Complement — tapping into new sites and effector systems

Nature Reviews Immunology | Perspectives | Opinion

Martin Kolev,^{1, 2,} Gaelle Le Friec^{1, 2,} & Claudia Kemper^1,

Published online 14 November 2014

Abstract

Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

Introduction

 The complement system, which was discovered more than 100 years ago, is one of the oldest components of immunity and is central to the detection and destruction of invading pathogens^{1, 2, 3, 4, 5}. Complement is a system of fluid-phase proteins (found in the blood, lymph and interstitial fluids) and cell membrane-bound proteins. The serum-circulating proteins, which are generally synthesized in the liver, are mostly present in an inactive pro-enzyme state, and the membrane-bound proteins comprise receptors and regulators of complement activation fragments. The detection of microorganisms that have breached the host environmental barriers by fluid-phase complement components leads to the activation of the complement cascade and the elimination of the microbial target (Fig. 1a). The complement cascade can be activated by three pathways: the classical, alternative and lectin pathways^{1, 2, 3, 4, 5} (Fig. 1a). All activation pathways lead to the generation of the C3 and C5 convertase enzyme complexes, which cleave C3 into the anaphylatoxin C3a and the opsonin C3b, and C5 into the anaphylatoxin C5a and into C5b, respectively. Deposition of C5b onto a target initiates membrane attack complex (MAC) formation and target lysis¹. The opsonins and anaphylatoxins promote phagocytic uptake of pathogens by scavenger cells, and activate neutrophils, monocytes and mast cells, respectively^{1, 2, 3, 4, 5}. On the basis of these effector functions, complement has long been considered as an innate immune pathway.

 

Figure 1: Distinct location-directed functions of complement activation.

a | Liver-derived, systemically circulating complement forms the first line of defence against invading pathogens and can be activated through three pathways: the classical pathway, the lectin pathway and the alternative pathway, with the initial deposition of C3b on a surface also initiating a feedback amplification loop. Through the formation of C3 convertases (C4bC2a for the classical and lectin pathways, and C3bBb for the alternative pathway), these pathways culminate in the generation of the opsonin C3b and the anaphylatoxin C3a. Subsequent C5 convertase formation (C4bC2aC3b for the classical and lectin pathways, and C3bBbC3b for the alternative pathway) leads to C5b and anaphylatoxin C5a generation, with C5b initiating the formation of the membrane attack complex (MAC) and its insertion into target membranes. C3 and C5 can also be activated directly via activating proteases (see Box 1). Self tissue is protected from complement deposition through fluid-phase and cell-bound regulators; C1 inhibitor (C1-INH) inhibits the functions of C1r, C1s and mannan-binding lectin-associated serine protease 2 (MASP2). C3b (and C4b) are inactivated by the serine protease complement Factor I and one of several cofactor proteins (surface-bound CD46 and complement receptor type 1 (CR1) or fluid-phase Factor H and C4b-binding protein (C4BP)). Convertases are regulated through disassembly by regulators that have decay-accelerating activity — surface-bound CD55 and CR1 or fluid-phase Factor H and C4BP — and the formation of the MAC is controlled by CD59 and vitronectin (also known as S protein)¹¹³. b | Locally occurring complement activation is triggered when a cell-activating signal (such as T cell receptor (TCR) stimulation) initiates the generation and secretion of C3, C5, Factor B (FB) and Factor D (FD), leading to C3 and C5 convertase formation in the extracellular space and/or on the cell surface, and ultimately to the generation of the complement activation fragments C3a, C3b, C5b and C5a. C3a, C3b and C5a bind to their respective receptors on the T cell and induce cellular responses. Intracellular complement activation in resting CD4⁺ T cells (and possibly other cell types) occurs continuously through the action of the C3-cleaving protease cathepsin L. The resulting C3a fragment engages the intracellular lysosome-localized receptor C3aR, which sustains tonic mammalian target of rapamycin (mTOR) activation and T cell survival (resting T cells express C3aR only intracellularly). TCR activation induces cell-surface translocation (shuttling) of this intracellular C3 activation system (indicated by the dashed arrows), where engagement of surface C3aR and CD46 induce intracellular signalling events (for details on these signalling events, see Refs 11,35) that ultimately mediate upregulation of key growth factor receptors — including the receptors for interleukin-2 (IL-2), IL-7 and IL-12 (IL-2R, IL-7R and IL-12R, respectively) — as well as proliferation and the induction of effector function. Autocrine complement receptor activation in antigen-presenting cells (APCs) is triggered by Toll-like receptor (TLR) activation and mediates APC maturation and the expression of MHC class II and co-stimulatory molecules, as well as cytokine production. The sum of autocrine and paracrine effects of local complement activation during cognate APC and T cell interactions defines the functional outcome of T cell activity. Although not depicted here, the cell-surface expression of complement regulators affects these processes by regulating local complement activation^{9, 30, 36}. Furthermore, the C3 activation fragments inactive C3b (iC3b) and C3dg are deposited extracellularly on apoptotic cells and are then taken up by APCs; here, they regulate lysosomal fusion, processing of apoptotic cell debris and subsequent antigen presentation by an as yet undefined mechanism⁴⁸. MBL, mannose-binding lectin; P, properdin; T_H, T helper.

