Lactate-driven macrophage polarization in the inflammatory microenvironment alleviates intestinal inflammation.

Background: Lactate has long been considered an intermediate by-product of glucose metabolism. However, in recent years, accumulating evidence reveals that lactate has unique biological activities. In previous studies, lactate signaling was shown to inhibit inflammation. Furthermore, in vitro experiments have shown that lactate can promote the transformation of pro-inflammatory macrophages into anti-inflammatory macrophages. However, no in vivo studies have shown whether lactate can alleviate inflammation. Methods: RAW 264.7 macrophages were stimulated by LPS to induce an M1 phenotype, and cultured with low and high concentrations of lactate. The cells were then observed for phenotypic transformations and expression of inflammatory mediators and surface markers. The expression of inflammatory factors was also analyzed in the cell-free supernatant fraction. Further, a mouse model of DSS-induced colitis was established and treated with lactate. Colonic tissue injury was monitored by histopathological examinations. Results: The in vitro experiments showed that lactate promoted the transformation of activated macrophages to M2 phenotype and decreased the expression of TLR4-mediated NF-κB signaling proteins and inflammatory factors. In the DSS-induced colitis mouse model, lactate promoted the phenotypic transformation of macrophages in colonic tissue, reduced inflammation and organ damage, inhibited the activation of TLR4/NF-κB signaling pathway, decreased the serum levels of pro-inflammatory factors, increased the expression of anti-inflammatory factors, promoted the repair of the intestinal mucosal barrier and reduced the severity of colitis. Conclusions: Lactate inhibits the TLR/NF-κB signaling pathway and the production of pro-inflammatory factors by promoting polarization of macrophages. In addition, lactate promotesthe repair of the intestinal mucosal barrier and protects intestinal tissue in inflammation. Furthermore, lactate is relatively safe. Therefore, lactate is a promising and effective drug for treating inflammation through immunometabolism regulation.

Analysis of the Implementation Effect of College Curriculum Ideological and Political under the Background of Ecological Sustainable Development.

Our country is paying more and more attention to ecological issues. How to put ecological sustainable development in real life is a key problem. This article discusses the significance of integrating ecological sustainable development and college ideological political courses. Therefore, an experiment was designed to analyse the model of ecology and other courses. The final experiment showed the following: (1) The ecological sustainable development model can be well integrated into the students in their courses. Students can freely choose to study some quality assurance thought courses and reinforce their spiritual level. The mentality of this model can also self-optimize to solve students' troubles in choosing courses and consider the level of students intimately. (2) According to the experimental data of the figures and tables, it is concluded that the form "other professional courses" at my country is still very serious, and the popularization of ideological and political courses is not common, so it is a little troublesome to carry out work, but our system model is very characteristic. With its help, we have obtained the effect of the integration of ecological sustainable development and students' curriculum. The state has vigorously provided economic support for their development. Under their influence, students have gradually maintained their awareness of protecting the environment. Socioeconomic and environmental conditions are also improving. However, in the future, the times will change, and we will continue to update the system model to adapt to more challenges.

17-Year Follow-up of Comparing Mastoscopic and Conventional Axillary Dissection in Breast Cancer: A Multicenter, Randomized Controlled Trial.

INTRODUCTION: Longer follow-up was necessary to determine the exact value of mastoscopic axillary lymph node dissection (MALND). METHODS: From January 1, 2003, to December 31, 2005, 1027 patients with breast cancer were randomly assigned to two groups: MALND and CALND (conventional axillary lymph node dissection); 996 eligible patients were enrolled. RESULTS: The final cohort of 996 patients was followed for an average of 198 months. Events other than death differed significantly between the two cohorts (p = 0.0311; 46.3% in MALND and 53.2% in CALND, respectively). The sum of events other than death and deaths from other causes was much higher in the CALND (59.6%) than MALND (53.4%) group (p = 0.0494). The 17-year disease-free survival DFS rates were 36.7% for the MALND and 33.6% for the CALND group, respectively. There was a significant difference between the groups (p = 0.0306). Overall survival (OS) rates were 53.2% after MALND and 46.0% after CALND (p = 0.0119). MALND patients had much less axillary pain (p = 0.0000), numbness or paresthesia (p = 0.0000), arm mobility (p = 0.0000) and arm swelling on the operated side (p = 0.0000). Aesthetic appearance of the axilla was much better in the MALND than CALND group (p = 0.0000) at an average follow-up of 17 years. CONCLUSIONS: The use of MALND in breast cancer surgery not only decreases the relapse and arm complications but also improves long-term survival of patients. Therefore, MALND should be one of the preferred approaches for breast cancer surgery when ALND is needed. TRIAL REGISTRATION INFORMATION: The comparison of long-term outcomes of mastoscopic and conventional axillary lymph node dissection in breast cancer: a multicenter randomized control trial. ChiCTR-TRC-11001477, CHiCTR. First registration 08/14/2011.

