Macromolecular diffusion plays a fundamental role in biological processes. Here, we give an overview of recent methodological advances and some of the challenges for understanding how molecular diffusional properties influence biological function that were highlighted at a recent workshop, BDBDB2, the second Biological Diffusion and Brownian Dynamics Brainstorm. BDBDB2 was held on October 11-13, 2010 at the Heidelberg Institute for Theoretical Studies (HITS), with live evening videoconferencing sessions to the University of California, San Diego. The workshop brought together in Heidelberg about 40 theoreticians and experimentalists from around the world and a further 15 scientists in San Diego.In biological systems, macromolecules are constantly moving around by diffusion. How do the molecules find their binding partners? How do they fold to form a particular shape? How do they diffuse through the crowded environment of the cell interior? How does the presence of many diffusing macromolecules in a cell affect the function of the individual molecules? These are just some of the questions that are being pursued with the experimental and theoretical approaches that were discussed at the second Biological Diffusion and Brownian Dynamics Brainstorm  is a computational technique that allows the diffusive motion of molecules in solvent to be simulated, and a particular focus of the meeting was the discussion of current developments in this simulation methodology. Here we describe the main themes of the meeting, which provide a snapshot of the current state-of-the-art of studies of macromolecular diffusion, with a particular focus on BD simulation methods. We first discuss theories and methods for computer simulations for studying diffusional processes. In the next section, we describe theoretical and experimental studies on diffusion-influenced biochemical reactions. In the final section, experimental approaches for investigating diffusional processes BD and Langevin dynamics (LD) simulation methods may be applied for the purpose of studying the motion and the interactions of biological macromolecules in solvent. The macromolecules are modelled as particles diffusing in a solvent that is modelled as a continuum that exerts frictional and random, stochastic forces on the particles. Common to these methods is the possibility of accessing phenomena whose time-scale is much greater than that usually achievable in atomistic molecular dynamics simulations. A range of methodological developments were presented at the BDBDB2 meeting and these have been implemented in a number of new software packages, as well as in existing packages. The approaches differ in the strategies used to minimize computational cost and, at the same time, retain sufficient detail and accuracy for studying the process of interest. The strategies used to extend the space and time-scale of the simulations can be divided into three categories: physical approximation, smart algorithms and parallelization. The different software packages take advantage of one or more of these strategies.Escherichia coli [Many physical approximations have been described to reduce the complexity of the system simulated. When the internal degrees of freedom are not fundamental for describing the process studied, the macromolecules can be considered as rigid bodies. This approximation, which greatly reduces the complexity, allows the atomic details of the macromolecules to be retained. Atomically detailed rigid-body BD simulations have been implemented, for example, in Macrodox , UHBD 22 and SDAhia coli ,6.One can further reduce the level of detail by keeping the rigid body representation and coarse-graining the atomistic details. For example, the representation of a molecule by a simple sphere with an excluded volume interaction or a sphere with a reactive patch interacting with a Coulomb potential, has been employed for analysing diffusional association processes .In many cases, such as macromolecular folding processes or binding by induced fit or conformational selection, the rigid body approximation breaks down and a strategy that explicitly treats internal flexibility is required. A coarse grained representation is frequently used. Typically, groups of atoms are represented as beads interacting via a set of interactions that have been parameterized using more accurate methods or experimental information. Coarse grained models are implemented in the BD simulation codes: UHBD , BD_BOX Smart algorithms are important for achieving computational efficiency. Gary Huber , for example, described several algorithms implemented in BrownDye , includiParallelization and making use of state-of-the art hardware is equally important. In the BD_BOX software, Maciej Dlugosz  has made extensive use of GPU programming and parallel programming with the Message Passing Interface (MPI) and the shared memory openMP approaches. BD_BOX is intended to be an engine that allows the simulation of very large biomolecular systems treated as coarse grained polymers in implicit solvent.In BD simulations, the solvent is treated implicitly, that is, the solvent granularity is neglected. In some cases, particular attention should be paid to the treatment of solvent-solute interactions. For example, Daria Kokh  showed that, to properly describe the adsorption of proteins to metal surfaces with a continuum model using BD simulations, specific properties of the hydration shell on metal surfaces should be accounted for by including additional, semi-empirically parameterized terms in the protein-surface forces ,12. OfteUnderstanding the effects of HI on the diffusion and association of macromolecules in complex environments (e.g. within the cell) is non-trivial since the importance of HI strongly depends on the properties of the system itself . Adrian Elcock found that HI are fundamental for reproducing experimental diffusional properties  of flexible interconnected polymer chains by performing simulations of folding proteins with and without HI . MoreoveE. coli cytoplasm successfully describe the relative thermodynamic stabilities of proteins measured in E. coli.For dilute solutions (< 0.1 volume fraction) of interacting unbound proteins, the effect of HI on diffusional properties is less crucial. Paolo Mereghetti showed how, in dilute regimes, the concentration dependent diffusion coefficient of lysozyme and BPTI solutions can be reproduced without explicitly including HI. These results agree with those obtained previously in similar simulations by McGuffee and Elcock . Further3) problem (N is the number of particles). Efficient procedures to reduce the computational time were discussed by Jose Garcia de la Torre  [2 and is valid for HI that are not too strong [Implementing HI in simulations is challenging as the canonical approach requires the factorization of a 3N × 3N diffusion tensor, which is an O ; and Thio strong ,16. ThisE. coli cytoplasm [In a dense environment, the correct reproduction of dynamic properties can be expected to depend on accurate modelling of HI. Indeed, beside the far-field part of HI, usually modelled with the Rotne-Prager  Yamakawaytoplasm  and founBD treats the primary solute species explicitly, and the solvent implicitly. That is, BD is based on a Langevin type formulation of time-dependent statistical mechanics . As has The continuum level treatments of diffusion have both advantages and disadvantages relative to BD treatments. Continuum level treatments offer computational efficiencies when very large numbers of simple  solutes are involved. Indeed, such descriptions are often amenable to analytical study. One familiar result is the Smoluchowski second-order rate constant for solute reaction with a perfectly absorbing, spherical target . More coOn the other hand, BD treatments offer advantages when the solute molecules are substantially non-spherical, are flexible, or have anisotropic interactions. The number of coordinates required to describe such systems in continuum terms grows rapidly as these factors are added in. Also, in the case of low concentrations of solute particles in critical regions, the Brownian treatments account for