However, the discovery that receptors for complement activation fragments are expressed by almost all immune cells — including B cells and T cells — and that these cells can sense and convert the levels of complement activation into tailored responses⁶ led to the appreciation that complement directs both innate and adaptive immune responses. For example, complement receptor activation lowers the threshold for B cell activation, directs antigen handling by follicular dendritic cells (FDCs) and contributes to the maintenance of B cell tolerance and memory^{7, 8}. Similarly, complement has a non-redundant role in CD4⁺ and CD8⁺ T cell activation and function, either directly through stimulating complement receptor-mediated signalling events in T cells or indirectly through modulating antigen-presenting cell (APC) function^{9, 10, 11}. The appreciation of the role of complement in adaptive immunity coincided with the understanding that complement detects not only pathogenic microorganisms but also potentially harmful self molecules, such as those that are exposed by stressed, injured, apoptotic or necrotic tissues and cells⁴. The discovery that complement aids in the disposal of cellular debris and instructs the adaptive immune system provided the missing mechanistic explanations for the long-known but poorly understood finding that complement deficiencies predispose to autoimmune disease^{12, 13, 14, 15}.

 

Recent studies are also providing a new dimension to our understanding of complement. Unexpectedly, it was shown that complement can be activated not only at the cell surface, as traditionally thought, but also in intracellular compartments¹⁶. Moreover, it is now becoming clear that systemic serum complement has different functions from local immune cell-derived complement. Rather than being a mostly pro-inflammatory effector system, complement is emerging as a central player in cell and tissue development, homeostasis and repair. Studies of the molecular mechanisms underlying these new functions of complement have led to the discovery of new crosstalk between complement components and other cell effector systems, including growth factor receptors, inflammasomes, metabolic sensors and the Notch system. In this Opinion article, we propose a model to explain how the different locations of complement activation dictate its diverse functions and how complement engages other effector systems at these locations to regulate immune-related and non-immune-related processes.

 

Figure 2: Functional crosstalk between complement and other cell effector systems.

The functional crosstalk between the complement system and Toll-like receptors (TLRs) and the coagulation cascade has long been acknowledged. The recent developments in the field have led to the discovery of additional direct crosstalk with key effector systems, including the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, carbohydrate receptors (such as dectin 1), Fc receptors for IgG (FcγRs), and cytokine and growth factor receptors, as well as the WNT and Notch systems. Cell populations in which this crosstalk occurs are indicated and, where identified, the signalling pathways driving the functional outcome of the crosstalk between complement and effector systems are shown. The regulation of the mammalian target of rapamycin (mTOR) metabolic sensing system by complement is not included here but the current knowledge about this crosstalk is summarized in Refs 23,39. '+' denotes upregulation; '−' denotes downregulation; AP-1, activator protein 1; APC, antigen-presenting cell; cAMP; cyclic AMP; DC, dendritic cell; DLL1, delta-like ligand 1; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; IL, interleukin; LRP, low-density lipoprotein receptor-related protein; MAC, membrane attack complex; MAPK, mitogen-activated protein kinase; MASP2, mannan-binding lectin-associated serine protease 2; MYD88, myeloid differentiation primary response protein 88; NF-κB, nuclear factor-κB; P2RX7, P2X purinoceptor 7; PI3K, phosphoinositide 3-kinase; SHIP, SH2 domain-containing inositol-5-phosphatase; SYK, spleen tyrosine kinase; R, receptor; SPAK, ST20/SPS1-related proline-alanine-rich protein kinase; T_H, T helper; TNF, tumour necrosis factor; T_Reg, regulatory T.

Figure 3: Complement at the nexus of the extensive crosstalk between cell effector systems.

The interaction between complement and other key cell effector systems involved in innate and adaptive immunity is multifactorial and in most cases bidirectional. Furthermore, the functional impact of complement on effector systems with primarily non-immune functions (for example, the Notch and WNT systems) is more substantial than previously thought, and indicates that complement contributes to normal development, and possibly to ageing and behaviour. We suggest that the emerging role of complement in core physiological metabolic pathways may be the crucial functional intersection point in this network. FcγR, Fc receptor for IgG; TLR, Toll-like receptor.

Conclusions and future perspectives

Complement has traditionally been defined as an innate and systemic system that functions in the defence against pathogens. However, it is now considered to be a central regulator of innate and adaptive immunity, with new functions that extend beyond protective immunity, including roles in cell generative, degenerative and regenerative processes. The finding that complement is activated within cells and not only engages intracellular complement receptors but also intersects with several other cell effector systems helps to explain its unexpectedly wide-reaching effects.

 

Nevertheless, there is still much to discover about this ancient system and key future questions include: how is intracellular complement generation and activation regulated? Does this novel pathway contribute to disease? Are additional complement components, including regulators, functionally active inside cells? In this regard, we have detected intracellular C5a (A. Fara and C. Kemper, unpublished observations) and several studies have reported intracellular expression of Factor D, complement receptor type 1 (CR1; also known as CD35) and the positive regulator properdin in resting cells^{89, 90}. Thus, one could envision the existence of an intracellular 'Complosome' — somewhat analogous to the inflammasome⁹¹ — that has novel functions in cell survival and activation. Furthermore, a unifying feature of the new roles and interactions for complement is their reliance on appropriate sensing of cellular integrity and balanced control of energy and substrate metabolism. Therefore, the emerging cooperation between complement and the metabolic pathway network may arise as a core intersection point for the diverse functions of complement in immunity and beyond (Fig. 3).

 

 

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DNA tape recorder stores a cell's memories

Latest News > Biology > DNA tape recorder stores a cell's memories

By Sarah C. P. Williams 13 November 2014 2:15 pm


Mojtaba Amin

SCRIBE, a new cellular memory system, uses DNA to store information the same way that a cassette

might record sounds.

If cells could talk, they’d have quite a story to tell: Their life history would include what molecules they’d seen passing by, which signals they’d sent to neighbors, and how they’d grown and changed. Researchers haven’t quite given cells a voice, but they have now furnished them with a memory of sorts—one that’s designed to record bits of their life history over the span of several weeks. The new method uses strands of DNA to store the data in a way that scientists can then read. Eventually, it could turn cells into environmental sensors, enabling them to report on their exposure to particular chemicals, among other applications.