Differential protein metabolism and regeneration in hypertrophic diaphragm and atrophic gastrocnemius muscles in hibernating Daurian ground squirrels.

NEW FINDINGS: What is the central question of this study? The aim was to investigate whether diaphragm hypertrophy and gastrocnemius atrophy during hibernation of Daurian ground squirrels involve differential regulation of protein metabolism and regeneration. What is the main finding and its importance? We clarified the differences in protein metabolism and muscle regenerative potential in the diaphragm and gastrocnemius of hibernating ground squirrels, reflecting the different adaptability of muscles. ABSTRACT: Are differences in the regulation of protein metabolism and regeneration involved in the different phenotypic adaptation mechanisms of muscle hypertrophy and atrophy in hibernators? Two fast-type muscles (diaphragm and gastrocnemius) in summer active and hibernating Daurian ground squirrels were selected to detect changes in cross-sectional area (CSA) and protein expression indicative of protein synthesis metabolism (protein expression of P-Akt, P-mTORC1, P-S6K1 and P-4E-BP1), protein degradation metabolism (MuRF1, atrogin-1, calpain-1, calpain-2, calpastatin, desmin, troponin T, Beclin1 and LC3-II) and muscle regeneration (MyoD, myogenin and myostatin). In the hibernation group compared with the summer active group, the CSA of the diaphragm muscle increased significantly by 26.1%, whereas the CSA of the gastrocnemius muscle decreased significantly by 20.4%. Our study also indicated that increased protein synthesis, decreased protein degradation and increased muscle regenerative potential contributed to diaphragm muscle hypertrophy, whereas decreased protein synthesis, increased protein degradation and decreased muscle regenerative potential contributed to gastrocnemius muscle atrophy. In conclusion, the differences in muscle regeneration and regulatory pattern of protein metabolism might contribute to the different adaptive changes observed in the diaphragm and gastrocnemius muscles of ground squirrels.

A Temporal Examination of Cytoplasmic Ca2 + Levels, Sarcoplasmic Reticulum Ca2 + Levels, and Ca2 + -Handling-Related Proteins in Different Skeletal Muscles of Hibernating Daurian Ground Squirrels.

To explore the possible mechanism of the sarcoplasmic reticulum (SR) in the maintenance of cytoplasmic calcium (Ca2+) homeostasis, we studied changes in cytoplasmic Ca2+, SR Ca2+, and Ca2+-handling proteins of slow-twitch muscle (soleus, SOL), fast-twitch muscle (extensor digitorum longus, EDL), and mixed muscle (gastrocnemius, GAS) in different stages in hibernating Daurian ground squirrels (Spermophilus dauricus). Results showed that the level of cytoplasmic Ca2+ increased and SR Ca2+ decreased in skeletal muscle fiber during late torpor (LT) and inter-bout arousal (IBA), but both returned to summer active levels when the animals aroused from and re-entered into torpor (early torpor, ET), suggesting that intracellular Ca2+ is dynamic during hibernation. The protein expression of ryanodine receptor1 (RyR1) increased in the LT, IBA, and ET groups, whereas the co-localization of calsequestrin1 (CSQ1) and RyR1 in GAS muscle decreased in the LT and ET groups, which may increase the possibility of RyR1 channel-mediated Ca2+ release. Furthermore, calcium pump (SR Ca2+-ATPase 1, SERCA1) protein expression increased in the LT, IBA, and ET groups, and the signaling pathway-related factors of SERCA activity [i.e., ß-adrenergic receptor2 protein expression (in GAS), phosphorylation levels of phospholamban (in GAS), and calmodulin kinase2 (in SOL)] all increased, suggesting that these factors may be involved in the up-regulation of SERCA1 activity in different groups. The increased protein expression of Ca2+-binding proteins CSQ1 and calmodulin (CaM) indicated that intracellular free Ca2+-binding ability also increased in the LT, IBA, ET, and POST groups. In brief, changes in cytoplasmic and SR Ca2+ concentrations, SR RyR1 and SERCA1 protein expression levels, and major RyR1 and SERCA1 signaling pathway-related factors were unexpectedly active in the torpor stage when metabolic functions were highly inhibited.

