论文信息 - C‐di‐GMP: the dawning of a novel bacterial signalling system

2006 - Circulation

Receptor for Advanced-Glycation End Products: Key Modulator of Myocardial Ischemic Injury

Background— The beneficial effects of reperfusion therapies have been limited by the amount of ischemic damage that occurs before reperfusion. To enable development of interventions to reduce cell injury, our research has focused on understanding mechanisms involved in cardiac cell death after ischemia/reperfusion (I/R) injury. In this context, our laboratory has been investigating the role of the receptor for advanced-glycation end products (RAGE) in myocardial I/R injury. Methods and Results— In this study we tested the hypothesis that RAGE is a key modulator of I/R injury in the myocardium. In ischemic rat hearts, expression of RAGE and its ligands was significantly enhanced. Pretreatment of rats with sRAGE, a decoy soluble part of RAGE receptor, reduced ischemic injury and improved functional recovery of myocardium. To specifically dissect the impact of RAGE, hearts from homozygous RAGE-null mice were isolated, perfused, and subjected to I/R. RAGE-null mice were strikingly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH, improved functional recovery, and increased adenosine triphosphate (ATP). In rats and mice, activation of the RAGE axis was associated with increases in inducible nitric oxide synthase expression and levels of nitric oxide, cyclic guanosine monophosphate (cGMP), and nitrotyrosine. Conclusions— These findings demonstrate novel and key roles for RAGE in I/R injury in the heart. The findings also demonstrate that the interaction of RAGE with advanced-glycation end products affects myocardial energy metabolism and function during I/R.

1994 - Endocrinology

1,25-Dihydroxyvitamin D3 and pancreatic beta-cell function: vitamin D receptors, gene expression, and insulin secretion.

Previous studies have indicated that the pancreas has receptors specific for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and that 1,25-(OH)2D3 increases insulin secretion in vitamin D-deficient rats. In this study we report that in vitamin D-replete, but calcium-deficient, rats in which 1,25-(OH)2D3 levels are elevated, insulin secretion is not altered. In addition, in in vitro studies 1,25-(OH)2D3 at concentrations of 10(-10)-10(-7) M was consistently found to inhibit insulin secretion from islets of vitamin D-replete rats or from the rat insulinoma beta-cell line RIN 1046-38. The RIN cell line was found to contain both vitamin D receptors and calbindin-D28k (CaBP-D28k) protein and mRNA. In RIN cells, treatment with sodium butyrate (2 mM for 3 days) induces a more islet phenotype, as indicated by increased insulin content and secretion and increased insulin gene expression. 1,25-(OH)2D3 treatment (50-100 nM for 48 or 72 h) had no effect on the enhanced levels of insulin secreted in the presence of butyrate. However, 2 mM sodium butyrate induced CaBP-D28k protein (4-fold; control, 0.8 +/- 0.2; sodium butyrate, 3.5 +/- 0.1 microgram/mg protein) and mRNA (3-fold) in the RIN cell line, in accord with the induction by butyrate of insulin content and secretion and beta-cell differentiation, suggesting a possible role for CaBP-D28k in these processes. Although 1,25-(OH)2D3, unlike butyrate, did not enhance insulin secretion, both 1,25-(OH)2D3 (100 nM) and butyrate (2 mM) inhibited RIN cell growth (to 69% and 28% of the control, respectively), and butyrate and 1,25-(OH)2D3 in combination led to a further inhibition of cell growth (to 13% of the control). In response to 1,25-(OH)2D3 (10 nM for 72 h), vitamin D receptors were up-regulated 313% in RIN cells [control, 37 +/- 2; 1,25-(OH)2D3 treated, 115 +/- 5 fmol/mg protein]. In conclusion, 1) contrary to previous studies in the vitamin D-deficient rat, our findings indicate that 1,25-(OH)2D3 action does not necessarily result in enhanced insulin secretion; 2) inhibition of cell growth and up-regulation of vitamin D receptors by 1,25-(OH)2D3 suggest that parameters in addition to insulin secretion can be affected by 1,25-(OH)2D3 in the beta-cell; 3) the RIN beta-cell line provides a novel in vitro system for studying the effect of the vitamin D endocrine system on pancreatic islet physiology.