 

“They’ve done a really exceptional job turning DNA into readable, writable memory inside living cells,” says Ahmad Khalil, a biomedical engineer at Boston University who was not involved in the new work. “I think it’s a very cool new direction for synthetic biology to take.”

 

In the past, researchers have turned cells into simple sensors by switching on or off the production of proteins in response to a stimulus. But each switch could record only one simple piece of information—whether the cell had been exposed to the stimulus—not the duration or magnitude of this exposure. And if the cell died, the information—encoded in a protein—would be lost.

 

“We wanted a system that would be easier to scale up to collect more than one piece of information,” says synthetic biologist Timothy Lu of the Massachusetts Institute of Technology in Cambridge. “So we started out, as engineers, thinking about what an ideal memory system would look like.”

Lu’s team settled on a biological program that rewrites a living cell’s DNA when the cell senses a signal—from a flash of light to the presence of a chemical. Once the DNA is altered, the information remains embedded in the genetic material even if the cell dies. By sequencing the genes of a population of cells that all contain the program, researchers can determine the magnitude and duration of the signal: The more cells have the genetic mutation, the stronger or longer the signal was.

 

The approach, dubbed Synthetic Cellular Recorders Integrating Biological Events (SCRIBE), relies on retrons—which make up a genetic system found naturally in some bacteria that produces single-stranded DNA that the bacteria normally use to alter their host. Lu’s team started with bacterial cells and inserted a retron that would be turned on—producing the unique DNA—only in response to a specific stimulus like a chemical. While the cell is in the process of copying its genetic material, the new DNA would then replace a nearly identical existing gene segment in the cell, changing it slightly.  

 

Lu tested SCRIBE on cells that he engineered to sense light, as well as others that responded to a common biological reagent. In one instance, he made the memory especially easy to read by engineering the cells to mutate an antibiotic resistance gene in response to light. When cells were then grown in the presence of the antibiotic, the researchers could immediately see which cells contained the new gene. The results were confirmed by sequencing the bacteria’s genomes. But SCRIBE, described online today in Science, could be designed to sense other stimuli and cause any desired genetic mutation in return.

 

“There are a bunch of potential applications of this system,” Lu says. “One is being able to do long-term recording of a cell’s environment.” For example, he says, living cells could be left in an area of water for a week, then collected. Sequencing the DNA from the cells could then reveal whether the cells had been exposed to certain bacteria or toxins in the water. SCRIBE could also be a boon to basic researchers, Lu adds. “During development, as you go from a single cell to a multicellular organism, each cell encounters different cues,” he says. SCRIBE could let researchers record what each cell encountered to shape its fate.

 

“What’s neat about this strategy is that you have a lot more diversity and flexibility than other methods to give cells memory,” Khalil says. Because scientists can choose the stimulus—or multiple different stimuli—that they want the cell to record, as well as what gene change they want to use as a marker, the possibilities for applications are wide, he says.

Posted in Biology 

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The Origins of Oversized Chromosomes

The Scientist »  The Nutshell

Researchers reconstruct the formation of the giant neochromosomes that contribute to some cancers.

By Molly Sharlach | November 12, 2014

 

Neochromosomes (green) found in some cancer calls

may be up to three times as long as normal

chromosomes (magenta).

MURDOCH CHILDREN’S RESEARCH INSTITUTE, OWEN MARSHALL

More than half a century ago, scientists noticed a distinctive abnormality in the karyotypes of some soft-tissue cancers: unusually large chromosomes, now referred to as neochromosomes. In a new analysis reported this week (November 10) in Cancer Cell, a team of Australian researchers have uncovered the origins of neochromosomes and revealed mechanisms that could guide therapies to block the chromosomes’ construction.

 

Using next-generation sequencing and mathematical modeling to investigate the development of neochromosomes, David Thomas of the Garvan Institute in Sydney and his colleagues found that the process appears to begin with the splintering and rearrangement of chromosome 12, followed by breakage-fusion-bridge cycles that lead to the amplification of oncogenes. Notably, neochromosomes often contain dozens of copies of the MDM2 and CDK4 genes, which are involved in cell cycle regulation.

 

Neochromosomes, which may be up to 700 million base pairs long (three times the size of the longest normal chromosome), also contain bits and pieces of material from all of the cell’s chromosomes, particularly in their telomeres, which are stitched on at later stages.

 

“These cancers manipulate the normal replication process in an ingenious way, creating a monster that can selectively steal and amplify the genes it needs to grow and survive,” Thomas said in a statement. “In some liposarcoma cell lines, DNA from every chromosome in the cell was found in the neochromosome, with between 60 and 100 copies of key oncogenes. Patient tumours also exhibited similar gene rearrangement.”

 

“The life history that emerges . . . is apparently a tale of disaster upon disaster,” wrote Joshua Waterfall and Paul Meltzer of the US National Cancer Institute in a commentary on the study. “It is quite surprising that anything functional, let alone beneficial for the cell, can be so created.”

Tags

oncogene, genetics & genomics, chromosomes and cancer

 

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New Models and Tools for Studying Synaptic Development and Function

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Circular Chromosomes Straightened

The Scientist » News & Opinion » Daily News

A newly described method linearizes circular chromosomes in yeast and caps them with telomeres to mimic natural chromosomes.