The role of the globular heads of the C1q receptor in TcdA-induced human colonic epithelial cell apoptosis via a mitochondria-dependent pathway.

BACKGROUND: Clostridioides (formerly Clostridium) difficile infection is the leading cause of antibiotic-associated colitis. Studies have demonstrated that C. difficile toxin A (TcdA) can cause apoptosis of many human cell types. The purpose of this study was to investigate the relationships among exposure to TcdA, the role of the receptor for the globular heads of C1q (gC1qR) gene and the underlying intracellular apoptotic mechanism in human colonic epithelial cells (NCM 460). In this study, gC1qR expression was examined using real-time polymerase chain reaction (PCR), western blotting and immunohistochemical staining. Cell viability was assessed by the water-soluble tetrazolium salt (WST-1) assay, and cell apoptosis was assessed by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Mitochondrial function was assessed based on reactive oxygen species (ROS) generation, changes in the mitochondrial membrane potential (ΔΨm) and the content of ATP. RESULTS: Our study demonstrated that increasing the concentration of TcdA from 10 ng/ml to 20 ng/ml inhibited cell viability and induced cell apoptosis (p < 0.01). Moreover, the TcdA-induced gC1qR expression and enhanced expression of gC1qR caused mitochondrial dysfunction (including production of ROS and decreases in the ΔΨm and the content of ATP) and cell apoptosis. However, silencing of the gC1qR gene reversed TcdA-induced cell apoptosis and mitochondrial dysfunction. CONCLUSION: These data support a mechanism by which gC1qR plays a crucial role in TcdA-induced apoptosis of human colonic epithelial cells in a mitochondria-dependent manner.

Freestanding Na3V2O2(PO4)2F/Graphene Aerogels as High-Performance Cathodes of Sodium-Ion Full Batteries.

Although sodium vanadium fluorophosphate, Na3(VO1-xPO4)2F1+2x (0 ≤ x ≤ 1), is a highly promising cathode candidate for sodium-ion batteries because of its stable structure and high working voltage, the low charge diffusion dynamics and the inactive materials used in traditional coating electrodes reduce the energy density of a sodium-ion full battery. Hence, Na3V2O2(PO4)2F/graphene aerogels (NVPF/GAs) with a three-dimensional continuous porous network are first prepared by coassembly and freeze-drying. The three-dimensional porous network helps to obtain a high NVPF content of 81 wt %, relieve the volume change for improving the cyclability, and enhance the wettability of the electrode with the electrolyte for accelerating the diffusion dynamics of sodium ions and electrons. As a directly used freestanding cathode without the use of any binder/collector, an optimized freestanding NVPF/GA electrode exhibits excellent cycling and rate performances compared to traditional coating electrodes. The average capacities at current densities of 0.2, 0.5, 1.0, 2.0, and 5.0 C are 135.4, 128.0, 125.1, 121.9, and 115.1 mA h g-1, respectively. Especially, it maintains a capacity retention of 100% after 1000 cycles at an ultrahigh current of 40 C. A sodium-ion full battery with the NVPF/GA cathode and the Sb/graphene/carbon anode attains a of 82.1 mA h g-1 without an obvious decline after 100 cycles.

Autophagy and Akt-mTOR signaling display periodic oscillations during torpor-arousal cycles in oxidative skeletal muscle of Daurian ground squirrels (Spermophilus dauricus).

