The role of exo-(1-->4)-beta-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination

BACKGROUND AND AIMS: The cotyledons of Lupinus angustifolius contain large amounts of cell wall storage polysaccharide (CWSP) composed mainly of (1-->4)-beta-linked D-galactose residues in the form of branches attached to a rhamnogalacturonan core molecule. An exo-(1-->4)-beta-galactanase with a very high specificity towards (1-->4)-beta-linked D-galactan has been isolated from L. angustifolius cotyledons, and shown to vary (activity and specific protein) in step with CWSP mobilization. This work aimed to confirm the hypothesis that galactan is the main polymer retrieved from the wall during mobilization at the ultrastructural level, using the purified exo-galactanase as a probe. METHODS: Storage mesophyll cell walls ('ghosts') were isolated from the cotyledons of imbibed but ungerminated lupin seeds, and also from cotyledons of seedlings after the mobilization of the CWSP. The pure exo-(1-->4)-beta-galactanase was coupled to colloidal gold particles and shown to be a specific probe for (1-->4)-beta-D-galactan. They were used to localize galactan in ultrathin sections of L. angustifolius cotyledonary mesophyll tissue during CWSP mobilization. KEY RESULTS: On comparing the morphologies of isolated cell walls, the post-mobilization 'ghosts' did not have the massive wall-thickenings of pre-mobilization walls. Compositional analysis showed that the post-mobilization walls were depleted in galactose and, to a lesser extent, in arabinose. When pre-mobilization ghosts were treated with the pure exo-galactanase, they became morphologically similar to the post-mobilization ghosts. They were depleted of approximately 70% of the galactose residues that would have been mobilized in vivo, and retained all the other sugar residues originally present. Sharply defined electron-transparent wall zones or pockets are associated with CWSP mobilization, being totally free of galactan, whereas wall areas immediately adjacent to them were apparently undepleted. CONCLUSIONS: The exo-(1-->4)-beta-galactanase is the principal enzyme involved in CWSP mobilization in lupin cotyledons in vivo. The storage walls dramatically change their texture during mobilization as most of the galactan is hydrolysed during seedling development.     

A Risk Prediction Model for the Assessment and Triage of Women with Hypertensive Disorders of Pregnancy in Low-Resourced Settings: The miniPIERS (Pre-eclampsia Integrated Estimate of RiSk) Multi-country Prospective Cohort Study

Background

Pre-eclampsia/eclampsia are leading causes of maternal mortality and morbidity, particularly in low- and middle- income countries (LMICs). We developed the miniPIERS risk prediction model to provide a simple, evidence-based tool to identify pregnant women in LMICs at increased risk of death or major hypertensive-related complications.

Methods and Findings

From 1 July 2008 to 31 March 2012, in five LMICs, data were collected prospectively on 2,081 women with any hypertensive disorder of pregnancy admitted to a participating centre. Candidate predictors collected within 24 hours of admission were entered into a step-wise backward elimination logistic regression model to predict a composite adverse maternal outcome within 48 hours of admission. Model internal validation was accomplished by bootstrapping and external validation was completed using data from 1,300 women in the Pre-eclampsia Integrated Estimate of RiSk (fullPIERS) dataset. Predictive performance was assessed for calibration, discrimination, and stratification capacity. The final miniPIERS model included: parity (nulliparous versus multiparous); gestational age on admission; headache/visual disturbances; chest pain/dyspnoea; vaginal bleeding with abdominal pain; systolic blood pressure; and dipstick proteinuria. The miniPIERS model was well-calibrated and had an area under the receiver operating characteristic curve (AUC ROC) of 0.768 (95% CI 0.735–0.801) with an average optimism of 0.037. External validation AUC ROC was 0.713 (95% CI 0.658–0.768). A predicted probability ≥25% to define a positive test classified women with 85.5% accuracy. Limitations of this study include the composite outcome and the broad inclusion criteria of any hypertensive disorder of pregnancy. This broad approach was used to optimize model generalizability.

Conclusions

The miniPIERS model shows reasonable ability to identify women at increased risk of adverse maternal outcomes associated with the hypertensive disorders of pregnancy. It could be used in LMICs to identify women who would benefit most from interventions such as magnesium sulphate, antihypertensives, or transportation to a higher level of care. Please see later in the article for the Editors'' Summary     

A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.     

