Molecular characterization of a CXCL8-like protein from ayu and its effect on chemotaxis of neutrophils and monocytes/macrophages

CXCL8, a CXC-type chemokine, plays a crucial role in acute inflammation by recruiting and mediating neutrophils and other cells. In this study, the cDNA and genomic DNA sequence of a CXCL8-like protein (PaCXCL8l) from ayu (Plecoglossus altivelis) was determined. Sequence analysis showed that PaCXCL8l represented the typical structure of animal CXCL8s. Phylogenetic tree analysis indicated that PaCXCL8l was closest to CXCL8 of Atlantic cod (Gadus morhua). Constitutive expression of PaCXCL8l was detected in all tested tissues and monocytes/macrophages, and its expression dramatically increased upon Listonella anguillarum infection. In vitro, recombinant PaCXCL8l exhibited a significant chemotactic effect on neutrophils at 0.1 μg/ml and on monocytes/macrophages at 1.0 μg/ml. In vivo, the numbers of peritoneal neutrophils and monocytes/macrophages were both up-regulated following intraperitoneal administration of recombinant PaCXCL8l. These results suggest that PaCXCL8l is crucially involved in the immune response of ayu by mediating chemotaxis of neutrophils and monocytes/macrophages.     

Prognostic Value of Myeloid Differentiation Primary Response 88 and Toll-Like Receptor 4 in Breast Cancer Patients

Purpose

Breast cancer remains a major cause of death in women worldwide, and tumor metastasis is the leading cause of death in breast cancer patients after conventional treatment. Chronic inflammation is often related to the occurrence and growth of various malignancies. This study evaluated the prognosis of breast cancer patients based on contributors to the innate immune response: myeloid differentiation primary response 88 (MyD88) and Toll-like receptor 4 (TLR4).

Methods

We analyzed data from 205 breast invasive ductal carcinoma (IDC) patients who were treated at the Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Fudan University Shanghai Cancer Center, from 2002 to 2006. Overall survival (OS) and disease-free survival (DFS) were compared.

Results

In total, 152 patients (74.15%) were disease-free without relapse or metastasis, whereas 53 (25.85%) patients developed recurrence or metastasis. A significant positive correlation was observed between MyD88 and TLR4 expression (p<0.001). Patients with high expression were more likely to experience death and recurrence/metastasis events (p<0.05). Patients with low MyD88 or TLR4 expression levels had better DFS and OS than patients with high expression levels (log-rank test: p<0.001). Patients with low MyD88 and TLR4 expression levels had better DFS and OS than patients with high expression levels of either (log-rank test: p<0.001). In a multivariate analysis, high MyD88 expression was an independent predictive factor for decreased DFS (adjusted HR, 3.324; 95% CI, 1.663–6.641; p = 0.001) and OS (adjusted HR, 4.500; 95% CI, 1.546–13.098; p = 0.006).

Conclusions

TLR4-MyD88 signaling pathway activation or MyD88 activation alone may be a risk factor for poor prognosis in breast cancer. Therefore, TLR4-MyD88 signaling pathway activation in tumor biology provides a novel potential target for breast cancer therapy.     

蕨类植物碳氮磷化学计量特征及其与土壤养分的关系

为探讨蕨类植物碳氮磷化学计量特征与土壤养分的关系,对福建省亚热带森林林下芒萁和乌毛蕨地上部分和地下部分的碳、氮、磷(C、N、P)含量和0~10 cm和10~20 cm两个土层的养分含量进行了测定。结果表明,无论是芒萁还是乌毛蕨,地上部分的N、P含量均高于地下部分,而C含量则无显著差异,导致地上部分的C∶N和C∶P均低于地下部分。与乌毛蕨相比,芒萁地上部分的N、P含量更低,地上和地下部分的C含量、C∶N和C∶P以及N、P含量的变异系数和表型可塑性指数则更高,表明芒萁采取了较高的养分利用效率和"表现最大化"的策略,而乌毛蕨则选择了较低的养分利用效率和"表现维持"的方式。两种蕨类植物地上和地下部分的N含量与土壤N含量(0~20 cm)均无显著相关。芒萁两个部位的P含量则均与土壤P含量(0~10 cm和10~20 cm)呈显著正相关,乌毛蕨P含量总体上与土壤P含量的相关性不显著(除地下部分的P含量与10~20 cm土层的P含量呈弱的正相关外)。这表明芒萁具有作为亚热带森林土壤P库指示植物的潜力。     