2015 - The Journal of clinical investigation

Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.

2003 - Journal of bacteriology

Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays

ABSTRACT Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA “pathoarray” developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.

1991 - European journal of biochemistry

'Crosstalk': a pivotal role for protein kinase C in modulating relationships between signal transduction pathways.

Over the past 20 years we have seen a phenomenal increase in our understanding of the diversity and the molecular mechanism of action of signal transduction systems. Now we are faced with a cell being provided with a myriad of cell surface receptors, each connected to particular signal transduction systems. The very complexity of such systems, together with the need of the cell to monitor and respond to a variety of external stimuli, poses the question as to what overall controls are placed upon these various transduction mechanisms and how they might relate to each other. At one extreme, we can envisage that pathways might be isolated completely from each other and thus function in an an apparent ‘vacuum’. On the other hand, we can envisage ‘cross-talk’ between the various pathways linking each into an adaptable network array. Such a network, linking distinct signalling systems, would offer the cell a sophisticated ability to sense multiple environmental signals impinging upon it, thus providing a means of adapting or regulating its response to a particular range of effectors.

2010 - Annual review of pathology

Mammalian sirtuins: biological insights and disease relevance.

Aging is accompanied by a decline in the healthy function of multiple organ systems, leading to increased incidence and mortality from diseases such as type II diabetes mellitus, neurodegenerative diseases, cancer, and cardiovascular disease. Historically, researchers have focused on investigating individual pathways in isolated organs as a strategy to identify the root cause of a disease, with hopes of designing better drugs. Studies of aging in yeast led to the discovery of a family of conserved enzymes known as the sirtuins, which affect multiple pathways that increase the life span and the overall health of organisms. Since the discovery of the first known mammalian sirtuin, SIRT1, 10 years ago, there have been major advances in our understanding of the enzymology of sirtuins, their regulation, and their ability to broadly improve mammalian physiology and health span. This review summarizes and discusses the advances of the past decade and the challenges that will confront the field in the coming years.

1993 - Molecular microbiology

PilS and PilR, a two‐component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa

Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12 700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial‐specific bacteriophage P04. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the Sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage‐resistant mutants was identified in which the transposon had inserted near sequences that cross‐hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN‐dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild‐type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72–75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA–D. PilS and PilR clearly belong to the family of two‐component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.

1997 - Critical care medicine

Randomized, controlled trial of selective digestive decontamination in 600 mechanically ventilated patients in a multidisciplinary intensive care unit.

OBJECTIVE To evaluate the efficacy of two regimens of selective decontamination of the digestive tract in mechanically ventilated patients. DESIGN Prospective, randomized, concurrent trial. SETTING Multidisciplinary intensive care unit (ICU) in a 1,800-bed university hospital. PATIENTS Consecutive patients (n = 660) who were likely to require mechanical ventilation for at least 48 hrs were randomized to one of three groups: conventional antibiotic regimen (control group A); oral and enteral ofloxacin-amphotericin B (group B); and oral and enteral polymyxin E-tobramycin-amphotericin B (group C). Both treatment groups received systemic antibiotics for 4 days (ofloxacin in group B and cefotaxime in group C). INTERVENTIONS Patients were randomized to receive standard treatment (control group A, n = 220), selective decontamination regimen B (group B, n = 220), and selective decontamination regimen C (group C, n = 220). After early deaths and exclusions from the study, 185 controls (group A) and 193 (group B)/200 (group C) selective decontamination regimen patients were available for analysis. MEASUREMENTS AND MAIN RESULTS Measurements included colonization and primary/secondary infection rate, ICU mortality rate, emergence of antibiotic resistance, length of ICU stay, and antimicrobial agent costs. The study duration was 19 months. The patient groups were fully comparable for age, diagnostic category, and severity of illness. One third of patients in each group suffered a nosocomial infection at the time of admission. There was a significant difference between treatment group B and control group A in the number of infected patients (odds ratio of 0.42, 95% confidence interval of 0.27 to 0.64), secondary lower respiratory tract infection (odds ratio of 0.47, 95% confidence interval of 0.26 to 0.82), and urinary tract infection (odds ratio of 0.47, 95% confidence interval of 0.27 to 0.81). Significantly more Gram-positive bacteremias occurred in treatment group C vs. group A (odds ratio of 1.22, 95% confidence interval 0.72 to 2.08). Infection at the time of admission proved to be the most significant risk factor for subsequent infection in control and both treatment groups. ICU mortality rate was almost identical (group A 16.8%, group B 17.6%, and group C 15.5%) and was not significantly related to primary or secondary infection. Increased antimicrobial resistance was recorded in both treatment groups: tobramycin-resistant enterobacteriaceae (group C 48% vs. group A 14%, p < .01), ofloxacin-resistant enterobacteriaceae (group B 50% vs. group A 11%, p < .02), ofloxacin-resistant nonfermenters (group B 81% vs. group A 52%, p < .02), and methicillin-resistant Staphylococcus aureus (group C 83% vs. group A 55%, p < .05). Antimicrobial agent costs were comparable in control and group C patients; one third less was spent for group B patients. CONCLUSIONS In cases of high colonization and infection rates at the time of ICU admission, the preventive benefit of selective decontamination is highly debatable. Emergence of multiple antibiotic-resistant microorganisms creates a clinical problem and a definite change in the ecology of environmental, colonizing, and infecting bacteria. The selection of multiple antibiotic-resistant Gram-positive cocci is particularly hazardous. No beneficial effect on survival is observed. Moreover, selective decontamination adds substantially to the cost of ICU care.

