Classical dot–blot format implemented as an amperometric hybridisation genosensor

A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot–blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method—that increases the genosensor sensitivity—are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin–DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot–blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot–blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.     

Tribological Behavior of Gampsocleis Gratiosa Foot Pad Against Vertical Flat Surfaces

Some tribological behavior between mature Gampsocleis gratiosa foot pads and vertical flats of different materials were studied in this work. stereomicroscope （SMS） and scanning electron microscope （SEM） were used to measure the morphology of the Gampsocleis gratiosa foot pads. An atomic force microscope （AFM） was used to measure the morphologies of the surfaces of glass and a wall doped with calcium carbonate material. The attaching behavior of Gampsocleis gratiosa feet on the two vertical surfaces was observed. The attaching force （perpendicular to the vertical surface） and the static frictional force （along the direction of gravitation） of Gampsocleis gratiosa foot pads on a vertical glass were measured. It was shown that the average attaching force is 50.59 mN and the static frictional force is 259.10 mN. The physical models of the attaching interface between Gampsocleis gratiosa foot pads and the two vertical surfaces were proposed. It was observed that the foot pads are smooth in macroscale; however, the pad surface is composed by approximate hexagonal units with sizes of 3 μm to 7 μm in microscale; the adjacent units are separated by nanoscale grooves. The Observations showed that the Gampsocleis gratiosa can not climb the vertical calcium carbonate wall; in contrast, they can easily climb the vertical glass surface. Based on the features of the geometrical morphologies of the foot pads and the glass surface, we speculate that the attaching force and strong static frictional force are attributed to the interinlays between the deformable Gampsocleis gratiosa foot pads and the nanoscale sharp tips of the glass surface.     

The proliferation of mouse mammary epithelial cells in response to specific mitogens is modulated by the mammary fat pad in vitro

Summary The ability of the murine mammary fat pad to directly stimulate the growth of mammary epithelial cells and to modulate the effects of various mammogenic agents has been investigated in a newly described, hormone- and serum-free coculture system. COMMA-1D mouse mammary epithelial cells were cultured for 5 or 7 d with various supplements in the absence or presence of epithelium-free mammary fat pad explants from virgin female BALB/c mice. Cocultured fat pad stimulated increases in the DNA content of COMMA-1D cultures by two- to threefold or six-to eightfold after 5 or 7 d, respectively. The mitogenic effect was additive to that of 10% fetal calf serum and could not be attributed to the release of prostaglandin E₂ or synthesis of prostaglandins by epithelial cells. In addition, bovine serum albumin attenuated (P<0.05) the mitogenic effect of cocultured mammary fat pad. Added alone, insulinlike growth factor-I, epidermal growth factor, and insulin increased (P<0.05) total DNA of COMMA-1D cultures by 2.5-, 3.7-, and 2.3-fold, respectively. Cocultured mammary fat pad markedly interacted (P<0.01) with these mitogens to yield final DNA values that were 21.2-, 13.3-, and 22.1-fold greater than in basal medium only. Associated with this proliferation was the formation of numerous domes above the COMMA-1D monolayer. There was no proliferative response to growth hormone or prolactin in the absence or presence of cocultured fat pad (P>0.05). Whereas hydrocortisone did not alter cell number, it attenuated (P<0.05) the mitogenic effect of cocultured mammary fat pad. These results indicate that the murine mammary fat pad is not only a direct source of mitogenic activity, but also modulates the response of mammary epithelial cells to certain mammogens.     