stochastic effects in the most natural way.An appealing prospect for future work is the development of hybrid models, in which continuum type treatments can be used in some parts of space and BD treatments in other parts of space . An early effort in this direction has been described by the Helms group . AnotherThe speed of binding and reaction events is crucial to protein functionality in many biological processes. Bimolecular association kinetics can be represented by a two-step process with an intermediate state (AB)* referred to as a transient (or encounter) complex:5-106 M-1s-1). Whereas slower binding is generally associated with conformational rearrangements of the binding partners during the second reaction step [The first reaction step is driven by the relative diffusion of the molecules, A and B, and long-range electrostatic forces. It enables the binding partners to orient specifically and to go on to form a bound (native) complex, C . When prion step . Among vion step , Huan-Xiion step  and to iion step . Gideon ion step . Barry Gion step .E. coli phosphate binding protein [In an alternative approach, Martin Held  showed how transition path theory  can be u protein . The metAn important problem in the modelling of protein binding kinetics is the influence of protein conformational changes on the rate constant of the binding process. In particular, many enzymatic reactions cannot be understood from the rigid- protein viewpoint since conformational changes provide a mechanism for achieving enzyme specificity. This fact motivates development of kinetic models that take into account dynamic modulation of the active site accessibility. Andy McCammon  demonstrated how the kinetics of binding could be coupled to the kinetics of the conformational transitions of the protein by using a "gating" model ,40. ApplAn analytical approach to the binding rate constant problem for the "induced-fit" and "conformational selection" protein-ligand binding models was presented by Zhou . He propThe widely used standard bimolecular kinetic relations are strictly valid only under dilute conditions and when the concentration of one component is considerably smaller than that of the other . At highA diverse range of methods is being developed for spatiotemporal modelling of multi-step cellular processes. Johannes Seibert  described BD simulations to study the effects of membrane geometry on primary rod vision signal transduction. Protein diffusion and binding/dissociation processes in a disc vesicle of the primary rod for vision were studied by BD simulations of sphere models of rhodopsin and G-protein molecules.Elfriede Friedmann  presented a numerical model with a mixed system of differential equations to investigate the effect of cell shape on the Janus kinase (JAK) signal transduction and activator of transcription (STAT) pathway in different cell types. A new numerical algorithm was introduced to reduce the long computational time caused by the fine mesh and small time step which were necessary due to the combination of fast diffusion with the slow activation and deactivation kinetics of STAT5 .Johan Elf  discussed how different reactions may require different spatial or temporal discretization approaches. Specifically, processes in the reaction network cannot be considered on the same footing when macroscopic average rates in the coarse-grained states include many microscopic rebinding events between molecules. The latter reactions should be resolved in order to correctly calculate the probability of coarse-grained events. Elf described a new algorithm that allowed the transition to be made from the macroscopic to the microscopic limit in diffusion simulations and enabled a molecular level of process description . The metin vivo, so far, two studies have been carried out to simulate intracellular conditions at a molecular level of detail permitting direct comparison to experimental data. McGuffee and Elcock [in vivo translational diffusion coefficient of the green fluorescent protein, using simulations with two different detailed models containing a heterogeneous mixture of macromolecules from the E. coli cytoplasm.Considering macromolecular diffusion d Elcock  and Andod Elcock  reproducE. coli cells and the interaction with DNA transcription sites.Advances in techniques for single-molecule experiments are providing new insights into the diffusional properties of proteins in cells. Single-molecule kinetic experiments that allow the tracking of the free diffusion of a protein molecule in a living bacterial cell with millisecond time resolution and sub-diffraction spatial precision were presented by Johan Elf . It was 15N in-cell NMR in E. coli was reported by Gary Pielak, suggesting that intrinsically disordered proteins in the cell have a different dynamical response to macromolecular crowding compared to structurally ordered proteins [Fluorescence correlation spectroscopy (FCS) was used to study subdiffusive motion of proteins in different parts of the cell by Matthias Weiss  and Joerg Langowski . Anomalous subdiffusion due to crowding was found to be common, and also to affect the rates for protein-protein binding . 15N in-proteins .in vivo conditions. Particular attention is being paid to hydrodynamic interactions and macromolecular flexibility. Experimentally, advances are, in particular, being achieved in single molecule kinetics and in vivo diffusion experiments. This snapshot of research on biological diffusion and BD simulations shows a vibrant field with growing synergy between experimental and computational studies from which many interesting results can be expected in the next few years.In this short overview, it has not been possible to cover all the interesting advances presented at BDBDB2, so we have aimed to highlight some of the main areas where rapid advances are being made, as well as some of the challenges that demand further development of both experimental and computational methods. From the computational and theoretical perspective, notable effort is being devoted to developing models to simulate


Plantar fasciitis is the most commonly reported cause of chronic pain beneath the heel. Management of this condition commonly involves the use of corticosteroid injection in cases where less invasive treatments have failed. However, despite widespread use, only two randomised trials have tested the effect of this treatment in comparison to placebo. These trials currently offer the best available evidence by which to guide clinical practice, though both were limited by methodological issues such as insufficient statistical power. Therefore, the aim of this randomised trial is to compare the effect of ultrasound-guided corticosteroid injection versus placebo for treatment of plantar fasciitis.The trial will be conducted at the La Trobe University Podiatry Clinic and will recruit 80 community-dwelling participants. Diagnostic ultrasound will be used to diagnose plantar fasciitis and participants will be required to meet a range of selection criteria. Participants will be randomly allocated to one of two treatment arms: (i) ultrasound-guided injection of the plantar fascia with 1 mL of 4 mg/mL dexamethasone sodium phosphate , or (ii) ultrasound-guided injection of the plantar fascia with 1 mL normal saline (control group). Blinding will be applied to participants and the investigator performing procedures, measuring outcomes and analysing data. Primary outcomes will be pain measured by the Foot Health Status Questionnaire and plantar fascia thickness measured by ultrasound at 4, 8 and 12 weeks. All data analyses will be conducted on an intention-to-treat basis.Data collection for the trial will be finalised in February 2011. Following this, statistical analyses of the trial data will be performed and the findings prepared for presentation.This will be a randomised trial investigating the effect of dexamethasone injection on pre-specified treatment outcomes in people with plantar fasciitis. Within the parameters of this protocol, the trial findings will be used to make evidence-based recommendations regarding the use of corticosteroid injection for treatment of this condition.