2007

1 Origins and Principles of Translational Control

Proteins occupy a position high on the list of molecules important for life processes. They account for a large fraction of biological macromolecules—about 44% of the human body’s dry weight, for example (Davidson et al. 1973)—they catalyze most of the reactions on which life depends, and they serve numerous structural, transport, regulatory, and other roles in all organisms. Accordingly, a large proportion of the cell’s resources is devoted to translation. The magnitude of this commitment can be appreciated in genetic, biochemical, and cell biological terms. Translation is a sophisticated process requiring extensive biological machinery. One way to gauge the amount of genetic information needed to assemble the protein synthetic machinery is to compile a “parts list” of essential proteins and RNAs. Analyses of the genomes of several microorganisms have converged on similar estimates (Hutchison et al. 1999; Tamas et al. 2002; Kobayashi et al. 2003; Waters et al. 2003). These organisms get by with about 130 genes for components of the translation machinery, including about 90 protein-coding genes (specifying 50–60 ribosomal proteins, about 20 aminoacyl-tRNA synthetases, and 10–15 translation factors) and about 40 genes for ribosomal and transfer RNAs (rRNA and tRNAs). A somewhat larger number of genes are involved in eukaryotes, which have more ribosomal proteins and initiation factors, for example. Discounting genes that are dispensable for growth in the laboratory, it can be calculated that approximately 40% of the genes in a theoretical minimal cellular genome are devoted to the translation apparatus. This heavy...

2018 - Physiological reviews

Neuroimmune Interactions: From the Brain to the Immune System and Vice Versa.

Because of the compartmentalization of disciplines that shaped the academic landscape of biology and biomedical sciences in the past, physiological systems have long been studied in isolation from each other. This has particularly been the case for the immune system. As a consequence of its ties with pathology and microbiology, immunology as a discipline has largely grown independently of physiology. Accordingly, it has taken a long time for immunologists to accept the concept that the immune system is not self-regulated but functions in close association with the nervous system. These associations are present at different levels of organization. At the local level, there is clear evidence for the production and use of immune factors by the central nervous system and for the production and use of neuroendocrine mediators by the immune system. Short-range interactions between immune cells and peripheral nerve endings innervating immune organs allow the immune system to recruit local neuronal elements for fine tuning of the immune response. Reciprocally, immune cells and mediators play a regulatory role in the nervous system and participate in the elimination and plasticity of synapses during development as well as in synaptic plasticity at adulthood. At the whole organism level, long-range interactions between immune cells and the central nervous system allow the immune system to engage the rest of the body in the fight against infection from pathogenic microorganisms and permit the nervous system to regulate immune functioning. Alterations in communication pathways between the immune system and the nervous system can account for many pathological conditions that were initially attributed to strict organ dysfunction. This applies in particular to psychiatric disorders and several immune-mediated diseases. This review will show how our understanding of this balance between long-range and short-range interactions between the immune system and the central nervous system has evolved over time, since the first demonstrations of immune influences on brain functions. The necessary complementarity of these two modes of communication will then be discussed. Finally, a few examples will illustrate how dysfunction in these communication pathways results in what was formerly considered in psychiatry and immunology to be strict organ pathologies.

