entire active season. The snails were allowed to deposit eggs after which we assessed the level of paternity in their hatched offspring. Individuals mated throughout the reproductive season, whereas egg production – the major task of the female function – was restricted to the first half of the season. Snails collected in autumn were allowed to hibernate under laboratory conditions. As a result, we found that premature individuals began to mate late in the reproductive season, but did not start to produce eggs before emerging from hibernation. Our results demonstrate a temporal shift of reproductive activities; the egg production and oviposition occur mainly in the first half of the season, while sperm production and mating occur over the entire season. In subadult and adult snails, sperm obtained from several partners in the second part of the reproductive season are stored during hibernation for the fertilization of eggs in the successive years. These results extend our understanding of the influence of both natural and sexual selection on reproductive strategies in hermaphrodites.     

A Framework to Explore the Knowledge Structure of Multidisciplinary Research Fields

Understanding emerging areas of a multidisciplinary research field is crucial for researchers, policymakers and other stakeholders. For them a knowledge structure based on longitudinal bibliographic data can be an effective instrument. But with the vast amount of available online information it is often hard to understand the knowledge structure for data. In this paper, we present a novel approach for retrieving online bibliographic data and propose a framework for exploring knowledge structure. We also present several longitudinal analyses to interpret and visualize the last 20 years of published obesity research data.     

Sleep Duration,Schedule and Quality among Urban Chinese Children and Adolescents: Associations with Routine After-School Activities

Background

With rapid urbanization accompanied by lifestyle changes, children and adolescents living in metropolitan areas are faced with many time use choices that compete with sleep. This study reports on the sleep hygiene of urban Chinese school students, and investigates the relationship between habitual after-school activities and sleep duration, schedule and quality on a regular school day.

Methods

Cross-sectional, school-based survey of school children (Grades 4–8) living in Shanghai, China, conducted in 2011. Self-reported data were collected on students’ sleep duration and timing, sleep quality, habitual after-school activities (i.e. homework, leisure-time physical activity, recreational screen time and school commuting time), and potential correlates.

Results

Mean sleep duration of this sample (mean age: 11.5-years; 48.6% girls) was 9 hours. Nearly 30% of students reported daytime tiredness. On school nights, girls slept less (p<0.001) and went to bed later (p<0.001), a sex difference that was more pronounced in older students. Age by sex interactions were observed for both sleep duration (p=0.005) and bedtime (p=0.002). Prolonged time spent on homework and mobile phone playing was related to shorter sleep duration and later bedtime. Adjusting for all other factors, with each additional hour of mobile phone playing, the odds of daytime tiredness and having difficulty maintaining sleep increased by 30% and 27% among secondary students, respectively.

Conclusion

There are sex differences in sleep duration, schedule and quality. Habitual activities had small but significant associations with sleep hygiene outcomes especially among secondary school students. Intervention strategies such as limiting children’s use of electronic screen devices after school are implicated.