Differentiating hemorrhagic infarct from parenchymal intracerebral hemorrhage can be difficult. The immediate and long-term management of the two conditions are different and hence the importance of accurate diagnosis. Using a series of intracerebral hemorrhage cases presented to our stroke unit, we aim to highlight the clues that may be helpful in distinguishing the two entities. The main clue to the presence of hemorrhagic infarct on computed tomography scan is the topographic distribution of the stroke. Additional imaging modalities such as computed tomography angiogram, perfusion, and magnetic resonance imaging may provide additional information in differentiating hemorrhagic infarct from primary hemorrhages. In acute stroke, the differential diagnosis of hemorrhage detected on computed tomography (CT) scan ranges from hemorrhagic infarct (HI), primary intracerebral hemorrhage (ICH) to hemorrhage from venous infarction. The differentiation between the first two conditions can be difficult, and there are currently no radiological criteria to assist in this regard. It is, therefore, not surprising that previous investigators have found poor agreement in making a diagnosis of HI or ICH .HI, or hemorrhagic transformation of an infarct, occurs in approximately one-third of cases of ischaemic stroke . When anHI occurs more commonly in elderly patients and those with larger infarcts . Among pHI typically happens within 1-2 weeks after stroke onset, less commonly (~9%) in the first 24 hours . The occA classification of HI based on the topography and intensity of hemorrhage on CT has previously been proposed by Moulin et al. in 1993 [ICH involving the caudate nucleus is uncommon , 13 Fig and invoThe hypodense region of oedema surrounding the hematoma in ICH usually radiate centripetally, and it does not follow the topography of an arterial territory. Similar pattern of oedema is seen in hemorrhages resulting from venous infarction Figures  and 8. IThe magnetic resonance (MR) imaging features of HI on diffusion weighted imaging (DWI) sequence have a mixed appearance. Within the hemorrhagic area, the appearance between HI and ICH is indistinguishable. However, the presence of an ischaemic process may be evidenced by discrete regions of restricted diffusion remote from the hemorrhagic area Figures –14. ThesTime-of-flight MR angiography can show the presence of occlusive intracranial disease and hence aids in confirming the diagnosis of HI Figures -14. AlthThe presence of “microbleeds” on gradient-echo (GRE) or susceptibility weighted imaging (SWI) sequence suggests the presence of blood product but does not necessarily indicate that the lesion in question is HI or ICH . In eldeCT angiography (CTA) is often used as a screening tool to exclude the possibility of aneurysmal bleed. It can also be used to concurrently evaluate the possibility of arterial occlusion and potential intra-arterial therapy. Given the additional risk of radiation exposure and iodinated contrast agents, further studies are required to evaluate the usefulness of this modality for determining arterial occlusion in patients with isolated putaminal or thalamic hemorrhage.CT perfusion (CTP) with cerebral blood flow, cerebral blood volume, and mean transit time is usually performed at the same time as CTA in tertiary stroke centres. When this is available, it can help with differentiation between HI and ICH. In contrast to ischaemic stroke, a large perfusion defect around an ICH has not yet been reported. The presence of such a mismatch may point to the possibility of HI.Differentiating HI from ICH can be difficult. Careful examination of the topography of the stroke on the initial CT in different sections may distinguish the two conditions. Signs compatible with an infarct such as dense artery sign and insular ribbon sign should be actively looked for. Advanced imaging technique such as CTA, CTP, and MR imaging may be particularly helpful in difficult cases, looking for perfusion deficit, arterial occlusion, and diffusion restriction remote from the site of hemorrhages. Distinguishing HI from ICH is important given the difference in acute and long term management.


Today, there is increasing awareness of the multiple dynamic roles of lipids in cell life. Knowing how lipid molecular species are organized, interact with proteins, and change with environmental stress and metabolic state is crucial to understanding the membrane structure and the cellular functions.The present view of lipid biology arises from the availability of technologies able to detect even minor lipid components with short lifetimes. In the late 1990s, the technical innovation in mass spectrometry led to the development of “lipidomics” as an evolution of lipid biochemistry. Furthermore, progress in microscopy and the availability of many types of fluorescent probes, together with genetic engineering, presently offer the possibility to move quickly towards lipid systems biology. As a consequence, besides being able to analyze major and minor lipids of all structures and sizes by mass spectrometry and nuclear magnetic resonance, we can study topology, structural organization, and dynamics of lipids by microscopy and fluorescent probes in living cells and microbes. Lipids are among the taxonomic traits that can be used to clearly delineate the archaea from all other organisms. Archaeal phospholipids are built on glycero-1-phosphate and contain ether-linked isoprenoid chains, while bacterial and eukaryal lipids are constituted of fatty acids ester-linked to glycero-3-phosphate. The radical structural differences between lipids of archaea and bacteria or eukaryotes raise many questions about early evolution of cell membranes.This special issue contains selected papers dealing with archaeal phospholipid biosynthetic pathways, physical chemical properties and biotechnological applications of archaeal lipids, mass spectrometry lipid analyses and lipids of the archaeal viruses.E. coli resulting in the production of archaeal-type lipids in the bacterium; in the future such engineered E. coli cells may serve to test the properties of mixed membranes constituted of both archaeal and bacterial phospholipids.An updated phylogenic analysis of enzymes involved in archaeal phospholipid biosynthetic pathways is presented by the group of D. Moreira. The biosynthetic pathways have also been analyzed from the experimental point of view in a study of the group of H. Hemmii, which has expressed 4 genes involved in the biosynthesis of archaeal phospholipids in Pyrococcus furiosus have been analyzed by MALDI/TOF-MS coupled to TLC in a study of S. Lobasso et al.; while lipids of uncultured methanogens present in geological samples have been detailed characterized by ESI-MS by M. Y. Yoshinaga et al.Lipid components of the membranes of The ability of archaeal lipids to act as adjuvant in vaccines designed to give protection against solid tumor has been examined in a study by G. D. Sprott et al.A review article by E. Roine and D. H. Bamford describes the specific lipid components of viral membranes of archaeal viruses, in the frame of their possible functional roles in the mechanism of infection.The chemical physical properties of the lipid core of membranes constituted of archaeal diether lipids have been examined in a study by A. Ota et al. by using fluorescence steady-state anisotropy and electron paramagnetic resonance measurements and in a review article of P. L. Chong et al. where the role of cyclopentane rings in the lipid chains of tetraether lipids on membrane fluidity is examined.Finally an interesting contribution of Y. Koga comparatively examines the different mode of adaptation to high temperature of archaea and bacteria pointing out the role that differences in chemical physical properties of archaeal and bacterial lipids might have played in the differentiation of archaea and bacteria during evolution.We hope that this issue will contribute to the understanding of the multiple roles of lipids in the cell biology of archaea and will stimulate further investigation in this field, opened by the pioneering studies of Morris Kates in the 60s.Angela CorcelliParkson Lee-Gau ChongYosuke Koga