1999 - Biotechnology progress

Biocommodity Engineering

The application of biotechnology to the production of commodity products (fuels, chemicals, and materials) offering benefits in terms of sustainable resource supply and environmental quality is an emergent area of intellectual endeavor and industrial practice with great promise. Such “biocommodity engineering” is distinct from biotechnology motivated by health care at multiple levels, including economic driving forces, the importance of feedstocks and cost‐motivated process engineering, and the scale of application. Plant biomass represents both the dominant foreseeable source of feedstocks for biotechnological processes as well as the only foreseeable sustainable source of organic fuels, chemicals, and materials. A variety of forms of biomass, notably many cellulosic feedstocks, are potentially available at a large scale and are cost‐competitive with low‐cost petroleum whether considered on a mass or energy basis, and in terms of price defined on a purchase or net basis for both current and projected mature technology, and on a transfer basis for mature technology. Thus the central, and we believe surmountable, impediment to more widespread application of biocommodity engineering is the general absence of low‐cost processing technology. Technological and research challenges associated with converting plant biomass into commodity products are considered relative to overcoming the recalcitrance of cellulosic biomass (converting cellulosic biomass into reactive intermediates) and product diversification (converting reactive intermediates into useful products). Advances are needed in pretreatment technology to make cellulosic materials accessible to enzymatic hydrolysis, with increased attention to the fundamental chemistry operative in pretreatment processes likely to accelerate progress. Important biotechnological challenges related to the utilization of cellulosic biomass include developing cellulase enzymes and microorganisms to produce them, fermentation of xylose and other nonglucose sugars, and “consolidated bioprocessing” in which cellulase production, cellulose hydrolysis, and fermentation of soluble carbohydrates to desired products occur in a single process step. With respect to product diversification, a distinction is made between replacement of a fossil resource‐derived chemical with a biomass‐derived chemical of identical composition and substitution of a biomass‐derived chemical with equivalent functional characteristics but distinct composition. The substitution strategy involves larger transition issues but is seen as more promising in the long term. Metabolic engineering pursuant to the production of biocommodity products requires host organisms with properties such as the ability to use low‐cost substrates, high product yield, competitive fitness, and robustness in industrial environments. In many cases, it is likely to be more successful to engineer a desired pathway into an organism having useful industrial properties rather than trying to engineer such often multi‐gene properties into host organisms that do not have them naturally. Identification of host organisms with useful industrial properties and development of genetic systems for these organisms is a research challenge distinctive to biocommodity engineering. Chemical catalysis and separations technologies have important roles to play in downstream processing of biocommodity products and involve a distinctive set of challenges relative to petrochemical processing. At its current nascent state of development, the definition and advancement of the biocommodity field can benefit from integration at multiple levels. These include technical issues associated with integrating unit operations with each other, integrating production of individual products into a multi‐product biorefinery, and integrating biorefineries into the broader resource, economic, and environmental systems in which they function. We anticipate that coproduction of multiple products, for example, production of fuels, chemicals, power, and/or feed, is likely to be essential for economic viability. Lifecycle analysis is necessary to verify the sustainability and environmental quality benefits of a particular biocommodity product or process. We see biocommodity engineering as a legitimate focus for graduate study, which is responsive to an established personnel demand in an industry that is expected to grow in the future. Graduate study in biocommodity engineering is supported by a distinctive blend of intellectual elements, including biotechnology, process engineering, and resource and environmental systems.

