Adaptation of two-dimensional electrophoresis for muscle tissue analysis

It is important to understand the molecular mechanisms that take place in muscle tissues and to predict meat quality characteristics. One of the most popular methods is two-dimensional electrophoresis, which allows us to visualize, share and identify different molecules, including meat proteins. However, the standard conditions of this method are not universal for all types of raw material, so the authors suggest a new variation of two-dimensional electrophoresis for muscle tissue analysis. Samples were tested by the classical version of isoelectric focusing (cathode buffer in the top and anode buffer in the bottom chamber of the electrophoresis cell) and its variation (anode buffer in the top and cathode buffer in the bottom chamber of the electrophoresis cell). Next, extruded gels were incubated in two different buffer systems: the first was equilibration buffer I (6 M urea, 20% w/v glycerol, 2% w/v SDS and 1% w/v Ditiothreitol in 375 mM Tris-HCl buffer, pH 8.8) followed by equilibration buffer II (6 M urea, 20% w/v glycerol, 2% w/v SDS and 4% w/v iodoacetamide in 375 mM Tris-HCl buffer pH 8.8 and the second, buffer А, consisting of 5 M urea, 2% w/v SDS, 5% v/v mercaptoethanol, 62.5 mM Tris-HCl buffer, pH 6.8 and 0.01% w/v bromophenol blue. Electrophoretic studies of muscle tissue revealed the best protein separation after changing the direction of the current (authors' variation), while no differences were detected after changing incubation buffers.

Numerical Optimization for Bottom Structure of ZGM Medium Speed Mill

The inner flow fields in the ZGM95 medium speed mill are numerically simulated by employing the commercial code of FLUENT. The results show that the static pressure and the flow rate at each nozzle ring is different from those of others due to the primary air entering the mill only from one side. Some baffles are proposed to be added in the bottom chamber of the mill to optimize the performance of medium speed mill, and the modified models are also simulated. A couple of fan-shaped baffles, which are arranged symmetrically in the bottom chamber, can improve the distribution of the flow fields efficiently in the mill while few pressure drop of the mill will increase. The structure with fan-shaped baffles is recommended to be an important choice of the structural optimization of the medium speed mill. The numerical investigations also indicate that the structural modification of mill should be as simple as possible.

Contribution of Decreased Migration of Monocytes to Impaired Inflammation in Gaucher Disease