We have made comparisons between hair follicles (HFs) and antler units (AUs)—two seemingly unrelated mammalian organs. HFs are tiny and concealed within skin, whereas AUs are gigantic and grown externally for visual display. However, these two organs share some striking similarities. Both consist of permanent and cyclic/temporary components and undergo stem-cell-based organogenesis and cyclic regeneration. Stem cells of both organs reside in the permanent part and the growth centres are located in the temporary part of each respective organ. Organogenesis and regeneration of both organs depend on epithelial-mesenchymal interactions. Establishment of these interactions requires stem cells and reactive/niche cells  to be juxtaposed, which is achieved through destruction of the cyclic part to bring the reactive cells into close proximity to the respective stem cell niche. Developments of HFs and AUs are regulated by similar endocrine (particularly testosterone) and paracrine (particularly IGF1) factors. Interestingly, these two organs come to interplay during antlerogenesis. In conclusion, we believe that investigators from the fields of both HF and AU biology could greatly benefit from a comprehensive comparison between these two organs. Hair follicles (HFs) and deer antlers are the only two mammalian organs capable of stem-cell-mediated cyclic regeneration in adult life , 2. AfteA typical mature HF  can be dIn this review, we define antler unit (AU) as a term for both antler proper and antler pedicle , whereasIn summary, both HF and AU have permanent and cyclic components. The permanent component of each organ harbours its respective stem cells, and the cyclic component contains the growth centre for the formation/regeneration of each organ. The entire HF organ is ensheathed in a mesoderm-derived connective tissue, whereas the AU is in an epithelium-derived epidermis.The ontogeny of both HF and AU includes organogenesis and cyclic regeneration.Based on morphological features, Paus et al.  classifiRenewal of the follicle and replacement of the pelage occur subsequently, at varying times depending on the follicle type and species, with the cyclic HF components rapidly degenerating via a process involving apoptosis. This stage of the follicle growth cycle is termed catagen. An epithelial strand surrounded by the retracting basement membrane draws the DP upward, where it comes to rest just below the bulge . Upon coPedicles develop from the presumptive region of the frontal bone (behind and above the eye) when male deer approach puberty , 16. IniDuring antler growth, the AU consists of an internal bony component and an external skin component. Formation of the internal component proceeds through four histologically distinguishable stages . The firPedicle skin forms from the skin overlying the frontal crest and proceeds through three distinctive stages: (1) compression of the subcutaneous loose connective tissue at the transitional stage, (2) stretching of the undulated apical epidermis at the early pedicle endochondral ossification stage, and (3) neogenesis of the skin and the associated HFs at the mid pedicle endochondral ossification stage . The traIn summary, both HF and AU undergo organogenesis to generate permanent and cyclic components. The permanent component of each organ is formed first and then the cyclic component is formed. It should be noted that in the HF, the distal part is permanent and the proximal part is cyclic, while the converse is true for the AU because the HF grows inwards and AU grows outwards. Organogenesis of both HF and the AU involves two principle types of cells: epithelium and mesenchyme. However, the HF is essentially an epithelial structure, while the AU is essentially a mesenchymal outgrowth. Each tissue is formed primarily from the cell type that is destined to constitute the final appendage, that is, hair in HF and antler in AU.Each cycle of hair regeneration begins when proliferating hair germ cells emerge from the bulge at the end of telogen to commence the active growth phase (anagen). The shedding of the existing hair fibre (exogen), at or following anagen onset, was initially thought to be due to the outward movement of the nascent hair fibre , but it At anagen, matrix cells, the transient amplifying cells, derived from HF stem cells of the bulge in human HF, proliferate at an astonishing rate  migrate to the proximity of the stem-cell niche (bulge in HF or PP in AU) to form a close association with their respective stem cells. Shedding of hairs or casting of hard antlers requires active proteolysis. Histologically, regeneration of each organ recapitulates the process of its respective organogenesis. Generally, loss of hair or hard antlers coincides with the onset of regeneration of each new organ.The cyclic components of HF and AU periodically regress and regenerate. For this to occur, there must be a population of stem cells residing in the permanent component of each organ. Furthermore, as the HF is principally an epithelial structure and AU is a mesenchymal structure, the tissue-specific stem cells required should be of the same lineage, that is, epithelial and mesenchymal, respectively. Tissue/cell deletion and transplantation experiments have played an important role in discovering and characterising the tissue-specific stem cells for both organs.Studies that have either deleted or transplanted key components have played an important role in elucidating the tissue-specific stem cells for both HF and AU . Oliver μm in thickness, unpublished) transplanted either subcutaneously or intradermally can initiate growth of an ectopic antler [In AU, when the periosteum overlying a frontal crest (AP) or enveloping a pedicle (PP) is removed, no AU is formed and antlers cannot regenerate. When the AP is transplanted elsewhere subcutaneously, an ectopic AU will form and subsequent antler regeneration will ensue –44 Figu. In expec antler , 47. Notc antler , 48. Theβ-galactosidase) have further confirmed that the stem cells required for development of the mouse HF are located in the bulge [β-galactosidase-positive cells gradually migrated down the HF shaft from the bulge and became juxtaposed to the proximal end of the follicle. Six weeks after transplantation, the β-galactosidase-positive cells had migrated to the bulb region  in the subsequent developed AU including reserve mesenchyme  and the overlying skin (particularly the epithelium-derived epidermis) are separated by a wide and loose layer of subcutaneous connective tissue . A pedicE-M interactions are also periodically reactivated throughout adult life as components of the developmental program reoccur during the onset of each cyclic regeneration of HF or antler. In the early phase of anagen in HF, the DP is progressively separated from the bulge due to rapid expansion of the hair germ-derived cell mass, until the establishment of the mature anagen follicle . At the During early antler regeneration, the skin (particularly epidermis) that covers the posterior (the site where the main beam will form) and anterior (brow tine) edges of a pedicle stump is rapidly displaced from the stem-cell niche, the distal region of PP Figures , due to To experimentally confirm that the DP in HF or the epidermis in AU is indispensible to the organogenesis and regeneration of each respective organ, both tissue deletion and transplantation methods have been employed. Unexpectedly, the tissue deletion approach was ineffective in preventing regeneration of both HF (rat