2012 - Nature Reviews Drug Discovery

Targeting the TGFβ signalling pathway in disease

Many drugs that target transforming growth factor-β (TGFβ) signalling have been developed, some of which have reached Phase III clinical trials for a number of disease applications. Preclinical and clinical studies indicate the utility of these agents in fibrosis and oncology, particularly in augmentation of existing cancer therapies, such as radiation and chemotherapy, as well as in tumour vaccines. There are also reports of specialized applications, such as the reduction of vascular symptoms of Marfan syndrome. Here, we consider why the TGFβ signalling pathway is a drug target, the potential clinical applications of TGFβ inhibition, the issues arising with anti-TGFβ therapy and how these might be tackled using personalized approaches to dosing, monitoring of biomarkers as well as brief and/or localized drug-dosing regimens.

2000 - American journal of human genetics

Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole.

The origin of human triploidy is controversial. Early cytogenetic studies found the majority of cases to be paternal in origin; however, recent molecular analyses have challenged these findings, suggesting that digynic triploidy is the most common source of triploidy. To resolve this dispute, we examined 91 cases of human triploid spontaneous abortions to (1) determine the mechanism of origin of the additional haploid set, and (2) assess the effect of origin on the phenotype of the conceptus. Our results indicate that the majority of cases were diandric in origin because of dispermy, whereas the maternally-derived cases mainly originated through errors in meiosis II. Furthermore, our results indicate a complex relationship between phenotype and parental origin: paternally-derived cases predominate among "typical" spontaneous abortions, whereas maternally-derived cases are associated with either early embryonic demise or with relatively late demise involving a well-formed fetus. As the cytogenetic studies relied on analyses of the former type of material and the molecular studies on the latter sources, the discrepancies between the data sets are explained by differences in ascertainment. In studies correlating the origin of the extra haploid set with histological phenotype, we observed an association between paternal-but not maternal-triploidy and the development of partial hydatidiform moles. However, only a proportion of paternally derived cases developed a partial molar phenotype, indicating that the mere presence of two paternal genomes is not sufficient for molar development.

2006 - Journal of bacteriology

Genome Sequence of Avery's Virulent Serotype 2 Strain D39 of Streptococcus pneumoniae and Comparison with That of Unencapsulated Laboratory Strain R6

ABSTRACT Streptococcus pneumoniae (pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenesis. To date, the complete genome sequences have been reported for only two S. pneumoniae strains: TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report here the genome sequences and new annotation of two different isolates of strain D39 and the corrected sequence of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only five relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single-base-pair changes, six deletions, and four insertions and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, the relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed.

2005 - Annals of the New York Academy of Sciences

The Miniature Pig as an Animal Model in Biomedical Research

Abstract: Crucial prerequisites for the development of safe preclinical protocols in biomedical research are suitable animal models that would allow for human‐related validation of valuable research information gathered from experimentation with lower mammals. In this sense, the miniature pig, sharing many physiological similarities with humans, offers several breeding and handling advantages (when compared to non‐human primates), making it an optimal species for preclinical experimentation. The present review offers several examples taken from current research in the hope of convincing the reader that the porcine animal model has gained massively in importance in biomedical research during the last few years. The adduced examples are taken from the following fields of investigation: (a) the physiology of reproduction, where pig oocytes are being used to study chromosomal abnormalities (aneuploidy) in the adult human oocyte; (b) the generation of suitable organs for xenotransplantation using transgene expression in pig tissues; (c) the skin physiology and the treatment of skin defects using cell therapy‐based approaches that take advantage of similarities between pig and human epidermis; and (d) neurotransplantation using porcine neural stem cells grafted into inbred miniature pigs as an alternative model to non‐human primates xenografted with human cells.

2001 - The Journal of Biological Chemistry

Repair of Oxidized Proteins

Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. ThemsrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitrostudies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min−1 and 20 min−1, respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.