By Kerry Grens | November 6, 2014

 

FLICKR, AJ CANN

Synthetic biologists often work with circular chromosomes to engineer genetic material because they’re stable and easy to manipulate, but they don’t resemble the natural shape of chromosomes in eukaryotes. Reporting in PNAS this week (November 5), Jef Boeke of NYU Langone Medical Center and postdoc Leslie Mitchell designed a tool, which they dubbed the telomerator, that straightens circular yeast chromosomes and adds telomeres to either end.

“To convert circular DNA to something more akin to a natural chromosome is appealing,” said Timothy Lu, a synthetic biologist at MIT who was not involved in the study. Lu said the telomerator could help advance a number of goals, from designing artificial chromosomes that encode complex pathways to testing the significance of telomere location in the genome. “It’s really a platform technology for downstream applications.”

 

The telomerator includes an endonuclease target—the site where the DNA loop will be severed—flanked by telomere seed sequences that form the basis of telomere construction. The telomerator is inserted into a gene of interest in the circular chromosomes, and when an endonuclease cuts the sequence at the recognition site, each exposed end carries a seed sequence on which to build telomeres. “The reason you can actually linearize the molecule and produce a stable molecule is because this telomere seed sequence gets exposed,” explained Mitchell. She and Boeke engineered the telomerator so that it would be induced only in the presence of galactose, giving scientists an easy way to turn it on by simply changing the growth medium.

 

The researchers tested the telomerator on a synthetic chromosome designed a few years earlier, cutting it at 54 different genes. For 51 of the permutations, “we got pretty happy yeast at the end of the experiment,” said Boeke. For another three, it appeared that the proximity of essential genes to the new telomere interfered with their expression, a phenomenon known as telomeric silencing. Blocking telomeric silencing rescued the cells’ growth. “I would say we’ve worked out most of the kinks for yeast,” said Boeke. “All you need to decide right now is where you want to put it.”

 

Yo Suzuki, a synthetic biologist at the J. Craig Venter Institute, said that linearizing DNA is important to avoiding problems in meiotic recombination that can emerge with circular chromosomes. In particular, crossover events between two circular chromosomes can result in a chromosome with two centromeres, which would break during chromosome segregation. “If you have a linear molecule in yeast, you don’t have that problem,” Suzuki said.

 

Suzuki envisions that the telomerator will open up more opportunities for chromosome engineering, such as splitting chromosomes into customized fragments, fitting them back together, and then delivering the genetic material to recipient cells such as bacteria. Suzuki and colleagues have developed an approach of moving genomes into new cells via cell-to-cell transfer, and being able to do all the chromosome engineering within the yeast cell before transferring the material to a recipient will accelerate progress in the field, he said. “Boeke’s approach of linearizing is the first step,” he told The Scientist.

 

Alina Chan, a postdoc in Pam Silver’s lab at Harvard University, said it will be informative to explore the characteristics of the telomeres on the linearized chromosomes. “That’s something that would be interesting to look at in an artificial context: how long telomere seeds get extended and whether they get truncated over time,” Chan told The Scientist. Additionally, she said, the telomerator will be useful to study why eukaryotes have linear chromosomes. “What are the evolutionary advantages of the linear format?” she asked.

 

Boeke said his next goal is to apply the telomerator to mammalian cells. “We’d like to be able to build large molecules in the form of circles because they’re relatively easy to move around, sequence, et cetera,” he said. “But when we deliver them to mammalian cells we’d like to deliver a linear chromosome that looks like a native chromosome. And we think the telomerator allows us to do that.”

 

Chan pointed out that some of the technology the telomerator calls upon was developed two decades ago. And since then, others have induced chromosome splitting. Boeke’s advance was to achieve it in a circular chromosome. “It’s important to point out that various bits and widgets of this thing we pulled together are not unique to us, but it’s a combination of all these pieces that uniquely defines the telomerator,” Boeke said.

 

The name, too, was inspired by past successes in bioengineering, he noted. The repressilator is a genetic circuit developed more than a decade ago that cycles the expression of a fluorescent signal. The term telomerator “is a nod to the first synthetic biology device,” Boeke said.

 

L.A. Mitchell and J.D. Boeke, “Circular permutation of a synthetic eukaryotic chromosome with the telomerator,” PNAS, doi:10.1073/pnas.1414399111, 2014.

Tags

yeast, telomeres, telomerase, synthetic biology, genome, genetics & genomics and chromosomes

 

 

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The RNA World: molecular cooperation at the origins of life.

Abstract

The RNA World concept posits that there was a period of time in primitive Earth's history — about 4 billion years ago — when the primary living substance was RNA or something chemically similar. In the past 50 years, this idea has gone from speculation to a prevailing idea. In this Review, we summarize the key logic behind the RNA World and describe some of the most important recent advances that have been made to support and expand this logic. We also discuss the ways in which molecular cooperation involving RNAs would facilitate the emergence and early evolution of life. The immediate future of RNA World research should be a very dynamic one.

Key points

• Research into the RNA World paradigm is active, and new discoveries in synthetic organic chemistry and biochemistry routinely provide new insights.
• The field of prebiotic chemistry is increasingly discovering phenomena that provide solutions to multiple (as opposed to single) problems simultaneously.
• A key issue in RNA World research is how RNAs might have made copies of themselves (that is, how they replicated). There are now several possible mechanisms of this process, and increasing focus is being placed on those that display autocatalytic feedback.
• Cooperation among various molecules was probably a key aspect of the RNA World, and at least three types of molecular cooperation could have been at play during the origins of life.
• Chemical alternatives to RNA per se may have existed at some point in the Earth's earliest history, and many efforts are underway to find and evaluate such structures.
• Network establishment was another process that had a large impact on the organization of the living state, from small molecules to large molecules and cell-like structures.