whiskers) and AU. This is because the removal of the DP fails to stop HF organogenesis or regeneration, as the cells from the remnant outer root sheath and its adherent mesenchymal layer can compensate for this loss . LikewisIn contrast to the approach of tissue deletion, experiments involving the transplantation of cells or tissue have convincingly demonstrated that the DP is the key tissue component for the initiation of HF. Reynolds and Jahoda  reportedIn the case of AU, the importance of communication between the two tissues was demonstrated by inserting a thin membrane between the skin and periosteum to show the dependency on skin for antler organogenesis  and regeFurther examples illustrating the importance of this E-M interaction are tissue transplants into hairless mice, and the antlers grown by castrated male and female deer. In mutant hairless mice , 58, theIn summary, generation and regeneration of both HF and AU rely on interactions between the stem cells of each organ  and the other key cell types (DP cells in HF and skin cells in AU). To enable each process to occur, the two interactive tissue types must be intimately juxtaposed although the means through which this close association is achieved is different for each tissue: for HF by emerging together in organogenesis and by the destruction (via apoptosis) of the intervening suprabulbar strand during regeneration; and for AU by compression of the loose connective tissue layer during AU generation and by breaking the integrity of skin and PP through velvet shedding in antler regeneration. Induction of the DP in early anagen activates some of bulge stem cells, leading to the proliferation of these cells to form the epithelial-derived cyclic component of the HF. Feedback from the activated and rapidly proliferating stem cells drives the DP to undergo characteristic changes in its volume, histological appearance, and composition of the basement membrane , 63. LikCyclic regeneration of HF has evolved in mammals as a means for replacement (moulting), camouflage, temperature regulation, or social and sexual signalling . LikewisThe rationale for the first hypothesis is that the proliferative capacity of matrix cells is determined at the initiation of a new hair cycle and that new matrix cells are not generated throughout the entire growth phase. Because transient amplifying cells have a limited potential to proliferate and because the majority of matrix cells are actively involved in continuous replication , they evIt is well established that regenerating antlers cease growing due to extensive calcification caused by the sharp increase in concentrations of circulating androgen hormones . TherefoThe following experiments provide evidence that there is a marked difference between stem cells and the transient amplifying cells of HF or AU in their ability to proliferate. Cotsarelis et al.  found thP > 0.05) in antler length was detected between the first and the fifth sets. In contrast, the regenerative potential of the antler remnants was significantly decreased with successive cycles of removal and regeneration, and regrowth was almost totally exhausted after the third cycle when only small antlers were formed . Synchronisation is largely entrained by photoperiod and temperature. By artificially manipulating photoperiod, the frequency and amplitude of the growth cycles of these appendages can be profoundly affected. For example, thick winter or thin summer coats of mink can be readily achieved by artificially altering day length . Up to fOf the endocrine factors, androgen hormones are reported to be the most important for regulating the types of fibre produced by HFs in some species, including humans , 75. ChaThe connection between human hair or deer antlers and the testis was first noticed by Aristotle over 2000 years ago: boys castrated before puberty do not grow sexual hair (cited by ) and preClearly circannual variation in the growth of hair on the scalp, face, and thigh in human has been linked to seasonal changes in concentrations of androgens in blood . For exaThe annual growth of antlers (see the Ontogeny Section in this review) is strictly under the control of circulating androgen hormones, particularly testosterone. Each year, hard antlers are cast from their pedicles when concentrations of testosterone decrease to a certain threshold. Wound healing over the pedicle stump and antler regeneration take place while concentrations of testosterone remain low. Antler growth gradually ceases due to an increase in calcification caused by the rapid increase in concentrations of circulating testosterone and this is accompanied by shedding of the velvet skin , 85.Because androgen receptors are only found in the DP cells in HFs , 87, it The growth of the HF and AU is also regulated by a number of potent growth factors. Among these factors, insulin-like growth factor 1 (IGF-1) has been particularly important for the growth of these organs with dose-dependent mitogenic effects on both HF  and AU sIt is currently understood that androgens act on HFs via androgen receptors within the DP cells and trigger the expression of hormone responsive genes. This then alters the paracrine factors produced by the DP cells which regulate the growth and activity of other cell types in the human HF. These paracrine factors could be soluble mitogenic factors or extracellular matrix components . We postβ/BMP, FGF, and TNF families [β-catenin signalling provides the master switch for determining the fate of HFs because expression of the Wnt inhibitor Dkk1 or lack of epidermal β-catenin resulted in the lack of induction of hair follicle growth [β-catenin causes an enhanced formation of placodes, and epidermal keratinocytes globally adopt an HF fate [β-catenin staining was detected in dividing, undifferentiated mesenchymal cells in the growth centre of early regenerating antler bud. When the canonical Wnt pathway was inhibited at the level of Lef/TCF, the number of antler cells decreased as a result of increased apoptosis. Activation of the Wnt pathway inhibited alkaline phosphate activity (a marker of antler progenitor cell differentiation) of these antler cells. Therefore, β-catenin plays an important role in the regulation of antler progenitor cell survival and cell fate; and hence, Wnt signalling is important for antlerogenesis.The paracrine factors responsible for mediating the communication between the epithelium and mesenchyme tissues during organogenesis of HF and regeneration include members of the Wnt/wingless family and the hedgehog family and of the TGF-families . Canonice growth , 96. Con HF fate , 98. Lik HF fate  that theSonic hedgehog, an epithelial placode cell factor, signals to the underlying mesenchyme during HF organogenesis to form the dermal condensate which subsequently gives rise to the DP . This faMaintenance of stem cells is ensured by slow cycling, which is controlled by low levels of c-myc in mammalian HFs . Antler Both generation and regeneration of the AU have been considered to be unique zoological phenomena , 109, paA histological examination indicates that one of the most obvious effects of AP/PP induction to the overlying skin during AU generation or antler regeneration is the formation of miniaturised HFs (velvet skin HFs produce the thinnest hairs on deer ). This hIf the AP restricts the development of HFs during antlerogenesis, what would happen if the skin associated with the AP is hairless? When transplanted underneath the hairless skin, such as the snout of a deer's nose  or the vDirect confirmation as to whether HFs truly mediate the interactions between the AP and skin during antlerogenesis, such as ablation of the HF to see if the skin still can participate in antlerogenesis, is not always practical. An alternative approach to test this hypothesis was to deliver minced AP tissue directly under the bulbs