2005 - PLoS Biology

Two-Component Signal Transduction Pathways Regulating Growth and Cell Cycle Progression in a Bacterium: A System-Level Analysis

Two-component signal transduction systems, comprised of histidine kinases and their response regulator substrates, are the predominant means by which bacteria sense and respond to extracellular signals. These systems allow cells to adapt to prevailing conditions by modifying cellular physiology, including initiating programs of gene expression, catalyzing reactions, or modifying protein–protein interactions. These signaling pathways have also been demonstrated to play a role in coordinating bacterial cell cycle progression and development. Here we report a system-level investigation of two-component pathways in the model organism Caulobacter crescentus. First, by a comprehensive deletion analysis we show that at least 39 of the 106 two-component genes are required for cell cycle progression, growth, or morphogenesis. These include nine genes essential for growth or viability of the organism. We then use a systematic biochemical approach, called phosphotransfer profiling, to map the connectivity of histidine kinases and response regulators. Combining these genetic and biochemical approaches, we identify a new, highly conserved essential signaling pathway from the histidine kinase CenK to the response regulator CenR, which plays a critical role in controlling cell envelope biogenesis and structure. Depletion of either cenK or cenR leads to an unusual, severe blebbing of cell envelope material, whereas constitutive activation of the pathway compromises cell envelope integrity, resulting in cell lysis and death. We propose that the CenK–CenR pathway may be a suitable target for new antibiotic development, given previous successes in targeting the bacterial cell wall. Finally, the ability of our in vitro phosphotransfer profiling method to identify signaling pathways that operate in vivo takes advantage of an observation that histidine kinases are endowed with a global kinetic preference for their cognate response regulators. We propose that this system-wide selectivity insulates two-component pathways from one another, preventing unwanted cross-talk.

2009 - PloS one

Assembling the Marine Metagenome, One Cell at a Time

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa.

1996 - Molecular biology and evolution

Asymmetric substitution patterns in the two DNA strands of bacteria.

Analyses of the genomes of three prokaryotes, Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, revealed a new type of genomic compartmentalization of base frequencies. There was a departure from intrastrand equifrequency between A and T or between C and G, showing that the substitution patterns of the two strands of DNA were asymmetric. The positions of the boundaries between these compartments were found to coincide with the origin and terminus of chromosome replication, and there were more A-T and C-G deviations in intergenic regions and third codon positions, suggesting that a mutational bias was responsible for this asymmetry. The strand asymmetry was found to be due to a difference in base compositions of transcripts in the leading and lagging strands. This difference is sufficient to affect codon usage, but it is small compared to the effects of gene expressivity and amino-acid composition.

2007 - Microbiology and Molecular Biology Reviews

Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum

SUMMARY Actinobacteria constitute one of the largest phyla among Bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.

C‐di‐GMP: the dawning of a novel bacterial signalling system

Crystallization and preliminary X-ray diffraction characterization of the XccFimX(EAL)-c-di-GMP and XccFimX(EAL)-c-di-GMP-XccPilZ complexes from Xanthomonas campestris.

Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

Xanthomonas spp. Specificity of Plant Pathogenic Offer Insight into Host and Tissue Two New Complete Genome Sequences

Genome-Wide Mutagenesis of Xanthomonas axonopodis pv. citri Reveals Novel Genetic Determinants and Regulation Mechanisms of Biofilm Formation

Transcriptional Tradeoff between Metabolic and Stress-response Programs in Pseudomonas putida KT2440 Cells Exposed to Toluene*

Transgenic expression of Gluconacetobacter xylinus strain ATCC 53582 cellulose synthase genes in the cyanobacterium Synechococcus leopoliensis strain UTCC 100

Biological insights from structures of two-component proteins.

Quorum Sensing Controls Bioﬁlm Formation in Vibrio cholerae through Modulation of Cyclic Di-GMP Levels and Repression of vpsT (cid:1)

Manganese transport and the role of manganese in virulence.

The second messenger bis‐(3′‐5′)‐cyclic‐GMP and its PilZ domain‐containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa

The biosynthesis and regulation of bacterial prodiginines

The Vibrio harveyi quorum-sensing system uses shared regulatory components to discriminate between multiple autoinducers.

Solving the Problem: Genome Annotation Standards before the Data Deluge

The Cell Wall of the Arabidopsis Pollen Tube—Spatial Distribution, Recycling, and Network Formation of Polysaccharides1[C][W][OA]

Cyclic diguanylate signaling in Gram-positive bacteria.

Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

Complete Genome Sequence of Uropathogenic Proteus mirabilis , a Master of both Adherence and Motility (cid:1) †

Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world

c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress

PIRSF Family Classification System for Protein Functional and Evolutionary Analysis