Whether hibernation accelerates or suppresses autophagy is still unknown. In the current study, we examined changes in autophagy in oxidative soleus (SOL) muscle in summer active (SA), pre-hibernation (PRE), torpor (TOR), interbout arousal (IBA), and post-hibernation groups of Daurian ground squirrels (Spermophilus dauricus). Here, the SOL muscle showed no significant atrophy during hibernation in regard to muscle wet weight, fiber cross-sectional area, or MuRF1 protein level. Autophagy-related proteins beclin1 and Atg7 increased significantly, whereas LC3-II decreased significantly in the PRE group compared with the SA group. However, neither the expression nor activity of cathepsin L showed any differences between the SA and PRE groups. In addition, beclin1, LC3-II, and the LC3-II/LC3-I ratio increased, p62 decreased, LC3 puncta increased, p62 puncta decreased, and cathepsin L activity increased in the TOR group compared with the PRE group. In contrast, beclin1, LC3-II, and the LC3-II/LC3-I ratio decreased, p62 increased, LC3 puncta decreased, p62 puncta increased, and cathepsin L activity declined in the IBA group compared with the TOR group. Moreover, the phosphorylation of Akt (Ser473) and mTOR (Ser2448) changed significantly during hibernation and showed an inverse relationship with autophagy changes. In conclusion, autophagy proteins displayed periodic oscillation in the torpor-arousal cycle, which may be advantageous in maintaining SOL muscle mass during the entire hibernation period. Furthermore, the Akt-mTOR signaling was decreased in TOR and increased in IBA group in the SOL muscle of Daurian ground squirrels during hibernation.

Differential activation of the calpain system involved in individualized adaptation of different fast-twitch muscles in hibernating Daurian ground squirrels.

We examined the lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) muscles to determine whether differential activation of the calpain system is related to the degree of atrophy in these fast-twitch skeletal muscles during hibernation in Daurian ground squirrels (Spermophilus dauricus). Results from morphological indices showed various degrees of atrophy in the order LG > PL > EDL. Furthermore, all three muscles underwent fast-to-slow fiber-type conversion in hibernation. In regard to the calpain system in the LG muscle, cytosolic Ca2+ increased significantly in hibernation, followed by recovery in posthibernation. Furthermore, calpastatin expression significantly decreased, and calpain 1 and 2 expression significantly increased, which may be responsible for the increased degradation of desmin during hibernation compared with that during summer activity. In the EDL muscle, Ca2+ overload was observed during interbout arousal, and calpastatin showed an increase during hibernation and interbout arousal, which could explain the increased levels of troponin T during both periods compared with levels during summer activity. These findings suggest that cytosolic Ca2+ overload and subsequent calpain 1 and 2 activation may be an important mechanism of LG muscle atrophy during hibernation. Cytosolic Ca2+ homeostasis and high expression of calpain inhibitor calpastatin during hibernation may also be an important mechanism for the EDL muscle to maintain muscle mass. Thus, the differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy of different fast-twitch muscles during hibernation.NEW & NOTEWORTHY We found that the extent of both muscle atrophy and calpain system activation differed in fast-twitch lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) skeletal muscles in hibernating Daurian ground squirrels, but similar hierarchies in the order of LG > PL > EDL. The differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy in different fast-twitch muscles during hibernation.

Closely related viruses of the marine picoeukaryotic alga Ostreococcus lucimarinus exhibit different ecological strategies.

In marine ecosystems, viruses are major disrupters of the direct flow of carbon and nutrients to higher trophic levels. Although the genetic diversity of several eukaryotic phytoplankton virus groups has been characterized, their infection dynamics are less understood, such that the physiological and ecological implications of their diversity remain unclear. We compared genomes and infection phenotypes of the two most closely related cultured phycodnaviruses infecting the widespread picoprasinophyte Ostreococcus lucimarinus under standard- (1.3 divisions per day) and limited-light (0.41 divisions per day) nutrient replete conditions. OlV7 infection caused early arrest of the host cell cycle, coinciding with a significantly higher proportion of infected cells than OlV1-amended treatments, regardless of host growth rate. OlV7 treatments showed a near-50-fold increase of progeny virions at the higher host growth rate, contrasting with OlV1's 16-fold increase. However, production of OlV7 virions was more sensitive than OlV1 production to reduced host growth rate, suggesting fitness trade-offs between infection efficiency and resilience to host physiology. Moreover, although organic matter released from OlV1- and OlV7-infected hosts had broadly similar chemical composition, some distinct molecular signatures were observed. Collectively, these results suggest that current views on viral relatedness through marker and core gene analyses underplay operational divergence and consequences for host ecology.