of HFs  to deterIn summary, antlerogenesis depends on interactions between AP/PP-derived tissue and the overlying skin. The available evidence indicates that these interactions are mediated by the HFs residing in the AP/PP associated skin. On the one hand, antlerogenesis requires the presence of HFs, but on the other hand, antlerogenesis produces skin that adorns with miniaturised HFs.In this review, we have made comparisons between HFs and AUs—two seemingly unrelated mammalian organs. HFs are tiny and are concealed within skin, whereas AUs are gigantic and are grown externally for visual display. However, these two organs share some striking similarities . Both orSince HFs adorn the integument of almost every mammalian species including humans, their organogenesis and cyclic regeneration have been intensively investigated and some of the molecular mechanisms underlying these developmental processes have been elucidated . In contResistance of stem cells in the mouse HF bulge to DNA-damage-induced cell death is a consequence of higher expression of the antiapoptotic protein Bcl-2, enhanced DNA repair activity, and the rapidly attenuated activity of p53 . ExpressIn HFs, telogen can be divided into a phase that is refractory to HF growth stimuli and that is characterized by upregulation and activation of BMP2/4 and a competent phase in which bulge stem cells become highly sensitive to anagen-inducing factors . In the Formation of the pedicle is independent of the E-M interactions and is solely triggered by the increase in concentrations of circulating androgen hormones . When thde novo. It would be interesting to know what chemical factors are involved in this induction. Interestingly, HFs that are formed in the velvet skin have much larger sebaceous glands, but there are no arrector pili muscles, and sweat glands [Nascent velvet skin contains HFs , indicatt glands , 123. Tht glands .Our studies showed that the E-M interactions in organogenesis and regeneration of AUs seem to be transient in nature because once antlers have transformed or regenerated from pedicles, physical separation of the two interactive tissue types does not stop antler generation  or regenThe close association between velvet epidermis and the PP in the AU does not immediately trigger antler regeneration, but rather the cycles enter a quiescent phase. This is because the endocrine factors (predominately androgens) override the outcome of the E-M interactions to suppress regeneration of antlers. Likewise, the close proximity of the DP and the bulge in HFs does not trigger regeneration of the temporal part of an HF, but the cycle enters a quiescent period called telogen, the length of which varies with species and follicle types. What factors suppress the outcome of the E-M interactions and determine the length of telogen for each HF type? If these overriding factors act in an endocrine or paracrine manner, then we must consider that different hair types may contain different receptors because HFs in different regions on a body have differing lengths of the telogen phase although they share the same systemic milieu. Factors involved in the BMP signalling pathway may also be implicated in this because there is increased activity in BMP signalling pathways to maintain HF stem cells in a quiescent state and these signals must be overcome to promote new tissue growth , 119. Fu


Imaging of the cochlea and internal auditory canals are increasingly important nowadays because of the growing number of cochlear implants being performed throughout the world. We report a case of a 4-year-old boy who was born deaf and was being evaluated in our service for possible cochlear implantation. Audiometry disclosed profound bilateral deafness. The magnetic resonance imaging revealed only two nerves in each inner auditory canal: one in the anterior superior quadrant, identified as the facial nerve, and one on the posterior quadrants, representing both the superior and inferior vestibular nerves. The semicircular canals were not seen and the vestibule had dysplastic morphology. The diagnosis was bilateral agenesis of the cochlear nerves and semicircular canals. Congenital sensorineural hearing loss (SNHL) has an incidence of approximately 1–3 cases per 1.000 live births .Imaging of the cochlea and internal auditory canal (IAC) is essential for a cochlear implant candidate in order to establish cochlear duct permeability and the presence of cochlear nerve in the IAC as well as for choosing the side to be implanted . There aUsing a three-dimensional (3D) constructive interference in steady state (CISS) MRI sequences, the vestibulocochlear and facial nerves can be thoroughly evaluated. Both nerves have similar cisternal and canalicular courses. They both emerge from the lateral aspect of the lower border of the pons and traverse the cerebellopontine angle cistern at an oblique angle. Next, the nerves cross the inner auditory canal (IAC) in its entire length . Within The knowledge is limited about etiology of congenital deafness. The radiologic images have become an important diagnostic tool in this regard. The purpose of this paper is to present a case of a 4-year-old boy congenitally deaf who was found to have bilateral agenesis of the cochlea nerve during preoperative workup for cochlear implantation. A 4-year-old boy congenitally deaf came from another state for evaluation at the Division of Cochlear Implantation of Brasilia University Medical School Hospital. He had the diagnosis of profound deafness since he was 5 months old, and he started using an external hearing device since then without any practical benefit.He was born by cesarean section with normal gestation age and normal birth weight. There was no history of familial deafness, and there was no consanguinity between the parents. He had no additional congenital malformations, and he had normal neuropsychomotor development except for the profound bilateral deafness. Adenotonsillectomy and bilateral ventilation tubes placement had been performed in the past.Audiometry disclosed profound bilateral deafness. The use of the external device improved performance in 500 Hz (15 db in the left ear and 20 db in the right) only. Transient and distortion product evoked otoacoustic emissions were absent on both ears. Brainstem-evoked auditory potentials were also absent on both sides.The MRI revealed only two nerves in each IAC, one in the anterior superior quadrant, identified as the facial nerve, and one on the posterior quadrants, representing both the superior and inferior vestibular nerves Figures . The sem Agenesis of the auditory nerve is estimated to be responsible for 1% of congenital deafness cases . It is cShelton et al.  report aA radiological classification of the hypoplasia and aplasia of the vestibulocochlear nerve (VCN) is suggested. It is based on the affected branch and presence of labyrinth dysplasia. Type I: the VCN is affected with presence of IAC stenosis and a dysplastic labyrinth may be present. Type IIa: alteration of the cochlear branch with labyrinth dysplasia (from minor dysplasia to common cavity). Type IIb: normal labyrinth associated to damage of the cochlear branch. Type III: illness of the initial branch of vestibular nerve . The hypThere are reports of cochlear implantation (CI) in patients with cochlear malformations and hypoplasia of CN. The performances of these patients are not as good compared to patients treated with a normal anatomy of the cochlea , 9. An aCongenital bilateral cochlear nerve aplasia is a rare entity. The image studies became important tools in the diagnosis of these lesions. CT and MRI can evaluate possible malformations of the labyrinth and vestibulocochlear nerve. In the presence of aplasia of the CN, the cochlear implant is formally contraindicated. Studies with brainstern implantation have showed promising results.