Introduction

 

The RNA World is the conceptual idea that there was a period in the early history of life on Earth when RNA, or something chemically very similar, carried out most of the information processing and metabolic transformations needed for biology to emerge from chemistry. This scenario, if it indeed existed, took place some 4 billion years ago. By contrast, the realization that RNA is a good candidate for the emergence of life is an idea that is only ~50 years old. It was recognized early on by Crick¹, Orgel² and others that RNA has both a genotype and a phenotype, and that a system based on RNA would be a plausible precursor to the much more complex system of DNA–RNA–proteins on which current life is based. It was also realized that the ribonucleotide coenzymes now used by many proteins may be molecular 'fossils' from an RNA-based metabolism³. Discoveries of naturally occurring ribozyme catalysts, such as self-splicing introns⁴ and the ribonuclease P catalyst⁵, were made in the 1980s and, with the demonstration that ribosomal RNA catalyses peptide bond formation in the ribosome⁶, the credentials of RNA as a catalyst became firmly established.

 

Spiegelman's classic experiments with the bacteriophage Qβ showed how viral RNA could evolve over time in response to selection⁷. This study gave rise to the field of in vitro evolution⁸ by demonstrating that RNA could behave in a Darwinian manner in the absence of cells. Once this realization has been made, pioneers such as Orgel⁹, Eigen¹⁰, Joyce^{11, 12}, Gold¹³ and Szostak¹⁴ fully demonstrated the evolutionary capabilities of RNA and made it difficult to ignore the possibility that life started with RNA. However, additional pieces of data from both new and old angles then became available. The catalytic repertoire of RNA was shown to be diverse¹⁵, RNA riboswitches were detected in bacteria and shown to be widespread in biology¹⁶, and an autocatalytic cycle based on RNA ligases was found¹⁷. Furthermore, polymerase ribozymes that can use another sequence as a template were selected and improved^{18, 19, 20} (see below).

 

Different research questions are gradually being brought together to assemble a complete picture of the emergence of life via the RNA World scenario (Fig. 1). New routes of chemical synthesis of ribonucleotides that could operate prebiotically are being studied, and there are also further experiments to isolate ribozymes from random RNA sequences. These areas are reviewed below. Theoretical models of RNA action are being developed to describe the origin and evolution of replicating systems. A point that emerges both from theoretical work and from laboratory experiments is that cooperation at the molecular level is essential for the survival of replicating sequences. A key aim of this Review is to describe the different senses in which cooperation is relevant in the RNA World. We argue that RNA replication must also fit into a broader thermodynamic and biological context if this were to form the basis of life (Fig. 1). What was the energy source that drove the synthesis of large macromolecules on early Earth? What were the environmental conditions at the location where these molecules were forming? How did RNA replication become associated with growth and division of protocells? Current ideas on some of these questions are considered later in this Review.

 

Figure 1: Research in different fields is coming together to assemble a more complete picture of the way the RNA World began and operated.

a | Progress in organic chemistry helps to show how nucleotides and RNA oligomers could have been synthesized before life. b | In vitro evolution studies discover functional ribozymes in the very large RNA sequence space. c | Theoretic…

 

Figure 2: RNA polymerases as altruistic cooperators.

 

a | A trans-acting RNA polymerase (or replicase) such as the R18 polymerase¹⁸ is a likely mechanism for supporting replication in the RNA World. b | Such a polymerase can use a template that is either another copy of itself (red) or an unrelated sequence (grey). Well-mixed systems of altruistic replicators are destroyed by parasites. There are two ways in which cooperators can resist parasites. c | First, survival of replicases is possible in two-dimensional models on surfaces. A simulation of altruistic replicators (red) diffusing on a surface in the presence of parasites (grey) demonstrates survival of the replicators as a result of spatial clustering⁸². d | Second, when small groups of molecules are packaged in compartments, group selection can occur. Functional RNAs (red and blue) are shown replicating in a protocell compartment in the presence of parasites (grey). Random segregation creates cells of varying composition. Cells with an excess of parasites are unviable, and this prevents parasites from over-running the system, even if the parasites multiply at a higher rate within a cell. Part a reproduced from Ref. 50, Nature Publishing Group. Part c reprinted from J. Theor. Biol., Vol. 364, Shay, J. A., Huynh, C. & Higgs, P. G., The origin and spread of a cooperative replicase in a prebiotic chemical system, 249–259, Copyright (2014), with permission from Elsevier.

 

Figure 3: Different senses of molecular cooperation.

 

a | Cooperation in networks of mutually dependent strands with different functions is shown. An autocatalytic set involves precursor molecules present in the environment (blue circles) and molecules synthesized by the cycle (green circles). Each reaction (red triangles) is catalysed by a molecule that is part of the set (dashed arrows). b | An autocatalytic set composed of two cross-catalytic ligases¹⁷ is shown. RNA A and RNA B are ligated together by ribozyme E′ to create ribozyme E, which can reciprocate and ligate RNA A′ and RNA B′ to create ribozyme E′. c |Cooperation between multiple strands that assemble to perform a single function is shown. Ribozymes, such as the Azoarcus recombinase⁵⁷, can be made from several short strands that assemble as a result of RNA secondary structure formation and information contained in internal guide sequences (IGSs) and complementary targets (grey). d | While strands can cooperate to form ribozymes, these ribozymes can then potentially cooperate at an even higher level to construct an autocatalytic set, such as a three-membered cycle⁶⁰. In this scheme, the Azoarcus ribozyme is fragmented into two pieces in three different ways (that is, at three different junctions). The IGS of the ribozyme in E₁ is adjusted in such a way that it can only covalently assemble the E₂ ribozyme and so on, such that for assembly of all the ribozymes an obligatory cooperative process must (and does) occur. Part b from Lincoln, T. A. & Joyce, G. F. Self-sustained replication of an RNA enzyme. Science 323, 1229–1232 (2009). Reprinted with permission from AAAS. Parts c and d adapted from Ref. 60, Nature Publishing Group.