铅、铬胁迫对大弹涂鱼血细胞遗传损伤的彗星实验检测

通过彗星实验研究重金属Pb、Cr对大弹涂鱼的外周血细胞的影响。用不同浓度的Pb、Cr对大弹涂鱼外周血细胞进行1 h的染毒后通过单细胞凝聚电泳检测DNA损伤情况。结果表明国标浓度的Pb（0.005 mg/L）、Cr（0.05 mg/L）对大弹涂鱼外周血细胞均无明显影响;而国标10倍、100倍和1000倍浓度的Pb或Cr胁迫均会造成血细胞DNA损伤,且离子浓度与血细胞DNA的损伤程度间均存在＂剂量-效应＂,即浓度越高,DNA损伤越严重。因此大弹涂鱼血细胞可作为评价重金属遗传损伤毒性效应的敏感性生物标志物。     

Effects of High-Pressure Oxygen Therapy on Brain Tissue Water Content and AQP4 Expression in Rabbits with Cerebral Hemorrhage

To investigate the effects of different atmosphere absolutes (ATA) of high-pressure oxygen (HPO) on brain tissue water content and Aquaporin-4 (AQP4) expression in rabbits with cerebral hemorrhage. 180 New Zealand white rabbits were selected and randomly divided into normal group (n = 30), control group (n = 30) and cerebral hemorrhage group (n = 120), and cerebral hemorrhage group was divided into group A, B, C and D with 30 rabbits in each group. The groups received 1.0, 1.8, 2.0 and 2.2 ATA of HPO treatments, respectively. Ten rabbits in each group were killed at first, third and fifth day to detect the brain tissue water content and change of AQP4 expression. In cerebral hemorrhage group, brain tissue water content and AQP4 expression after model establishment were first increased, then decreased and reached the maximum on third day (p < 0.05). Brain tissue water content and AQP4 expression in control group and cerebral hemorrhage group were significantly higher than normal group at different time points (p < 0.05). In contrast, brain tissue water content and AQP4 expression in group C were significantly lower than in group A, group B, group D and control group (p < 0.05). In control group, AQP4-positive cells significantly increased after model establishment, which reached maximum on third day, and positive cells in group C were significantly less than in group A, group B and group D. We also found that AQP4 expression were positively correlated with brain tissue water content (r = 0.719, p < 0.05) demonstrated by significantly increased AQP4 expression along with increased brain tissue water content. In conclusion, HPO can decrease AQP4 expression in brain tissue of rabbits with cerebral hemorrhage to suppress the progression of brain edema and promote repairing of injured tissue. 2.0 ATA HPO exerts best effects, which provides an experimental basis for ATA selection of HPO in treating cerebral hemorrhage.     

微小RNA miR-21在皮肤创伤愈合中的研究进展

微小RNA是一类真核细胞中广泛存在的内源性转录后调控分子,其在细胞的增殖、分化、凋亡、迁移等过程中发挥了重要的调控作用。皮肤创伤修复涉及复杂的细胞与分子的相互作用网络。近年来研究表明micro RNAs在皮肤创伤修复中发挥调控作用,引人关注。miR-21作为重要的癌基因是目前研究的最多的miRNAs分子之一,其在皮肤创伤修复中的作用研究也越来越受到重视。研究表明miR-21参与了细胞增殖与迁移、炎症反应、血管生成和细胞外基质合成等重要修复相关事件的调控。因此,阐明miR-21分子在正常皮肤创伤愈合中的作用,厘清miR-21表达失调在修复不足和修复过度中的功能,将深化我们对于皮肤创伤愈合基本理论的认识,并为促进创面愈合与防治修复不足和过度提供潜在的治疗靶点。本文就miR-21分子在正常皮肤创伤修复、慢性难愈性创面和增生性瘢痕中作用的研究进展进行综述展望。     

Dynamic process of phagocytosis and forms of macrophage cell death induced by ingestion of apoptotic neutrophils

Clearance of apoptotic neutrophils by macrophages is important for both the successful resolution of acute inflammation and homeostasis.However,the dynamic process of phagocytosis of apoptotic neutrophils by macrophages and the fate of macrophages after the ingestion of apoptotic neutrophils has not been well documented.In the present study,we staged the recognition and tethering,internalization,digestion and exocytosis steps of phagocytosis of apoptotic neutrophils.Furthermore,we found that after the ingestion of apoptotic cells,a subset of macrophages underwent cell death by autophagy,apoptosis or oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes.The percentage of autophagic,apoptotic and oncotic macrophages were 8.00%±2.00%,12.33%±2.08%,and 3.66%±1.50%,respectively.These results indicated that after ingestion of apoptotic neutrophils,a subset of macrophages undergoes autophagy and apoptosis.We propose that autophagy of macrophages after the ingestion of apoptotic cells may be a new mechanism present in the resolution of inflammation.     