One of the major limitations of engineering large tissue structures is cell death resulting from the inability of nutrients to diffuse across large distances inside a scaffold. This task, carried out by the vasculature inside the body, has largely been described as one of the foremost important challenges in engineering 3D tissues since it remains one of the key steps for both in vitro production of tissue engineered construct and the in vivo integration of a transplanted tissue. This short review highlights the important challenges for vascularization and control of the microcirculatory system within engineered tissues, with particular emphasis on the use of microfabrication approaches.Recent advances in medicine and healthcare allow people to live longer, increasing the need for the number of organ transplants. However, the number of organ donors has not been able to meet the demand, resulting in an organ shortage. The field of tissue engineering has emerged to produce organs to overcome this limitation. While tissue engineering of connective tissues such as skin and blood vessels have currently reached clinical studies, more complex organs are still far away from commercial availability due to pending challenges with  Beyond this distance, diffusion is not effective and tends to reduce cell survival and function. Therefore, the inability to adequately vascularize engineered tissues results in inefficient transport of nutrients and metabolites and often leads to cell death and tissue necrosis. Moreover, vascularization of engineered tissues plays an important role in graft perfusion and is also crucial in facilitating the integration of the implanted material with the host vasculature [Progress in the development of large tissue-engineered organs has so far been limited by the inability to generate sophisticated three dimensional 3D) structures comprised of a functional vasculature. The vascular system is a dynamic environment comprised of a variety of cell types that constantly remodels itself under the influence of endothelial, immune, nervous and endocrine cells . Vasculaculature ,3.D structuin vivo. However, the fabrication of complex organs such as the heart or liver requires adequate vascular supply to ensure the survival of specialized cells within their 3D structure. To achieve this level of functionality, it is necessary to integrate a network of vessels ranging from a few microns to several millimeters within engineered tissues. The incorporation of this microcirculation into tissues and organs represents a considerable challenge which includes the engineering of vascular conduits having micron scale dimensions. It also requires a functional endothelium to improve vascular activity and avoid thrombosis, as well as other specialized cell types performing the physiological tissue function of interest. Various approaches have been proposed to design scaffolds comprised of a vascular network analogous to capillaries. These approaches rely on the release of growth factors [in vitro.The fabrication of tissue constructs often requires cell seeding of 3D scaffolds. These scaffolds are generally made of gels, foams or fibrous meshes and usually have basic macroscale properties that enable cell adhesion, migration and proliferation . Althoug factors ,15 from  factors  , basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), can trigger the formation of vascular structures following antation -22.Both natural and synthetic scaffolding materials have been loaded with these pro-angiogenic molecules, leading to the sprouting of capillary beds within the constructs. However, the lack of directed growth of blood vessels to enable interconnectivity between the capillary networks still remains the biggest challenge of this technology.in vitro. The resulting angiogenesis phenomenon can be explained by the remodeling that occurs within the construct, which is driven by the endothelial network that activates the release of pro-angiogenic factors in the construct. The remodeling of the extracellular matrix (ECM) allowing the formation of vasculature is driven by matrix-metalloproteases (MMPs), regulating cell proliferation and migration within the tissue [in vivo transplantation [in vivo, where a stable tree-like structure of a branched network was observed for an extended period of time [in vitro and in vivo conditions [Microvascular structures incorporated in tissue engineering scaffolds prior to their implantation can also be obtained by the co-culture of endothelial cells with the cell types of interest regarding tissue function. This cell-based approach uses the ability of endothelial cells to release growth factors and promotes the formation of capillaries e tissue . Using aantation ,16,23-25antation , cardiacantation , bone [2antation  and blooantation , all of  of time . Severalnditions ,32.In vivo studies also demonstrated that the presence of vascular structures formed in vitro greatly accelerated inosculation of the implanted tissue with the host vasculature. Results from L'Heureux et al. have shown that the formation of a vasa vasorum in the wall of a tissue engineered blood vessel (TEBV) occurred 3 months following implantation in vivo [ in vivo .et al. showed that a TEBV comprised of a vasa vasorum engineered in vitro (TEBVwVV) allowed for complete tissue integration and functional vasa vasorum activity after only 2 weeks in vivo [in vivo. Therefore, rapid formation of a vascular network between the engineered tissue and the host at the transplanted site, similar to the process observed during wound healing, represents a promising solution to provide implanted cells with adequate supply of oxygen and nutrients. However, this strategy does not provide a sustainable solution that can enable both perfusion in vitro and inosculation in vivo. In addition, these approaches did not result in the creation of organ scale constructs in vitro. In an attempt to engineer vasculature into engineered tissues and organs, microscale technologies and microfluidics systems have emerged as efficient tools to create easily perfusable channels in biocompatible and biodegradable 3D scaffolding materials.Similarly, Guillemette  in vivo . These sin vitro studies. Unlike angiogenesis and inosculation approaches, microfabricated devices with integrated microvasculature can be optimized to provide a uniform distribution of flow and mass transfer across the scaffolding material and thus provide the cells with an adequate supply of nutrients.The application of microtechnologies to biomaterials can be used to reproduce the capillary network and allow the flow of culture medium through a construct during in vitro [Soft lithographic and micromolding processes have been used to create microfluidic devices consisting of branched networks that can be connected to perfusion systems in vitro . These tin vitro -37. Thesin vivo [These properties are of utmost importance since they greatly influence the stability, oxygen and nutrient distribution and therefore the functionality of engineered tissues. Microfluidic systems can effectively transport solutes in capillary channels ranging from a few millimeters down to micrometers . The conin vivo .in vitro, there is actually no available method to consecutively branch multi-dimensional channels inside a scaffold [Even though previous work has shown that microscale channels can be engineered scaffold . Top-dowscaffold . Moreovescaffold ,44,45. Fscaffold ,46, but scaffold -50. HoweCells in the body are in contact with a complex 3D environment comprised of a combination of soluble factors as well as the ECM and basement membrane proteins found in the tissue in which they reside. Most tissues consist of multiple cell types organized into hierarchical structures that allow them to regulate their function. Modular tissue engineering, or bottom-up approaches, have recently emerged as powerful fabrication methods to generate 3D structures that mimic this organization