Conclusions and perspectives

 

Contemporary studies both in the laboratory and by simulation are beginning to reveal the cooperative nature of the RNA World, as well as how various types of cooperation and conflict probably guided the earliest evolutionary processes. The basics of the RNA World concept are well established, but the details continue to evolve. A powerful theme has begun to emerge from many of these new approaches and their results. One way to describe this is systems chemistry¹⁰⁰. By focusing not on single reactions in isolation but on the collective set of processes that must all occur contemporaneously, systems chemistry can lead to the discovery of 'multi-fers' in which the system as a whole can establish itself through the shared use of common conditions, reactants or products. Another way to look at this process, one that we highlighted in this Review, is that of chemical cooperation (Box 1). This newer view, embedded in the broader perspective of intermolecular cooperation and conflict, is pervading other aspects of study of the RNA World, as we explored above.

 

Key questions for the next decade of research include the following. How long were the first ribozymes? In other words, what is the extent of the error threshold problem? How specific were the first ribozymes? Was life sparked by a single polymerase or a random autocatalytic set? How far can the RNA World go without being encapsulated in a cell, or were there cells already at the earliest stage? What was the energy source for the RNA World? Thus, how was it possible to link a stable energy supply to a metabolic synthesis of RNA? The RNA World idea emphasizes replication, but thermodynamic driving force is still needed for synthesis. We anticipate that the answers to many of these questions will not only be within our conceptual reach in the next decade or two, but will also invoke the insights gained from a more systematic appreciation of how conflict and cooperation can influence molecular processes. 

Nature Reviews Genetics | Review
Article series: Non-coding RNA
Paul G. Higgs^1, & Niles Lehman^2,

Year published (2014)

 

 

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DNA Extraction Kits Contaminated

The Scientist » News & Opinion » Daily News

Sequencing study reveals low levels of microbes in lab reagents that can create big problems for some microbiome studies.

By Kerry Grens | November 11, 2014

 

FLICKR, CIMMYT

Researchers studying microbiomes can do their best to prevent contamination, but a new study reveals widespread, low-level contamination in DNA extraction kits. Reporting in BMC Biology today (November 11), Alan Walker of the University of Aberdeen in the U.K. and colleagues list dozens of contaminating taxa that can swamp out a sample’s true microbial signal, if starting concentrations are low.

 

“It’s really important to sequence a negative extraction control,” said Patrick Schloss, a microbiome researcher at the University of Michigan who did not participate in this study. “That’s something people should be doing and are not doing.”

 

Contamination or signal?

The presence of microbial DNA in laboratory reagents is nothing new. Studies have even found bacterial DNA in ultrapure water, and just a few weeks ago researchers pointed out ubiquitous contamination in next generation sequencing runs. In many cases such extraneous DNA may not be an issue, but for highly sensitive deep sequencing of amplified samples, contaminants can start to compete with signal, as Walker’s team found.

 

Walker’s group made serial dilutions of Salmonella bongori, beginning with 100 million cells and reducing the sample down to 1,000 cells. When the sample of S. bongori was 10,000 cells or fewer, the abundance of DNA from other microbial taxa exceeded 50 percent of the sequences. This happened using four different commercial extraction kits. Bradyrhizobiaceae, Burkholderiaceae, Pseudomonadaceae, and dozens of other bacterial groups were present, although each kit had a different profile.

 

“The assumption has been they are sterile,” said Walker, adding that, to be fair, DNA extraction kits aren’t marketed as such. “What [researchers] need to do is go back and do some negative controls and with a bit of confidence say, ‘they really are in the samples,’ in cases where maybe there are potentially suspicious results,” he said. Schloss made the point that investigators should not rely on the list Walker’s team produced as a comprehensive catalog of potential contaminants, but be sure to do their own controls given that contamination can vary from batch to batch.

Safeguarding samples

 

Walker’s paper pointed to a couple of studies in which human diseases have been linked with unexpected microbes. In one, for example, Delphine Lee of the John Wayne Cancer Institute in Santa Monica, California, and colleagues found different microbial profiles in breast tissue from cancer patients and normal controls, namely, different abundances of Methylobacterium radiotolerans and Sphingomonas yanoikuyae. Although Methylobacteriaceae and Shingomonadaceae popped up in some of the DNA extraction kits Walker’s group tested, Lee said that she and her team are very aware of contamination concerns—especially given that her work involves low biomass samples—and that they performed the right controls.

 

“With low biomass the tiniest little change can be detected,” Lee told The Scientist. In contrast, a fecal sample would have many more microbial cells. “You could sneeze all over it, you’re not going to affect the findings.”

 

Another study, by Matthew Meyerson of Harvard Medical School and colleagues, identified a novel bacterial species, Bradyrhizobium enterica, in patients with a colitis syndrome. Bradyrhizobium were also detected in Walker’s study, although Meyerson said his team did not detect any DNA from the organism in the reagents used. Additionally, his group performed in situ hybridization on the samples, from which they could see a signal from B. enterica in tissue from patients with colitis but not in controls. Meyerson said contamination is always a possibility, and performing additional cellular assays adds a very strong layer of confidence in the sequencing results.

 

Schloss has experienced his own problems with reagent contamination. In a study on cystic fibrosis, his team found Pseudomonas DNA present in a commercial kit. Such contamination is particularly problematic given that these bacteria are an important pathogen in the lung, especially among people with cystic fibrosis. “We need to be thoughtful in how we design our experiments [and determine] what types of controls are critical.”