Prenatal Exposure to Maternal Bereavement and Childbirths in the Offspring: A Population-Based Cohort Study

IntroductionThe decline in birth rates is a concern in public health. Fertility is partly determined before birth by the intrauterine environment and prenatal exposure to maternal stress could, through hormonal disturbance, play a role. There has been such evidence from animal studies but not from humans. We aimed to examine the association between prenatal stress due to maternal bereavement following the death of a relative and childbirths in the offspring.ResultsA total of 4,121,596 subjects were followed-up until up to 41 years of age. Of these subjects, 93,635 (2.3%) were exposed and 981,989 (23.8%) had at least one child during follow-up time. Compared to unexposed, the hazard ratio (HR) [95% confidence interval] of having at least one child for exposed males and females were 0.98 [0.96–1.01] and 1.01 [0.98–1.03], respectively. We found a slightly reduced probability of having children in females born to mothers who lost a parent with HR = 0.97 [0.94–0.99] and increased probability in females born to mothers who lost another child (HR = 1.09 [1.04–1.14]), the spouse (HR = 1.29 [1.12–1.48]) or a sibling (HR = 1.13 [1.01–1.27]).ConclusionsOur results suggested no overall association between prenatal exposure to maternal stress and having a child in early adulthood but a longer time of follow-up is necessary in order to reach a firmer conclusion.     

Evaluation of the Quality of Guidelines for Myasthenia Gravis with the AGREE II Instrument

Background

Clinical practice guidelines (CPGs) are systematically developed statements to assist practitioners in making decisions about appropriate healthcare in specific clinical circumstances. The methodological quality of CPGs for myasthenia gravis (MG) are unclear.

Objective

To critically evaluate the methodological quality of CPGs for MG using AGREE II instrument.

Method

A systematical search strategy on PubMed, EMBASE, DynaMed, the National Guideline Clearinghouse (NGC) and the Chinese Biomedical Literature database (CBM) was performed on September 20th 2013. All guidelines related to MG were evaluated with AGREE II. The software used for analysis was SPSS 17.0.

Results

A total of 15 CPGs for MG met the inclusion criteria (12 CPGs in English, 3 CPGs in Chinese). The overall agreement among reviews was moderate or high (ICC >0.70). The mean scores (mean ± SD) for al six domains were presented as follows: scope and purpose (60.93% ±16.62%), stakeholder involvement (40.93% ±20.04%), rigor of development (37.22% ±30.46%), clarity of presentation (64.26% ±16.36%), applicability (28.19% ±20.56%) and editorial independence (27.78% ±28.28%). Compared with non-evidence-based CPGs, evidence-based CPGs had statistically significant higher quality scores for all AGREE II domains (P<0.05). All domain scores appear slightly higher for CPGs published after AGREE II instrument development and validation (P>0.05). The quality scores of CPGs developed by NGC/AAN were higher than the quality scores of CPGs developed by other organizations for all domains. The difference was statistically significant for all domains with the exception of clarity of presentation (P = 0.07).

Conclusions

The qualities of CPGs on MG were generally acceptable with several flaws. The AGREE II instrument should be adopted by guideline developers, particularly in China.     

Focus on Water: Polarity of Water Transport across Epidermal Cell Membranes in Tradescantia virginiana

Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (L_pOUT and L_pIN, respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [V_o], and hydraulic conductivity [L_p], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). V_o as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated V_o values of 1.5 to 4.9 nL. For the same cell, V_o and L_p estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher L_pOUT than L_pIN (L_pOUT/L_pIN ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in L_pOUT/L_pIN over a wide range of L_p values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex.The conductivity of membranes to water (hydraulic conductivity [L_p]) is an important property of the cells of all organisms, and whether plant cell membranes exhibit a polarity in this property has been debated for a number of decades (Dainty and Hope, 1959; Steudle, 1993). Most early evidence for polarity was based on transcellular osmotic experiments using giant algal cells in the Characeae, in which the relative areas of cell membrane exposed to conditions of osmotic inflow (endosmosis) or outflow (exosmosis) could be varied and, hence, L_p for both directions determined (Tazawa and Shimmen, 2001). Interpretation of these experiments is complicated by unstirred layer (USL) effects (Dainty, 1963), but even after accounting for these, it was concluded that inflow L_p (L_pIN) was higher than outflow L_p (L_pOUT) in these cells, with L_pOUT/L_pIN of about 0.65 (Dainty, 1963). When using osmotic driving forces in algal cells, L_pOUT/L_pIN values of between 0.5 and 0.91 have been reported in many studies (Steudle and Zimmermann, 1974; Steudle and Tyerman, 1983; Tazawa et al., 1996), and the same direction of polarity was also reported using osmotic driving forces in whole roots of maize (Zea mays; Steudle et al., 1987). When applying hydrostatic driving forces in algal cells using the pressure probe (Steudle, 1993), which is less influenced by USL effects (Steudle et al., 1980), L_pOUT/L_pIN has been closer to 1 (0.83–1; Steudle and Zimmermann, 1974; Steudle and Tyerman, 1983). However, in higher plant cells, an analysis of the data presented by Steudle et al. (1980, 1982) and Tomos et al. (1981) indicates the opposite polarity, with L_pOUT/L_pIN averaging from 1.2 to 1.4. Moore and Cosgrove (1991) used two contrasting hydrostatic methods to measure L_p in sugarcane (Saccharum spp.) stem cells: (1) the most commonly used pressure relaxation (PR) method, in which cell turgor pressure (P_cell) changes during the measurement, and (2) the more technically demanding pressure clamp (PC) method, in which P_cell is maintained constant. Consistent with other studies in higher plant cells, Moore and Cosgrove (1991) reported average L_pOUT/L_pIN from 1.15 (PC) to 1.65 (PR). Using the PR method in epidermal cells of barley (Hordeum vulgare), Fricke (2000) reported only a modest L_pOUT/L_pIN (based on reported half-time [T_1/2]) of 1.08. In view of the contribution of proteins (e.g. aquaporins) to overall membrane L_p, Tyerman et al. (2002) suggested that polarity may result either from asymmetry in the pores themselves or from an active regulation of the conductive state of the pores in response to the experimental conditions that cause inflow or outflow. Either of these mechanisms may explain the wide range of values reported in the literature for L_pOUT/L_pIN. Cosgrove and Steudle (1981) reported that a substantial (6-fold) and rapid (within 20 s) reduction in L_p could occur in the same cell, and in hindsight, this presumably reflected the influence of aquaporins. Cosgrove and Steudle (1981) did not consider the lower L_p as indicative of the L_p in situ, and Wan et al. (2004) reported that a reduction in L_p was associated with perturbations to P_cell on the order of 0.1 MPa. Hence, if measured membrane L_p itself can exhibit substantial changes over relatively short periods of time in the same cell, then further study of systematic differences between L_pOUT and L_pIN will require a robust hydrostatic methodology (PC or PR) that can reversibly and reproducibly apply small perturbations in pressure (P) to individual cells over short periods of time.For the PR method, a T_1/2 of water exchange is measured by fitting an exponential curve to the observed decay in P_cell over time following a step change in volume, and membrane L_p can be calculated if cell surface area (A), cell volume (V_o), and volumetric elastic modulus (ε) are known (Steudle, 1993). In practice, A and V_o are typically calculated from optical measurements of individual cell dimensions or estimates using average values, and ε is calculated based on V_o and an empirical change in pressure (dP) to change in volume (dV) relation for each cell (Steudle, 1993; Tomos and Leigh, 1999). In the PC method, first developed by Wendler and Zimmermann (1982), V_o (and, given reasonable assumptions about cell geometry, A) is estimated without the need for optical measurements, and L_p can be measured without the need to determine dP/dV or ε. However, this method is technically more demanding because it requires precise P control as well as a continuous record of the volume flow of water across the cell membrane (as measured by changes in the position of the cell solution/oil meniscus within the glass capillary over time) and has rarely been used (Wendler and Zimmermann, 1982, 1985; Cosgrove et al., 1987; Moore and Cosgrove, 1991; Zhang and Tyerman, 1991; Murphy and Smith, 1998). Since volume (V) is continuously changing over time, this approach may also be influenced by the hydraulic conductance of the capillary tip (K_h) used to make the measurements as well as surface tension effects due to the progressive changes in capillary diameter with meniscus position, and these influences have not been quantitatively addressed.Automation of the pressure probe operation, particularly automatic tracking of the meniscus location in the glass microcapillary tip, would address many of the above-mentioned issues, and to date, several attempts have been made to monitor the meniscus location using electrical resistance (Hüsken et al., 1978) or hardware-based image analysis (Cosgrove and Durachko, 1986; Murphy and Smith, 1998). Recently, Wong et al. (2009) redesigned the automated cell pressure probe (ACPP), originally proposed by Cosgrove and Durachko (1986), using a software-based meniscus detection system and a precise pressure control system. In the new ACPP system, both the position of the meniscus and oil pressure (P_oil) are recorded frequently (typically at 10 Hz), and P_oil is controlled with a resolution of ±0.002 MPa. We have combined the ACPP with a new technique to reproducibly fabricate microcapillary tips of known hydraulic properties (Wada et al., 2011) in order to correct for K_h and surface tension effects in both PC and PR estimates of the water relations parameters of Tradescantia virginiana epidermal cells and have determined the relation of L_pOUT to L_pIN in these cells.