Abstract 2162 Background: Gaucher disease (GD) is an autosomal recessively inherited lysosomal storage disorder caused by an inborn glucocerebrosidase (GBA) deficiency. This leads to the accumulation of glycolipids in macrophages, mainly in the bone marrow, spleen and liver, resulting in organ damage. However, recent studies suggest that clinical presentation of GD is primarily attributed to the impaired inflammatory process induced by “loaded macrophages”, rather than to their physical presence. This impairment could be explained by dysfunction of circulating monocytes, normally capable of migrating into sites of vascular injury and inflammation. The current in vitro study investigated potential changes in migratory capacity of GD-derived monocytes that would contribute to the inflammatory response. Methods: CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of untreated GD patients and healthy volunteers, using immunomagnetic bead separation (Miltenyi Biotec). Monocyte migration capacity was assessed with 5μm Polycarbonate Membrane Transwell inserts which contained either stromal cell-derived factor 1 (SDF1; CXCL12) (500 and 1000 ng/ml) or serum (0.25%) at the bottom chamber. Monocytes were allowed to migrate from the upper chamber through the membrane, over 4 hours. Migrating monocytes were collected from the bottom chamber and quantified by FACS. Blockage of the CXCR4 receptor was examined using AMD3100 (10μg/ml) added to the migration assay. Serum SDF-1 levels were evaluated by ELISA (Quntikine® human CXCL12/SDF-1α R&D systems). Results: Upon exposure to SDF-1, migration capacity of GD monocytes (n=7) was significantly lower than that observed in monocytes obtained from healthy volunteers (n=6), approaching 5.7% vs. 14%, p=0.02, for SDF1of 500 ng/ml, and 8.7% vs 21.8%, p=0.013, for SDF1of 1000 ng/ml). Similar results were observed in the presence of autologous serum: migration capacity of GD monocytes (n=7) was lower than demonstrated by healthy monocytes (n=5), (18.6% vs. 45%, p=0.05). Notably, substitution of “GD serum” (n=7) with “healthy serum” (n=7) resulted in significant improvement in migration capacity of GD-derived monocytes (from 18.6% to 33.6%, p=0.038), while a reciprocal substitution of the serum caused a marked reduction in migration of monocytes derived from healthy volunteers (34% instead of 45%). Blockage of the CXCR4 receptor led to a decreased migration capacity of healthy monocytes cultured in autologous serum (from 45.2% to 39.6%) (n=4). Remarkably, this blockage had no impact on migration of both healthy and GD-derived monocytes cultured with GD serum (n=6). However, GD monocytes treated with AMD3100 in the presence of healthy serum, exhibited a reduced migration rate compared to that observed in untreated cells (22.6% vs. 33.6%; p=0.06, n=7).CXCR4 expression on GD-derived monocytes appeared to be lower than that measured on cells derived from healthy donors [64.5% (n=6) vs. 83.6% (n=3)]. Of note, serum levels of SDF-1 were significantly increased in GD subjects compared to healthy ones (2603pg/ml vs 2039 pg/ml; p=0.004, n=10). Conclusions: GD patients exhibit an impaired SDF1-dependent migration of monocytes, despite expressing a significantly increased SDF-1serum level. The impaired migration capacity is partly explained by low levels of CXCR4 on monocyte surface. Additionally, the reduced migration appears to be dependent on as yet undetermined serum-derived factors, as demonstrated by the reversibility of this impairment with substitution of the GD serum for “healthy serum”. The observed reduction in migratory capacity may significantly contribute to the abnormal inflammation accounting for GD-associated vascular and bone complications. Further studies exploring the precise mechanisms involved in this process and their direct impact on GD-related organ damage are warranted. Disclosures: No relevant conflicts of interest to declare.

Placenta Derived Adherent Cells Modulate the Allogenic Mixed Lymphocyte Reaction Partially via an Indoleamine 2,3-Dioxygenase Mediated Mechanism.

Placenta Derived Adherent Cells (PDAC) are multipotent progenitor cells derived from human placental tissues. Previously we have reported that PDACs could suppress T-cell proliferation when added to in-vitro mixed lymphocyte reactions (PDAC-MLR) (Paludan C. et al, Blood. (ASH Annual Meeting Abstracts) 2006 108: Abstract 1737). Here we present aspects of the mechanism of this PDAC suppression property. We have found that PDACs modify cytokine production in the PDAC-MLR reaction in comparison to the MLR; TNF-α and IFN-γ are reduced 75% and 30% while TGF-β production is significantly increased. We have used a transwell assay system to investigate the cell-contact-dependency of the effects of PDACs on T-cell proliferation. The assay system comprised combinations of the MLR in the top chamber together with the PDAC-MLR, PDACs plus naive T cells or PDACs alone in the bottom chamber. Maximum inhibition of T cell proliferation of the MLR in the top insert could be achieved by placing the PDAC-MLR co-culture in the bottom chamber. Minimum suppression was obtained when placing PDACs plus naive T cells or PDACs alone in the bottom chamber. PDAC-MLR conditioned media could partially suppress the MLR reaction. Addition of L-tryptophan into the MLR with PDAC conditioned media completely abolished PDAC-induced suppression of T cell proliferation. Likewise, the addition of the 1-methyl tryptophan to the PDAC-MLR reaction could abolish the PDAC-induced suppression. These results suggested that the suppression of the MLR by PDACs was possibly due to the depletion of the essential amino acid L-tryptophan which could be due to up-regulation of indoleamine 2,3-dioxygenase (IDO). Quantitative RT-PCR analysis of IDO gene expression revealed that IDO was up-regulated by about 4000-fold when PDACs were co-cultured with activated T cells, but not when co-cultured with naive T cells. Experiments are ongoing to confirm the causative role of IDO, and other factors, in PDAC-suppression of T-cell proliferation. In summary, we believe that soluble factors including the production of pro-inflammatory cytokines may contribute to PDAC suppression of the MLR, that induction of soluble factors from PDACs is significantly augmented by T-cell activation and that IDO expression by PDACs during the PDAC-MLR reaction plays a significant and direct role in suppression of T cell proliferation by PDACs.