氨氯地平联合依那普利治疗原发性高血压的临床疗效及对患者左心室肥厚的影响

摘要目的：探讨氨氯地平联合依那普利治疗原发性高血压的临床效果,观察联合用药对左心室肥厚的影响。方法：选择本院收治的原发性高血压患者92例,随机分为观察组和对照组,各46例,对照组给予苯磺酸左旋氨氯地平5mg,1次／d,口服;观察组在对照组基础上加用马来酸依那普利10mg,2次／d,口服,疗程均为24周。观察两组治疗前后血压变化,应用超声心动图测量两组左心室厚度变化。结果：治疗后,观察组总有效率为91_3％;对照组总有效率为73．9％,观察组总有效率高于对照组（P〈0．05）。治疗前两组心率、血压比较无统计学差异（P〉0．05）,治疗后两组血压均明显降低,观察组收缩压、舒张压明显低于对照组（P〈O．05）;观察组心率明显低于对照组（P〈0．01）。治疗前两组左心室舒张末期室间隔厚度（Leaventricularend—diastolicventricularseptalthickness,IVST）、左心室后壁厚度（1eftventricularposteriorwallthickness,U,PwT）和左室射血分数（Leftventricularejectionfxaction,LVEF）比较无统计学差异（P〉0．05）;治疗后观察组IVST、L、,PwT明显低于对照组,LVEF明显高于对照组（P〈0．05）。结论：氨氯地平联合依那普利治疗原发性高血压能有效扭转左心室肥厚,降压效果较单独应用氨氯地平更佳。     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.     

Within‐plant distribution of 1,4‐benzoxazin‐3‐ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore‐foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within‐plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4‐benzoxazin‐3‐one derivatives (BXDs), affects the foraging behaviour of two leaf‐chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well‐adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD‐free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non‐adapted herbivores and thereby likely contribute to feeding niche differentiation.     

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold‐based and scaffold‐free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS—five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long‐term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick‐freeze/deep‐etch electron microscopy. Both the scaffold‐free and the collagen‐based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell‐derived matrix in the scaffold‐based constructs was significantly lower than that produced by scaffold‐free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell‐derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. Biotechnol. Bioeng. 2012; 109: 2683–2698. © 2012 Wiley Periodicals, Inc.     

Transformation of the oilseed crop Camelina sativa by Agrobacterium-mediated floral dip and simple large-scale screening of transformants

Camelina sativa is a promising under-exploited oilseed crop with potential to become a biofuel feedstock. The ability to transform C. sativa would allow for the rapid introduction of novel traits into this emerging crop. We report the development of an Agrobacterium-based floral dip transformation method, requiring no vacuum-infiltration step, with transformation efficiencies up to 0.8%. C. sativa cultivars Ames 26665, ??Calena?? A3U7761, Ames 1043, and ??Celine?? were tested using Agrobacterium tumefaciens strains GV3101, EHA105, and At503. Use of all strains and cultivars resulted in transformed plants; however, GV3101 was the only Agrobacterium strain and Ames 1043 the only C. sativa cultivar to yield transformed plants under all conditions tested. Progeny analysis revealed that in approximately 78% of the transformed plants, the transgene segregated as a single locus. Furthermore, a high-throughput, filter paper-based PCR method was developed to screen marker-free transformed plants. Together, these methods will allow for easier introduction of new genes into this promising oilseed crop.     

Total evidence, consensus, and bat phylogeny: A distance-based approach

Resolution of the total evidence (i.e., character congruence) versus consensus (i.e., taxonomic congruence) debate has been impeded by (1) a failure to employ validation methods consistently across both tree-building and consensus analyses, (2) the incomparability of methods for constructing as opposed to those for combining trees, and (3) indifference to aspects of trees other than their topologies. We demonstrate a uniform, distance-based approach which allows for comparability among the results of character- and taxonomic-congruence studies, whether or not an identical suite of taxa has been included in all contributing data sets. Our results indicate that total-evidence and consensus trees differ little in topology if branch lengths are taken into account when combining two or more trees. In addition, when character-state data are converted to distances, our method permits their combination with information produced by techniques which generate distances directly. Moreover, treating all data sets or trees as distance matrices avoids the problem that different numbers of characters in contributing studies may confound the conclusions of a total-evidence or consensus analysis. Our protocol is illustrated with an example involving bats, in which the three component studies based on serology, DNA hybridization, and anatomy imply distinct phylogenies. However, the total-evidence and consensus trees support a fourth, somewhat different, topology resolved at all but one node and which conforms closely to the currently accepted higher category classification of Chiroptera.