Muscle-specific activation of calpain system in hindlimb unloading rats and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse.

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.

Distinct molecular signatures in dissolved organic matter produced by viral lysis of marine cyanobacteria.

Dissolved organic matter (DOM) plays a central role in the microbial ecology and biogeochemistry of aquatic environments, yet little is known about how the mechanism of DOM release from its ultimate source, primary producer biomass, affects the molecular composition of the inputs to the dissolved pool. Here we used a model marine phytoplankton, the picocyanobacterium Synechococcus WH7803, to compare the composition of DOM released by three mechanisms: exudation, mechanical cell lysis and infection by the lytic phage S-SM1. A broad, untargeted analytical approach reveals the complexity of this freshly sourced DOM, and comparative analysis between DOM produced by the different mechanisms suggests that exudation and viral lysis are sources of unsaturated, oxygen-rich and possibly novel biomolecules. Furthermore, viral lysis of WH7803 by S-SM1 releases abundant peptides derived from specific proteolysis of the major light-harvesting protein phycoerythrin, raising the possibility that phage infection of these abundant cyanobacteria could be a significant source of high molecular weight dissolved organic nitrogen compounds.

Proteomic analysis reveals the distinct energy and protein metabolism characteristics involved in myofiber type conversion and resistance of atrophy in the extensor digitorum longus muscle of hibernating Daurian ground squirrels.

Previous hibernation studies demonstrated that such a natural model of skeletal muscle disuse causes limited muscle atrophy and a significant fast-to-slow fiber type shift. However, the underlying mechanism as defined in a large-scale analysis remains unclarified. Isobaric tags for relative and absolute quantification (iTRAQ) based quantitative analysis were used to examine proteomic changes in the fast extensor digitorum longus muscles (EDL) of Daurian ground squirrels (Spermophilus dauricus). Although the wet weights and fiber cross-sectional area of the EDL muscle showed no significant decrease, the percentage of slow type fiber was 61% greater (P < 0.01) in the hibernation group. Proteomics analysis identified 264 proteins that were significantly changed (ratio < 0.83 or >1.2-fold and P < 0.05) in the hibernation group, of which 23 proteins were categorized into energy production and conversion and translation and 22 proteins were categorized into ribosomal structure and biogenesis. Along with the validation by western blot, MAPKAP kinase 2, ATP5D, ACADSB, calcineurin, CSTB and EIF2S were up-regulated in the hibernation group, whereas PDK4, COX II and EIF3C were down-regulated in the hibernation group. MAPKAP kinase 2 and PDK4 were associated with glycolysis, COX II and ATP5D were associated with oxidative phosphorylation, ACADSB was associated with fatty acid metabolism, calcineurin and CSTB were associated with catabolism, and EIF2S and EIF3C were associated with anabolism. Moreover, the total proteolysis rate of EDL in the hibernation group was significantly inhibited compared with that in the pre-hibernation group. These distinct energy and protein metabolism characteristics may be involved in myofiber type conversion and resistance to atrophy in the EDL of hibernating Daurian ground squirrels.

Identification of the optimal dose and calpain system regulation of tetramethylpyrazine on the prevention of skeletal muscle atrophy in hindlimb unloading rats.