 

Clean kits

Martin Laurence, the founder of Montreal-based ShipShaw Labs, which develops bioinformatics tools, said that sequencing studies would greatly benefit from kits that are free of any microbial DNA. “That would simplify studies a lot,” said Laurence, who reported with colleagues earlier this year the presence of bacterial DNA in human genome sequencing reads.

 

In an e-mail to The Scientist, Qiagen spokesperson Przemek Jedrysik pointed out that the QIAamp DNA Stool Mini Kit, one of the four Walker tested, “was not designed to be DNA-free or to be used in low-biomass applications.” However, he added, Qiagen recognizes that certain analyses require cleaner reagents and the company has been developing products with ultra clean product (UCP) spin columns. “It has been confirmed by independent laboratories that the new UCP kits have a significantly lower background and thus meet requirements for high sensitivity analyses.”

 

It’s not just researchers and kit manufacturers with the responsibility of managing contamination issues, but peer reviewers as well, said Laurence. “Going forward, this article should make the peer-review process more strict for articles that find novel bugs and associate them with human disease.” In particular, that means ensuring studies include the appropriate controls. “Many, many studies have used these DNA extraction kits,” he said. “I’m very surprised that it took so long” for someone to report on the contamination.

 

S.J. Salter et al., “Reagent and laboratory contamination can critically impact sequence-based microbiome analyses,” BMC Biology, 12:87, 2014.

Tags

next-gen sequencing, microbiome, microbiology, genetics & genomics, DNA and contamination

 

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Incredible Flu Fighting Tea that Gives Fast Results

Healthy Food Style

Being healthy means being happy

If you are one of those always stocking up flu and cold medications before the cold weather sets in, then the time has come to rethink your strategy. Embrace all the remedies that nature gives and combine them to create a powerful flu fighting tea that gives incredible results.

Ginger stimulates circulation and has been used for centuries as a remedy for sore throat. It warms the body and gives you instant cold relief. It has antiviral and anti-inflammatory properties and is effective for people experiencing respiratory problems.

 

Lemon has citric acid and bioflavonoids that boost immunity and fight infection and is an excellent source of Vitamin C. Regular consumption of lemon helps you stay healthy and protects you from many ailments. It is widely known as strong natural antiseptic and when combined with ginger helps with sinuses.

 

Ginger-lemon combination was traditionally used by many ancient cultures to relieve symptoms accompanied by flu, cold and infections. Most common symptoms included runny nose, fever, cough, chills, sneezing, muscles aches, mild tiredness, headache etc.

 

Cinnamon is considered as a “wonder spice” when it comes to flu and cold treatment. Using cinnamon as part of warm drinks significantly reduces cold and flu effects.

 

The preparation of this tea is always recommended as a prevention of common cold or as a remedy to help your body get rid of flu fast and effectively.

 

Ingredients:

• a thumb of ginger
• 1 lemon
• 1/2 tbsp. cinnamon
• 2 cups water

Instructions:

Put the water in a teapot and bring it to boiling point. Then add the grated ginger and the cinnamon. Let it boil for 5 minutes. Put it aside until it cools a bit ( but not completely). Strain the mixture, squeeze the lemon and stir well. Drink immediately.

November 8, 2014

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Opinion: The Planet Needs More Plant Scientists

Academia is not producing sufficient PhDs in the plant sciences to solve the crop production challenges facing a rapidly growing population.

By Alan M. Jones | October 1, 2014

 

FLICKR, JOI ITOWhile the message is not new, the declaration of the flaws of the US biomedical research system by four prominent life scientists this spring captured everyone’s attention. Bruce Alberts, Marc Kirschner, Shirley Tilghman, and Harold Varmus wrote in PNAS of how “demands for research dollars grew much faster than the supply . . . [due to] perverse incentives [that] encourage grantee institutions to grow without making sufficient investments in their faculty and facilities.” Rather than devote money to faculty salaries, universities built infrastructure to house more self-paid researchers able to bring in more money via research grants, of which a large fraction was used as revenue (overhead) for the university. More labs required more students to fill them, leading to a dramatic rise of PhDs in the biomedical sciences, which then produced more researchers competing for dwindling grant dollars. In short, research institutions have no incentive to support individual faculty and instead have perverse incentives to encourage further research spending: more grants = more overhead = more buildings = more PIs = more PhDs in an increasingly out-of-control spiral. (See “PhDs in the U.S.”) This is not sustainable, and we are now experiencing the consequences, with the most despairing being the lack of adequate jobs for our postdocs and perceived insufficient funding for all of us.

 

PhDs in the U.S.: From 1982 to 2012, the total number of PhDs in the life sciences (blue) has grown dramatically. Most of these PhDs are in biological, biomedical, and health sciences (red), however; the number of PhDs in the agricultural and natural sciences (green) has remained flat over that same time period. The unsustainable rate of PhDs awarded per year in the biomedical sciences does not extrapolate to the rate of PhDs in other life sciences, however, especially the agricultural sciences, where the rate of PhDs per year has remained flat for decades. Since 1982, we have consistently trained only about 1,000 PhDs in applied agricultural and related sciences each year. And over the last decade, the U.S. has annually produced only 800 or so plant scientists working in applied agricultural science and only 100 with the skills for basic plant research. (See “Plant science stagnates.”) Given the global agricultural challenges we now face, this is a problem.

 

The Earth must support another 1 billion humans in the coming decade, and must do so with less arable land and in an unpredictable climate. This means we must find innovative ways to produce crops with higher yields and novel traits—a feat that will require the work of PhDs trained in agriculture and plant sciences. But at this point we are not producing enough plant scientists to lead us out of this Malthusian dilemma.