Plasmodium chabaudi: Adoptive transfer of immunity with different spleen cell populations and development of protective activity in the serum of lethally irradiated recipient mice

Groups of lethally X-irradiated NIH mice were injected with either glass wool-filtered (g.w.) immune spleen cells or nylon wool enriched immune T cells from syngeneic mice immune to Plasmodium chabaudi, or g.w. normal spleen cells. After cell recipients were infected with P. chabaudi the three groups reached similar mean peak parasitaemias on Day 11. In passive transfer tests serum obtained from mice sacrificed at this time gave little protection compared to normal serum. On Day 14 g.w. immune spleen cell recipients had subpatent infections and enriched immune T-cell recipients had a lower mean parasitaemia than g.w. normal spleen cell recipients. Serum obtained on Day 14 from g.w. immune spleen cell recipients gave better protection after passive transfer than sera from enriched immune T-cell or g.w. normal spleen cell recipients. Day 14 serum from enriched immune T-cell recipients, but not from g.w. normal spleen cell recipients, produced some initial protection after passive transfer. These results suggest that the transferred immune spleen cells contributed to the observed humoral immunity in lethally irradiated recipient mice.     

尼龙网固定化果胶酶的制备及其性质研究

用尼龙网作载体,经3-二甲氨基丙胺活化,用戊二醛将果胶酶固定化。所得固定化酶Km值与自然酶接近;对温度的稳定性有较大的提高,100℃保温30min才能使其失活。固定化酶在较宽的pH范围内能保持其正常活力,它对金属离子抑制剂的耐受性有较显著的提高,用0.5％果胶溶液作底物,重复使用10次后酶活力保留44％。固定化果胶酶与自然酶相比较,对不同果汁的澄清效果不同。固定化果胶酶在无保护剂存在的条件下,室温放置四个月活力不减少。     

High endocytotic and lysosomal activities in segments of rat myotubes differentiated in vitro

Summary The entire microvascular architecture in rat foot-pads including that of eccrine sweat glands was studied by scanning electron microscopy using a vascular corrosion-cast replication technique. In the central roofs of the pads, particularly elaborate capillary networks were arranged in rows perpendicular to the long axis of the foot. In the marginal regions of the pads, simple networks of capillaries were arranged in lamellar sheets parallel to the surface of the sole of the foot. Complex spongy networks of vascular trees were observed in the subcutaneous layer of the pads. These vessels were supplied by the pad artery, and then, after forming capillary networks in the roofs of the pads, they drained into the metatarsal vein. Rod-shaped cages of capillaries were observed around the eccrine sweat glands. One descending arteriole, arising from a connecting arteriole, and a few venules were connected with these capillary cages at their upper and lateral sides. Occasional arterio-venous and veno-venous anastomoses were also observed around the eccrine sweat glands. This microvascular architecture may adjust well to the mechanical and physiological conditions encountered in the foot-pads. The relation of the microvascular architecture around the eccrine sweat glands with their development is also discussed.     

Gene expression,protein profiling,and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.     

Development of functional glow-in-the-dark photoluminescence linen fabrics with ultraviolet sensing and shielding

Linen fibres were coated with a glow-in-the-dark photoluminescence, flame-retarding, and hydrophobic smart nanocomposite using the pad-dry-curing process. Ecologically friendly ammonium polyphosphate and lanthanide-activated strontium aluminium oxide (LSAO) nanoparticles were immobilized into linen fabric using eco-friendly room-temperature-vulcanizing silicone rubber. Different analytical techniques were used to examine the morphological characteristics and elemental compositions of LSAO nanoparticles and treated linen textiles. The self-extinguishing properties of the treated linen textiles were tested for their fire resistance. After 24 washing cycles, the coated linen samples retained their flame-retarding properties. The treated linen's superhydrophobicity rose in direct proportion to the LSAO concentration. After being excited at 365 nm, the colourless luminescent film that was coated on linen surface gave out an emission wavelength of 519 nm. The photoluminescent linen was monitored to create a range of different colours, including off-white in daytime light and green under ultraviolet (UV) light radiation, according to the Commission Internationale de l'éclairage laboratory colorimetric coordinates and photoluminescence spectra. Emission, excitation, and lifetime spectral analysis of the treated linen revealed persistent phosphorescence. For mechanical and comfort evaluation, the coated linen textiles' bending length and air permeability were assessed. Good UV light shielding and enhanced antibacterial activity were detected in the treated linens.     

Bisulfite-modified target DNA array for aberrant methylation analysis

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.