Previous studies in our lab have shown that tetramethylpyrazine (TMP) could effectively attenuate disuse induced muscle atrophy. In order to screening out the optimal dose of tetramethylpyrazine (TMP) for protection against disuse induced muscle atrophy in hindlimb unloading (HLU) rats, in this study, we compared effects of 4 TMP doses on muscle wet weight (MWW), the ratios of muscle wet weight/body weight (MWW/BW) and muscle wet weight/dry weight (MWW/DW), fiber type composition, as well as cross-sectional area (CSA) in soleus (SOL) muscle. Consequently, we quantified optimal dose effects on both functional properties and protein expression (calpain-1, calpain-2, calpastatin and MuRF1) in SOL and extensor digitorum longus (EDL) muscles. Data indicated that the protective potential of TMP was dose-dependent: 60mg/kg TMP was most effective in terms of atrophy prevention. This dose reduced SOL MWW, MWW/BW and CSA muscle loss by 60, 60 and 54% (P<0.001), respectively. HLU-induced slow-to-fast fiber transition was reduced by 17% (P<0.01). 60mg/kg TMP also significantly lessened the decrease of contractile force, the increase of shorting velocity and fatigability induced by HLU. Besides, it also attenuated expressions of calpain-1 (SOL -8.6%, P<0.05; EDL -10.9%, P<0.05), calpain-2 (SOL -60%, P<0.001; EDL -32%, P<0.01) and MuRF1 expression (SOL -21%, P<0.001; EDL -10%, P<0.01), promoted the expression of calpastatin by 18% (P<0.05) in SOL muscle. Taken together, present study demonstrated that 60mg/kg body weight was the optimal dose of TMP against disuse induced muscle atrophy which effectively protected muscle function by inhibiting calpain-1, calpain-2 and MuRF1 expression, promoted calpastatin expression, especially in slow-twitch muscle.

Comparison of mastoscopic and conventional axillary lymph node dissection in breast cancer: long-term results from a randomized, multicenter trial.

OBJECTIVE: To compare the long-term results of mastoscopic axillary lymph node dissection (MALND) and conventional axillary lymph node dissection (CALND). PATIENTS AND METHODS: From January 1, 2003, through December 31, 2005, a group of 1027 consecutive patients with operable breast cancer were randomly assigned to 1 of 2 study groups: MALND and CALND. The median follow-up was 63 months. The primary end points of the study were operative outcomes, complication reduction, function conservation, and cosmetics. The secondary end points were disease-free and overall survival. RESULTS: The mean operative blood loss in the MALND group was less than in the CALND group (P<.001). The patients who underwent MALND had less axillary pain, numbness or paresthesias, and arm swelling (P<.001). The aesthetic appearance of the axilla in the MALND group was much better than that in the CALND group (P=.001 at 6 months and P=.002 at 24 months). A significant difference was found between the 2 groups in distant metastasis (P=.04). The disease-free survival rate was 64.5% in the MALND group and 60.8% in the CALND group (P=.88). The overall survival rate was 81.7% in the MALND group and 78.6% in the CALND group (P=.95). CONCLUSION: Compared with CALND, MALND has advantages in operative outcomes, complication reduction, function conservation, and cosmetics.

Fast DNA hybridization on a microfluidic mixing device based on pneumatic driving.

A novel fluid mixing strategy was developed which significantly enhanced the efficiency of DNA hybridization. A pneumatic micro-mixing device consisting of two pneumatic chambers and an underneath DNA microarray chamber was built up. The fluid in the array chamber was pneumatically pumped alternately by the two pneumatic chambers. The chaotic oscillatory flow caused by the pumping greatly intensified the fluidic mixing. A homogeneous distribution of the tracer dye solution in the microarray chamber was observed after 2s mixing with a pumping frequency of 24 Hz. Microarray DNA hybridization was substantially accelerated using this device, and the fluorescence intensity showed a plateau after oscillating 30s at room temperature. The corresponding signal level of the dynamic hybridization was 12.5-fold higher than that of the static hybridization performed at 42°C. A signal-to-noise ratio of 117 was achieved and the nonspecific adsorption of the targets to the sample array was minimized, which might be attributed to the strong shearing force generated during the pneumatic mixing process.

[Biological characteristics of rabbit bone marrow mesenchymal stem cells and their response to different growth factors].

