Theoretical and experimental approaches to understand morphogen gradients

Morphogen gradients, which specify different fates for cells in a direct concentration‐dependent manner, are a highly influential framework in which pattern formation processes in developmental biology can be characterized. A common analysis approach is combining experimental and theoretical strategies, thereby fostering relevant data on the dynamics and transduction of gradients. The mechanisms of morphogen transport and conversion from graded information to binary responses are some of the topics on which these combined strategies have shed light. Herein, we review these data, emphasizing, on the one hand, how theoretical approaches have been helpful and, on the other hand, how these have been combined with experimental strategies. In addition, we discuss those cases in which gradient formation and gradient interpretation at the molecular and/or cellular level may influence each other within a mutual feedback loop. To understand this interplay and the features it yields, it becomes essential to take system‐level approaches that combine experimental and theoretical strategies.     

Phylogenetic approaches for studying diversification

Estimating rates of speciation and extinction, and understanding how and why they vary over evolutionary time, geographical space and species groups, is a key to understanding how ecological and evolutionary processes generate biological diversity. Such inferences will increasingly benefit from phylogenetic approaches given the ever‐accelerating rates of genetic sequencing. In the last few years, models designed to understand diversification from phylogenetic data have advanced significantly. Here, I review these approaches and what they have revealed about diversification in the natural world. I focus on key distinctions between different models, and I clarify the conclusions that can be drawn from each model. I identify promising areas for future research. A major challenge ahead is to develop models that more explicitly take into account ecology, in particular the interaction of species with each other and with their environment. This will not only improve our understanding of diversification; it will also present a new perspective to the use of phylogenies in community ecology, the science of interaction networks and conservation biology, and might shift the current focus in ecology on equilibrium biodiversity theories to non‐equilibrium theories recognising the crucial role of history.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Metagenomic approaches for defining the pathogenesis of inflammatory bowel diseases

The human gastrointestinal tract is home to immense and complex populations of microorganisms. Using recent technical innovations, the diversity present in this human body habitat is now being analyzed in detail. This review focuses on the microbial ecology of the gut in inflammatory bowel diseases and on how recent studies provide an impetus for using carefully designed, comparative metagenomic approaches to delve into the structure and activities of the gut microbial community and its interrelationship with the immune system.     

Proteomic approaches for studying chemoresistance in cancer

The role of various proteins involved in drug resistance in tumor cells is discussed in this review. Two types of studies are covered: those performed in the preproteomics era and those carried out with modern proteomic tools, namely 2D (electrophoretic) maps and 2D chromatography. In the preproteomic studies, one protein had generally been held responsible for a given chemoresistance. However, analysis via proteomic tools may reveal entire sets of proteins that are up- or downregulated (or switched on/off) in chemoresistant tumor cell lines compared with parental tumor lines. Therefore, it appears more realistic to expect that exposure of cells to drugs results in the activation of different mechanisms of resistance. Such investigations have led to the broadly shared opinion that exposure of cells to drugs results in the activation of different mechanisms of resistance, and that a specific drug-resistant phenotype consists of several molecular mechanisms that are simultaneously active. The proteomic papers reviewed clearly support the hypothesis that many metabolic pathways are affected during the resistance process. Although the modulation of expression levels of such proteins is not clear proof of their role in drug resistance per se, at least some of the themes are very likely to be involved in the resistance phenotype, and thus may be potential targets for new drugs. It is hoped that this review will bring new insight in this field and will stimulate novel and deeper searches with proteomic tools (including prefractionation of subcellular organelles, such as nuclei, to bring to the fore low-abundance proteins that might be responsible for the onset of drug resistance).     

Phylogenetic comparative approaches for studying niche conservatism

Analyses of phylogenetic niche conservatism (PNC) are becoming increasingly common. However, each analysis makes subtly different assumptions about the evolutionary mechanism that generates patterns of niche conservatism. To understand PNC, analyses should be conducted with reference to a clear underlying model, using appropriate methods. Here, we outline five macroevolutionary models that may underlie patterns of PNC (drift, niche retention, phylogenetic inertia, niche filling/shifting and evolutionary rates) and link these to published phylogenetic comparative methods. For each model, we give recent examples from the literature and suggest how the methods can be practically applied. We hope that this will help clarify the niche conservatism literature and encourage people to think about the evolutionary models underlying niche conservatism in their study group.     