 

The US Coalition for a Sustainable Agricultural Workforce recently completed a confidential survey among agricultural biotech companies to ascertain near-term needs for hiring domestic agricultural scientists. This survey generated an amazing result, given the tone of the PNAS perspective, predicting that by 2015, 1,000 new employees will be needed in the half-dozen largest plant-science companies in the US alone (Bayer Crop Science, Dow Agro Sciences, Dupont Pioneer Hybrid, Dupont Crop Protection, Monsanto, and Syngenta). Almost half of these anticipated new hires will hold PhDs. Unfortunately, with what appears to be a dwindling pool of qualified applicants applying to plant science PhD programs, we may not be keeping up with this demand.

 

Plant science stagnates: Selected subdisciplines relevant to a work force in plant industry (blue and green) have not increased this century, while selected biomedical subdisciplines (red) have grown steeply.

Biomedical subdisciplines include bioinformatics, biomedical sciences, biometrics and biostatistics, cancer biology, computational biology, developmental biology/embryology, neurosciences and neurobiology, structural biology, virology. Basic plant biology subdisciplines (green) include botany/plant biology, plant genetics, plant pathology/phytopathology, plant physiology. Agricultural research subdisciplines (blue) include agricultural and horticultural plant breeding, agricultural economics, agronomy and crop science, forest engineering, forest sciences and biology, forestry and related science, horticulture science, plant pathology/phytopathology (applied), plant sciences (other), soil chemistry/microbiology, soil sciences, entomology, plant genetics, plant pathology/phytopathology applied plant physiology. The growing world population needs to eat, and it is past due that we elevate basic, translational, and applied plant research to the priority given to biomedical research, or more boldly, to defense. Stabilizing food supplies in a changing environment is integral not only to the world population’s health, as an estimated 50 percent of childhood disease globally is attributed to malnourishment, but also to national security. Moreover, a recent study found that, around the world, the rate of return for investment in agricultural research is ten to one, bringing into question the scaling back of funding for agriculture research and development in many rich countries.   

Going forward, we must infuse more resources into plant biology research, to boost research output and to train tomorrow’s plant scientists. In the early 1980s, the National Science Foundation (NSF) established an 11-year postdoctoral fellowship program with the primary objective to nurture future leaders of plant biology research. By many accounts, this program was successful; among a cohort of 236 fellows, four are members of the National Academy of Sciences today, and more than 80 percent remained in plant biology. Of those, the majority stayed in academic institutions, while an impressive number (25 percent) went to industry, where many now hold corporate officer positions. Anticipating the need for leaders to alleviate hunger and to prevent global instability, we should reinstate this program to recruit our best talent to plant science and agricultural research.

 

In conclusion, it is important that the sirens of a glut of biomedical PhDs do not fallaciously harm other areas of science that are still in desperate need of young researchers and more research funding. This is especially true for the plant sciences, where the next generation of researchers must conquer significant challenges to feed a growing world population in a changing environment.

    

Acknowledgement: I thank Machi Dilworth for providing annotated data on the NSF Postdoctoral Fellowship program. Data source: NSF.

 

Alan M. Jones is a Kenan Distinguished Professor at the University of North Carolina, Chapel Hill.

Tags

plant science, plant biology, PhD, jobs and agriculture

 

 

 

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Best Drink to Boost Metabolism and Reduce Weight Naturally

Healthy Food Style

Being healthy means being happy

You may feel frustrated about all your attempts at slimming your waistline and shedding all those extra pounds you have been carrying around for some time. You tried your best and still no evident results.

No magical pill or electronic device can substitute the good old exercise and proper diet. Choosing the right combination is the key to healthy weight loss management without being afraid of the pounds bouncing back. Yellow and orange color foods are excellent solution for losing weight and they are extra delicious. Moreover, they are recommended for improving mood, so if you are disappointed after so many failed attempts, now is the time to take control of the situation, reduce weight and boost your mood at the same time.

 

Apples and pears are high in dietary fiber which aid the weight loss process by speeding up metabolism and keeping you full for longer period thus preventing intake of more calories. They also prevent constipation and regulate blood sugar.

 

Oranges and lemons are perfect addition to your weight loss plan owing to their abundance of vitamin C which acts as a natural fat burning compound. Vitamin C helps the body burn calories, flushes out toxins accumulated in the body which improves liver health necessary for appropriate metabolism of fats.

 

Metabolism is regarded as a complex process in which the body transforms the consumed food into fuel. Flax seeds and ginger are natural metabolism boosters and their regular consumption will not only help you reduce weight but also burn fats especially accumulated in the stomach area.

 

Mix these healthy ingredients to prepare incredibly delicious beverage that will keep you healthy and melt pounds naturally. Prevent weight gain, overweight or obesity, shape your body the way you have always dreamed about and improve your health. Your body will be grateful one day.

 

Always choose fresh organic ingredients in order to avoid pesticides and other harmful and toxic substances.

 

Consume this drink twice, early in the morning and 3 hours later. Then take your lunch and eat something light for dinner ( seasonal salad or vegetable soup). Drink this beverage regularly, boost your metabolism and start losing weight naturally.You will be amazing by the results.

Best Drink to Boost Metabolism and Reduce Weight Naturally

Serving Size-2

Ingredients:

• 2 pears
• 1 apple
• 1 banana
• 1 orange
• 1 lemon
• 1 tbsp. ground flax seeds
• a thumb of ginger

Instructions:

Combine all the listed ingredients in a blender and blend till you get creamy smoothie with incredible taste.

 

 

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