Synthesis and utilization of E. coli‐encapsulated PEG‐based microdroplet using a microfluidic chip for biological application

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single‐step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non‐toxic ways of fabricating microorganism‐encapsulated hydrogel microbeads. Due to these unique properties of micro‐sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl‐β‐D ‐thiogalactopyranoside induction, suggesting that there is no low‐molecular weight substrate transfer limitation inside of microbeads. Furthermore, non‐toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells. Biotechnol. Bioeng. 2010;107:747–751. © 2010 Wiley Periodicals, Inc.     

Increased Store-Operated Ca Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Store-operated Ca²⁺ entry (SOCE) contributes to Ca²⁺ handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn²⁺ fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca²⁺ store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39°C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca²⁺ permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca²⁺ as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca²⁺ entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca²⁺ signaling in MH.     

Quantification of Mineralized Bone Response to Prostate Cancer by Noninvasive In Vivo μCT and Non-Destructive Ex Vivo μCT and DXA in a Mouse Model

Background

To compare nondestructive in vivo and ex vivo micro-computed tomography (μCT) and ex vivo dual-energy-X-ray-absorptiometry (DXA) in characterizing mineralized cortical and trabecular bone response to prostate cancer involving the skeleton in a mouse model.

Methodology/Principal Findings

In vivo μCT was performed before and 10 weeks after implantation of human prostate cancer cells (MDA-PCa-2b) or vehicle into SCID mouse femora. After resection, femora were imaged by nondestructive ex vivo specimen μCT at three voxel sizes (31 µ, 16 µ, 8 µ) and DXA, and then sectioned for histomorphometric analysis of mineralized bone. Bone mineral density (BMD), trabecular parameters (number, TbN; separation, TbSp; thickness, TbTh) and mineralized bone volume/total bone volume (BV/TV) were compared and correlated among imaging methods and histomorphometry. Statistical tests were considered significant if P<0.05. Ten weeks post inoculation, diaphyseal BMD increased in the femur with tumor compared to the opposite femur by all modalities (p<0.005, n = 11). Diaphyseal BMD by in vivo μCT correlated with ex vivo 31 and 16 µm μCT and histomorphometry BV/TV (r = 0.91–0.94, P<0.001, n = 11). DXA BMD correlated less with bone histomorphometry (r = 0.73, P<0.001, n = 11) and DXA did not distinguish trabeculae from cortex. By in vivo and ex vivo μCT, trabecular BMD decreased (P<0.05, n = 11) as opposed to the cortex. Unlike BMD, trabecular morphologic parameters were threshold-dependent and when using “fixed-optimal-thresholds,” all except TbTh demonstrated trabecular loss with tumor and correlated with histomorphometry (r = 0.73–0.90, P<0.05, n = 11).

Conclusions/Significance

Prostate cancer involving the skeleton can elicit a host bone response that differentially affects the cortex compared to trabeculae and that can be quantified noninvasively in vivo and nondestructively ex vivo.     

Les larges vacuoles des têtes spermatiques sont-elles associées à des altérations du noyau ou de l’acrosome du spermatozoïde ?

Objective

The aim of this study was to detect acrosome and nucleus alterations in isolated spermatozoa with large vacuoles detected by MSOME (Motile Sperm Organelle Morphology Examination), named type 3 spermatozoa and defined by the presence of one or more vacuoles occupying more than 13% of the sperm head area.

Material and methods

Twenty infertile men were included in this study. Whole sperm and isolated spermatozoa were compared. Spermatozoa acrosome and nucleus were explored using 1) proacrosin immunostaining with a monoclonal antibody (4D4), 2) DNA fragmentation with TUNEL assay, 3) chromatin condensation with aniline blue staining, and 4) aneuploidy after fluorescence in situ hybridization (FISH) and analysis by electron transmission and confocal microscopy.

Results

Acrosome abnormalities were significantly increased in type 3 spermatozoa compared towhole sperm(77.8 ± 2.49% vs. 70.6 ± 2.62%). DNA fragmentation was similar in type 3 spermatozoa compared towhole sperm(14.5 ± 3.45%vs. 11.5 ± 1.25%). Chromatin condensation was significantly altered in isolated spermatozoa as well as aneuploidy frequencies (50.4 ± 3.10% vs. 26.5 ± 2.60% and 7.8 ± 1.98% vs. 1.3 ± 0.18%). Large vacuoles have an exclusive nuclear location, confirmed by electron and confocal microscopy.

Conclusion

Large vacuoles are probably due to sperm nucleus maturation dysfunction during spermiogenesis.     

Persistence of Genetically Modified Potatoes in the Field

Volunteers from genetically modified (GM) potatoes may pose an environmental problem if allowed to grow in the field after the annual crop is harvested. We tested whether they are more likely to produce volunteers than non-GM potatoes. Specifically, we compared the number of volunteers, number of tubers per plant, tuber size, and their vertical distribution in the soil. More volunteer plants came from non-GM potatoes than from GM potatoes, but the number and size of tubers were similar between the two. Vertical distribution of the tubers differed significantly, with most non-GM tubers being found in shallower soil (<2 cm deep). Our results suggest that spontaneous GM volunteers may emerge and produce tubers to a degree similar to that of the non-GM plants. No viable volunteers emerged from GM tubers in the next growing season, probably deterred by winter frost and a period of low soil temperatures (below −2°C) at our study site. However, in regions with warmer climates, such GM volunteers may survive Winter and produce more plants the following year.     

Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages

Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in?vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.     

Salt tolerance in <Emphasis Type="Italic">Solanum pennellii</Emphasis>: antioxidant response and related QTL

Background  

Excessive soil salinity is an important problem for agriculture, however, salt tolerance is a complex trait that is not easily bred into plants. Exposure of cultivated tomato to salt stress has been reported to result in increased antioxidant content and activity. Salt tolerance of the related wild species, Solanum pennellii, has also been associated with similar changes in antioxidants. In this work, S. lycopersicum M82, S. pennellii LA716 and a S. pennellii introgression line (IL) population were evaluated for growth and their levels of antioxidant activity (total water-soluble antioxidant activity), major antioxidant compounds (phenolic and flavonoid contents) and antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase) under both control and salt stress (150 mM NaCl) conditions. These data were then used to identify quantitative trait loci (QTL) responsible for controlling the antioxidant parameters under both stress and nonstress conditions.     

Development of strain-specific PCR primers for the identification of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T and subsp. vincentii ATCC 49256T

The objective of this study was to develop the strain-specific PCR primers for Fusobacterium nucleatum subsp. fusiforme ATCC 51190^T and F. nucleatum subsp. vincentii ATCC 49256^T based on the nucleotide sequence of the Fs17 and Fv35 DNA probes, respectively. The strain specificity was tested against 10 type strains of Fusobacterium spp. or subsp., 21 clinical isolates of F. nucleatum from Koreans, and five type strains of distinct Fusobacterium species. Primer sensitivity was determined by testing serial dilutions (4 ng–4 fg) of the purified genomic DNA from each of the type strains. PCR showed that two pairs of PCR primers, Fs17-F14/Fs17-R14 and Fv35-F1/Fv35-R1 primers, could produce strain-specific amplicons from F. nucleatum subsp. fusiforme ATCC 51190^T and F. nucleatum subsp. vincentii ATCC 49256^T, respectively. The two PCR primer sets could detect as little as 0.4 pg or 4 pg of the genomic DNA of each target strain. These results suggest that the two sets of PCR primers could be used to identify F. nucleatum subsp. fusiforme ATCC 51190^T and F. nucleatum subsp. vincentii ATCC 49256^T, particularly for ascertaining the authenticity of the strain.