Quantitative proteomic approaches for studying phosphotyrosine signaling

Protein tyrosine phosphorylation is a fundamental mechanism for controlling many aspects of cellular processes, as well as aspects of human health and diseases. Compared with phosphoserine and phosphothreonine, phosphotyrosine signaling is more tightly regulated, but often more challenging to characterize, due to significantly lower levels of tyrosine phosphorylation (i.e., a relative abundance of 1800:200:1 was estimated for phosphoserine/phosphothreonine/phosphotyrosine in vertebrate cells). In this review, we outline recent advances in analytical methodologies for enrichment, identification and accurate quantitation of tyrosine-phosphorylated proteins and peptides. Advances in antibody-based technologies, capillary liquid chromatography coupled with mass spectrometry, and various stable isotope labeling strategies are discussed, as well as non-mass spectrometry-based methods, such as those using protein/peptide arrays. As a result of these advances, powerful tools now have the power to crack signal transduction codes at the system level, and provide a basis for discovering novel drug targets for human diseases.     

Proteomic approaches for studying chemoresistance in cancer

The role of various proteins involved in drug resistance in tumor cells is discussed in this review. Two types of studies are covered: those performed in the preproteomics era and those carried out with modern proteomic tools, namely 2D (electrophoretic) maps and 2D chromatography. In the preproteomic studies, one protein had generally been held responsible for a given chemoresistance. However, analysis via proteomic tools may reveal entire sets of proteins that are up- or downregulated (or switched on/off) in chemoresistant tumor cell lines compared with parental tumor lines. Therefore, it appears more realistic to expect that exposure of cells to drugs results in the activation of different mechanisms of resistance. Such investigations have led to the broadly shared opinion that exposure of cells to drugs results in the activation of different mechanisms of resistance, and that a specific drug-resistant phenotype consists of several molecular mechanisms that are simultaneously active. The proteomic papers reviewed clearly support the hypothesis that many metabolic pathways are affected during the resistance process. Although the modulation of expression levels of such proteins is not clear proof of their role in drug resistance per se, at least some of the themes are very likely to be involved in the resistance phenotype, and thus may be potential targets for new drugs. It is hoped that this review will bring new insight in this field and will stimulate novel and deeper searches with proteomic tools (including prefractionation of subcellular organelles, such as nuclei, to bring to the fore low-abundance proteins that might be responsible for the onset of drug resistance).     

空气微生物群落解析方法:从培养到非培养

随着世界范围内流行性疾病以及我国空气雾霾事件的不断发生,空气生物性污染的研究开始受到高度重视,其研究方法也随着分子生物学技术的快速发展而不断更新,由早期以生化技术为基础的研究方法转变为以现代分子生物学技术为基础的研究方法。综述了空气微生物群落多样性解析方法从培养到非培养的发展过程,包括培养技术法、BIOLOG技术、生物标记法、基因指纹图谱技术、核酸杂交技术、实时荧光定量PCR、空气微生物宏基因组学及基因芯片技术,阐述了这些技术的基本原理,比较了各种技术的优缺点并重点介绍了它们在空气微生物群落多样性研究中的应用概况,最后展望了空气微生物学研究的发展方向。     

Biophysical approaches for studying the integrity and function of tight junctions

Cell-cell adhesion is an extremely important phenomenon as it influences several biologically important processes such as inflammation, cell migration, proliferation, differentiation and even cancer metastasis. Furthermore, proteins involved in cell-cell adhesion are also important from the perspective of facilitating better drug delivery across epithelia. The adhesion forces imparted by proteins involved in cell-cell adhesion have been the focus of research for sometime. However, with the advent of nanotechnological techniques such as the atomic force microscopy (AFM), we can now quantitatively probe these adhesion forces not only at the cellular but also molecular level. Here, we review the structure and function of tight junction proteins, highlighting some mechanistic studies performed to quantify the adhesion occurring between these proteins and where possible their association with human diseases. In particular, we will highlight two important experimental techniques, namely the micropipette step pressure technique and the AFM that allow us to quantify these adhesion forces at both the cellular and molecular levels, respectively.     