Chemokines and Cytokines Required for Migration of Mesechymal Stem Cells into Marrow or Injured Tissues.

Background: Intravenous infusion of mesenchymal stem cells (MSC) after chemotherapy are currently used to accelerate the hematopoietic recovery but their potential of migration and homing to bone marrow is poorly understood. Methods: Bone marrow MSC (BM-MSC) were isolated with the classical plastic adhesion method. Cultured MSC were identified through their expression of SH2, SH3, CD44, CD105 but lacked typical hematopoietic antigens: CD14, CD31, CD34, CD45 and CD62-e. The MSC migration was evaluated using matrigel invasion chamber assay. MSC were used at 5.105 cells/mL in the upper chamber. In the bottom chamber, different cytokines were tested for their chemoattractive properties: SDF-1 (100ng/mL), PDGF-bb (100ng/mL), IGF (0,5 μg/mL) and IL-6 (100ng/mL) in a serum free medium. BM-MSC conditionned medium (CM) at 10% was also used as chemoattractant. After 24 hours, cells that had not migrated were removed from the top of the insert by scrubbing with a cotton swab and cells that migrated through the membrane were counted after staining with 0,5% crystal violet. Results: We observed that MSC migration was greater in the presence of PDGF-bb, IL-6 or with 10% CM with respectively an increase of 6,6; 5,6 and 5 fold compared to spontaneous migration. Only a small number of MSC migrated in response to SDF-1, and this observation correlates with the very low expression of CXCR4 receptor (0,67±0,08%). Indeed, we demonstrated that CXCR4, the SDF-1 receptor was mainly intracytoplasmic expressed (74,9±1,3%). Conclusions: IL-6 and PDGF-bb are important cytokines produced by the bone marrow microenvironment or by injured tissues. In our study, we thus demonstrated that these chmokines could be involved in the migration of MSC to bone marrow as well as to damaged tissues. Mesenchymal stem cells migration Ctrl SDF-1 IGF-1 10%CM IL-6 PDGF-bb *number of cells evaluated/3 fields of view. Migrated MSC * 13,7±2,7 24,9±5 42,2±12,6 69±17,9 77,1±14,1 90,5±5,3

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

ABSTRACT A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.

Separating and Washing Coffee Harvested With Plastic Nets

A method was developed for cleaning and washing coffee harvested with plastic nets. The material collected was bulky due to large amounts of leaves. An air separator designed and constructed to separate foreign material from coffee beans and berries is described. It consists of a bladetype blower placed in a two-chamber tunnel. When the net-collected material is fed into the separator, the light components are separated in the upper chamber, while the heavier materials fall into the bottom chamber where a stronger air current separates most of the remaining foreign substances from the coffee. Separation of the material was more difficult when weather conditions were rainy or humid while the nets were on the ground. The separation was much easier when the material was collected during diy, windy weather. The capacity of the air separator for wet and sticky material was about 2 acres per 8-hour run; the capacity was about 4 acres per 8-hour run with dry material. The material obtained from the air separator consisted of coffee beans and berries, together with some extraneous matter. This material was fed into a horizontal washing machine where the remaining extraneous material was removed leaving the coffee ready for drying.

Improved Machine for Cleaning Coffee Harvested with Plastic Nets

This paper describes a new machine for cleaning coffee harvested with plastic nets, as well as the coupling of this machine to the horizontal washer. The machine consists of two blowers in two tunnel chambers. The material fed by a screw conveyor is taken by a rotating shaft and ejected by the action of the blower in the upper chamber. The separated material (coffee beans, berries, dirt and small twigs) which falls through the screen to the bottom chamber is carried by a water stream into the washing reel. Further separation of the above mentioned material is accomplished by the washer, the shaker screen, and the flotation tank. This machine, together with the washing reel, is capable of processing coffee from 4.3 acres in an 8-hour run with the screw conveyor set at a speed of 30 rpm. Reduction of the speed to 20 rpm also reduces the output to that of 1.9 acres in an 8-hour run.