This study was aimed to analyze the biological characteristics of rabbit bone marrow mesenchymal stem cells (rBM-MSCs) and their response to different growth factors. Rabbit BM-MSCs were separated from bone marrow mononuclear cells by using adherent cultivation. Biological characteristics were investigated by optical and electron microscopy. Immunophenotype of rBM-MSCs was measured by flow cytometry. The expression of collagen was detected by RT-PCR. Differentiation potential was identified by specific staining and RT-PCR. The response of rBM-MSCs to IL-1, 3, 8 and HGF with different concentrations were tested by MTT. The results showed that the rBM-MSCs gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology and could be cultured for over 15 passages. CD44 was highly expressed on F5 rBM-MSCs (32%) and CD45 was lowly expressed (4.7%). Type I collagen was highly expressed, while type II collagen was lowly expressed and type X collagen was not detected on rBM-MSCs using RT-PCR method. In various conditions inducting differentiation, rBM-MSCs could differentiate into the osteoblast, chondrocyte, adipocyte and neuron-like cells. The rBM-MSCs were sensitive to IL-3, even low concentration (10 ng/ml) of IL-3 could promote the proliferation of rBM-MSCs effectively (>32%, P < 0.01), whereas high concentration IL-3 inhibited it significantly. It is concluded that rabbit BM-MSCs were successfully isolated and culture-expanded. The biological characteristics of rabbit BM-MSCs are similar to those of human and rhesus BM-MSCs. IL-3 with low concentration can promote the proliferation of rBM-MSCs effectively, but high concentration of IL-3 can inhibit their proliferation.

High dose chemotherapy and transplantation of hematopoietic progenitors from murine D3 embryonic stem cells.

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. To date, characteristics of specific factors capable of influencing hematopoietic cell fate from ES cells remains elusive. We report that mMSC Feeder Layer and the combination of VEGF, SCF and TPO strongly promote hematopoietic differentiation. The results showed that the cells induced from ES-D3 expressed hematopoietic progenitor antigens (CD34 and CD117), myelocyte cell antigen (CD11b), erythrocyte cell antigen (Ter119), and transcription factors (Flk-1, GATA-2, SCL, beta-H1 and beta-major). Furthermore, those induced differentiated cells were injected into female C57BL/6 mice which were treated with high dose topotecan chemotherapy to restore part of their blood system function. We observed rapid white blood cell recovery, which suggested that the infusion of differentiated cells has a positive impact on hematopoiesis. The Sry gene in peripheral blood, bone marrow and spleen of transplanted female mice was confirmed by PCR analysis, which affirmed the existence of the chimera.

Double chimerism in recipient by transplantation of two allogeneic MHC-mismatched mouse fetal blood units.

We have constituted a mouse model for fetal blood transplantation (FBT) to cross over major histocompatibility complex (MHC) without causing serious GVHD. It seems that full matching at the MHC appears not necessary for FBT, while the nucleated cell dose is critical. Two fetal blood units were combined from different donors to increase the stem/progenitor cell dose so as to explore the possibility of MHC-mismatched allogeneic transplantation. 26 out of 40 mice in mixed FBT group survived in the observation period of 60 days after transplantation without obvious GVHD. Double chimerism was demonstrated by PCR and flow cytometric analysis; and skin transplantation test proved the induction of donor specific immune tolerance. Our data suggest that two MHC-mismatched allogeneic donor fetal blood units could simultaneously engraft and reconstitute immune and hematopoietic system in a mouse model. The result may be beneficial for the expansion of cord blood application and enables more patients to share the advantages of cord blood transplantation.

[Effects of HLA disparity of two umbilical cord blood units on human engraftment in SCID mice].

OBJECTIVE: To evaluate the feasibility and characteristics of human engraftment in HLA disparate cord blood transplantation. METHODS: Two human HLA-haploidentical or HLA-mismatched cord blood units were transplanted into sublethally irradiated severe combined immunodeficiency (SCID) mice. The characteristics of engraftment, hematopoietic and immunological reconstitution between the two groups were compared. RESULTS: Two mixed cord blood units can engraft in SCID mice with donor-recipient chimerism and reconstitute hematopoiesis and immunological functions. No unfavorable factors had been observed. Only one of the two cord blood units which had higher colony forming ability in vitro could engraft in most SCID mice as shown by HLA-DQB(1) gene detection. Two HLA-haploidentical cord blood units were simultaneously engrafted in 3 SCID mice. CONCLUSION: Double HLA-haploidentical or HLA-mismatched cord blood can engraft in SCID mice and reconstitute hematopoietic and immunological functions. HLA disparity has no significant effect on survival and engrafting rate. However, in less HLA disparity group, two cord blood units were prone to engraft simultaneously.