Molecular genetic and biochemical approaches for defining lipid-dependent membrane protein folding

We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long-range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually dependent on each other for functional organization of proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Culture-independent approaches for studying viruses from hypersaline environments

Hypersaline close-to-saturation environments harbor an extremely high concentration of virus-like particles, but the number of haloviruses isolated so far is still very low. Haloviruses can be directly studied from natural samples by using different culture-independent techniques that include transmission electron microscopy, pulsed-field gel electrophoresis, and different metagenomic approaches. Here, we review the findings of these studies, with a main focus on the metagenomic approaches. The analysis of bulk viral nucleic acids directly retrieved from the environment allows estimations of viral diversity, activity, and dynamics and tentative host assignment. Results point to a diverse and active viral community in constant interplay with its hosts and to a "hypersalineness" quality common to viral assemblages present in hypersaline environments that are thousands of kilometers away from each other.     

Proteomic approaches for studying human parenchymal lung diseases

Parenchymal lung diseases comprise a wide variety of diseases, with different etiologies, pathogeneses and prognoses. This perspective provides an overview of two different disease types: chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Chronic obstructive pulmonary disease, which is related to smoking, is one of the leading causes of chronic morbidity and mortality around the world, being characterized by airway obstruction and parenchymal lung damage (emphysema). Idiopathic pulmonary fibrosis of unknown etiology is classified as one of the most important idiopathic interstitial pneumonias and is connected to patchy but progressive lung fibrosis. Both diseases are generally diagnosed late and respond poorly to medical therapies. Although numerous biomarkers have been proposed for these diseases, they have not been validated or implemented into clinical practice. This perspective emphasizes some typical features of these diseases with different types of lung damage, how they are reflected in different samples, as well as potential advances and problems of current and future nonbiased proteomic approaches.     

Molecular genetic approaches for studying the etiology of diabetic nephropathy

A critical challenge faced by clinical nephrologists today is the escalating number of patients developing end stage renal disease, a major proportion of which is attributed to diabetic nephropathy (DN). The need for new measures to prevent and treat this disease cannot be overemphasized. To this end, modern genetic approaches provide powerful tools to investigate the etiology of DN. Human studies have already established the importance of genetic susceptibility for DN. Several major susceptibility loci have been identified using linkage studies. In addition, linkage studies in rodents have pinpointed promising chromosomal segments that influence renal traits. Besides augmenting our understanding of disease pathogenesis, these animal studies may facilitate the cloning of disease susceptibility genes in man through the identification of homologous regions that contribute to renal disease. In human diabetes, various genes have been evaluated for their risk contribution to DN. This widespread strategy has been propelled by our knowledge of the glucose-activated pathways underlying DN. Evidence has emerged that a true association does indeed exist for some candidate genes. Furthermore, the in vivo manipulation of gene expression has shown that these genes can modify features of DN in transgenic and knockout rodent models, thus corroborating the findings from human association studies. Still, the exact molecular mechanisms involving these genes remain to be fully elucidated. This formidable task may be accomplished by continuing to harness the synergy between human and experimental genetic approaches. In this respect, our review provides a first synthesis of the current literature to facilitate this challenging effort.     

Theoretical and experimental approaches to understand the biosynthesis of starch granules in a physiological context

Photosynthesis Research - Starch, a plant-derived insoluble carbohydrate composed of glucose polymers, is the principal carbohydrate in our diet and a valuable raw material for industry. The...     

New approaches for Helicobacter vaccine development--difficulties and progress.

Despite the enormous progress in understanding the process of bacterial pathogenesis and interactions of pathogens with eucaryotic cells the infectious diseases still remain the main cause of human premature deaths. It is now recognized that Helicobacter pylori infects about half of the world's population. Based on results of clinical studies the World Health Organization has assigned H. pylori as a class I carcinogen. The review presents new achievements aimed at construction efficient and safe anti-Helicobacter vaccine. We discuss the new global technologies such as immunoproteomics employed for selecting new candidates for vaccine construction as well as new vaccine delivery systems. The review presents also our knowledge concerning H. pylori interaction with immune system which might facilitate modulation of the host immune system by specific adjuvant included into vaccine.