Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

Herbivore abundance and grazing intensity on a Caribbean coral reef

Herbivory is a primary factor in determining the structure of coral reef communities. Spatial variation among reef habitats in the intensity of herbivory has been documented, but underlying variation in species composition and abundance within the herbivore guild has received little attention. The distribution and relative abundances of herbivorous fishes and sea urchins across several habitats were studied on the Belizean barrier reef off the Caribbean coast of Central America. Marked variation in total herbivore density as well as major changes in the composition of the herbivore guild were found across reef habitats. Acanthurids (surgeonfishes) predominated in shallow areas (< 5 m) while scarids (parrotfishes) were dominant in deeper habitats. Significant differences among habitats in an experimental assay of grazing intensity were strongly correlated with herbivore abundance. The spatial distribution of herbivorous fishes across reef habitats does not appear to be simply explained by differences in reef topography, but may depend on complex interactions among proximity to nearby shelter, predator abundance, density of territorial competitors, and local availability of food resources.     

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering strategies rely on suitable membranes and scaffolds, providing the necessary physicochemical stimuli to specific cells. This review summarizes the main results on piezoelectric polymers, in particular poly(vinylidene fluoride), for muscle and bone cell culture. Further, the relevance of polymer microstructure and surface charge on cell response is demonstrated. Together with the necessary biochemical cues, the proper design of piezoelectric polymers can open the way to novel and more reliable tissue engineering strategies for cells in which electromechanical stimuli are present in their environment.     

Regulation of matrix remodelling phenotype in gingival fibroblasts by substratum topography

Gingival connective tissue often has a composition resembling that of scar surrounding dental implant abutments. Increased cell adhesion, α‐smooth muscle actin (α‐SMA) expression and increased extracellular matrix deposition are a hallmark of fibrotic cells, but how topographic features influence gingival fibroblast adhesion and adoption of the α‐SMA positive myofibroblast phenotype associated with scarring is unknown. The purpose of the present study was to demonstrate whether implant topographies that limit adhesion formation would reduce myofibroblast differentiation and extracellular matrix deposition. Human gingival fibroblasts were cultured on PT (smooth) and SLA (roughened) titanium discs for varying time‐points. At 1 and 2 weeks after seeding, incorporation of α‐SMA into stress‐fibre bundles and fibronectin deposition was significantly higher on PT than SLA surfaces indicating differentiation of the cells towards a myofibroblast phenotype. Analysis of adhesion formation demonstrated that cells formed larger adhesions and more stable adhesions on PT, with more nascent adhesions observed on SLA. Gene expression analysis identified up‐regulation of 15 genes at 24 hrs on SLA versus PT associated with matrix remodelling. Pharmacological inhibition of Src/FAK signalling in gingival fibroblasts on PT reduced fibronectin deposition and CCN2 expression. We conclude that topographical features that reduce focal adhesion stability could be applied to inhibit myofibroblast differentiation in gingival fibroblasts.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

Tooth cusp sharpness as a dietary correlate in great apes

Mammalian molars have undergone heavy scrutiny to determine correlates between morphology and diet. Here, the relationship between one aspect of occlusal morphology, tooth cusp radius of curvature (RoC), and two broad dietary categories, folivory and frugivory, is analyzed in apes. The author hypothesizes that there is a relationship between tooth cusp RoC and diet, and that folivores have sharper teeth than frugivores, and further test the correlation between tooth cusp RoC and tooth cusp size. Eight measures of tooth cusp RoC (two RoCs per cusp) were taken from 53 M²s from four species and subspecies of frugivorous apes (Pongo pygmaeus, Pan troglodytes troglodytes, Pan troglodytes schweinfurthii, and Gorilla gorilla gorilla) and two subspecies of folivorous apes (Gorilla beringei beringei, and Gorilla beringei graueri). Phylogenetically corrected ANOVAs were run on the full dataset and several subsets of the full dataset, revealing that, when buccolingual RoCs are taken into account, tooth cusp RoCs can successfully differentiate folivores and frugivores. PCAs revealed that folivores consistently had duller teeth than frugivores. In addition, a weak, statistically significant positive correlation exists between tooth cusp size and tooth cusp RoC. The author hypothesizes differences in tooth cusp RoC are correlated with wear rates, where, per vertical unit of wear, duller cusps will have a longer length of exposed enamel ridge than sharper cusps. More data need to be gathered to determine if the correlation between tooth cusp RoC and tooth cusp size holds true when small primates are considered. Am J Phys Anthropol 153:226–235, 2014. © 2013 Wiley Periodicals, Inc.     

Spatial patterns of species diversity in the herb layer of early post-fire regeneration in mixed Pinus yunnanensis forests

Herb layer plays an important role in maintaining ecosystem functioning of forests. The aims of this study were to determine the pattern of species diversity in early post-fire regeneration of the herb layer in mixed Yunnan pine forests and to identify the effects of topography, fire severity, pre-fire vegetation and herb species life-history feature on post-fire regeneration.     

云南松混交林火烧迹地更新早期草本层物种多样性的空间格局

云南松(Pinus yunnanensis)林是我国西南地区最为广布的针叶林。火烧是云南松林常见的一种自然(或人为)干扰, 但对云南松林火后更新群落的物种多样性特征很少研究。该研究以云南禄丰县勤丰镇2013年“4.13”火灾后云南松混交林火烧迹地更新早期群落草本层为研究对象, 设置了47条1000 m²样带进行群落和环境因子调查, 探讨火烧后更新早期群落草本层的物种构成与丰富度的空间格局及其影响因素。结果表明: 火后更新草本层按物种构成可分为4个群落类型; 草本层物种丰富度受地形和火前群落冠层的影响显著, 与立木生物量和地形坡度正相关, 而与坡位和海拔负相关, 但与火烧强度没有显著的相关性。样带内草本群落β多样性与火前立木胸面积及火烧强度显著正相关, 而与海拔和坡位负相关。总体上, 生境地形特征、火前冠层和火烧强度构成了火烧后早期群落草本层物种构成与丰富度变化的主要控制因子。此外, 一年生和多年生草本物种的更新格局及其对环境的响应存在显著差异。     

长白山阔叶红松林3个常见树种径向生长的影响因素

为探究树木径向生长的影响因子,基于对长白山阔叶红松林25 hm²样地中3个常见树种红松、紫椴、蒙古栎连续5年的生长环监测数据,分析各树种的季节生长变化,比较树种间的生长速率差异,并分析初始径级、邻体树木竞争、土壤、地形因素等对不同树种径向生长的作用.结果表明: 3个树种径向生长的季节变化趋势一致,树木于5月末开始生长,7月是生长旺盛期,8月底生长开始减缓,于10月中下旬停止生长.3个树种的径向相对生长率存在显著差异,蒙古栎相对径向生长率显著高于红松和紫椴,该差异在小、中径级个体中表现更为显著.3个树种径向生长的主要影响因子不同,邻体树木的竞争显著抑制了红松和蒙古栎的径向生长,而紫椴的径向生长主要受初始径级、土壤和地形等因子的影响.