and that recapitulate tissue structure and spatial resolution -54. ThesMicrogels are microscale hydrogels fabricated by merging microscale fabrication and hydrogel chemistry. They exhibit properties similar to native ECM, can sustain cell encapsulation and have tunable geometrical, mechanical and biological properties which make them excellent candidates for tissue engineering applications. Based on these characteristics, cell-laden microgels were fabricated and then assembled into 3D tissue constructs to create precisein vivo structure of tissues and organs [in vitro [microarchitecture containing repeated functional units that mimic the d organs ,54,58,59d organs . Recent in vitro ,61. Arrain vitro. Maintaining physiological functionality and blood flow in a high-density vascular network with optimized oxygen transfer conditions is critical to keep the tissue in an appropriate state of homeostasis. This biomimetic approach can also be utilized in the design of organ-on-a-chip technologies aiming at the fabrication of precise and reliable small scale models that can later be used for drug screening and physiological in vitro studies [One of the challenges of directed self-assembly technology resides in the difficulty to scale-up the tissue produced. Larger structures have recently been built using lock-and-key principles and modular assembly . Ongoing studies .in vivo, but still cannot provide inlet and outlet vessels for in vitro perfusion. Despite all the advances in microfluidics, the use of microengineered 3D structures comprised of rationally designed and microfabricated channels offer limited functionality. These platforms do not provide a parenchymal space for cell types other than endothelial cells to grow within the constructs and present an integration problem with the host tissue. Modular and bottom-up approaches have recently emerged as promising biofabrication approaches in which functional microscale tissue building blocks can be assembled into 3D macroscale tissue constructs. These are relatively simple methods that allow the production of perfusable tissue, with precise control over the microscale features in a 3D construct. They are particularly promising in the case of organ engineering, where tissue requires perfusion and needs to perform a specialized physiological function. The precise design of microscale components in a high-throughput fashion combined with the capability to link these components together to generate larger structures represents a promising way to build vascularized 3D structures. Therefore, combining modular assembly methods with microfabrication technologies to engineer tissues and organs represent an effective method to control tissue architecture both at the micro and macroscale. This will be a major step forward in the field of tissue engineering that will not only result in the production of functional engineered tissues, but will also greatly help the translation of the technology from in vitro studies to in vivo applications.One of the major limiting factors in the field of tissue engineering is the difficulty to generate functional 3D tissues due to the inability to integrate vascular structures into scaffolds. Building networks of vessels branched together into a complex interconnected structure connecting across multiple length scales remain one of the greatest challenges in tissue engineering. Most cells in the human body are within a few hundred microns from a capillary, allowing the delivery of adequate nutrients and supplies to the tissues and organs. Since most tissue engineering scaffolds are unable to provide such proximity for continuous solutes and oxygen flow, the engineering of large tissues severely lacks from diffusion and transport properties. The methods currently investigated to generate vasculature in scaffolds mainly involve the use of proangiogenic growth factors and cell-based approaches, which have shown promising results The authors declare that they have no competing interests.All the authors have been involved in the writing process and have read and approved the final version of the manuscript.


Increasing incidence of diabetes has been reported in many countries and the disease burden related to diabetes to be distributed unevenly across the population. Patients with lower socioeconomic position have been reported to have higher diabetes prevalence, higher rates of diabetes related complications and excess mortality. This study examined trends in gender, age and socioeconomic differences in the burden of diabetes mortality in the Finnish population aged 35-80 and potential years of life lost (PYLL) due to diabetes.The data consist of an 11% random sample of Finnish residents in 1987-2007 and an 80% oversample of persons who died during those years. We examined diabetes both as underlying and contributory cause. We calculated age-specific and age-standardized diabetes death rates by gender and socioeconomic position using the direct method and PYLL due to diabetes related deaths for 2004-2007.Diabetes related mortality was higher among older Finns. A clear and systematic socioeconomic pattern was detected among both men and women: the higher the socioeconomic position the lower the mortality. The contribution of diabetes to PYLL was 8% among men and 6% among women. Among women, the contribution of diabetes to PYLL was lower in higher socioeconomic groups, whereas among men, the contribution was similar in all socioeconomic groups.In order to further reduce the burden of diabetes a better treatment balance to prevent diabetes complications would significantly decrease the burden of diabetes mortality. Use of underlying and contributory causes of death is useful in monitoring trends and sub-group differences in the burden of diabetes. Increasing incidence of especially type 2 diabetes has been reported in many countries worldwide . At the Earlier research on the burden of diabetes mortality has mainly examined mortality among patients with a diagnosis of diabetes and examined diabetes as the underlying cause of death along with other main causes. Whereas different strategies have been used to identify the total diabetes population , part of the diabetes population is likely to be always missed . Another method used for analyzing the burden of diabetes mortality has been to estimate the number of deaths attributable to diabetes in the general population. Traditionally only the underlying cause of death has been taken into account, but there are some relatively recent studies that also take into account contributory causes of death -15. Few The aim of this study is to examine the total burden of diabetes mortality in the general Finnish population by age, gender and socioeconomic position and trends in it from 1988 to 2007. We further aim to analyze socioeconomic differences in potential years of life lost due to diabetes. We assess diabetes both as the underlying and contributory cause of death for the total population aged 35-80 years. In order to evaluate the total burden of diabetes, we analyzed both underlying and contributory causes of death since diabetes associates with several potentially fatal chronic complications, including cardiovascular and renal disease.The data used in the study are based on information from different administrative registers linked with death records by Statistics Finland. These individual level data consist of an 11 percent random sample of the population residing in Finland in 1987-2007 and an 80 percent oversample of persons who died during those years. We used weights in the analyses to take account of this sampling design. Immigrants were dropped out and emigrants censored (1.2% of the sample). In all our analyses diabetes related deaths refer to diabetes as an underlying or a contributory cause, classified by Statistics Finland according to the International Classification of Diseases (ICD). The 9th revision of the classification was used for years 1988-1995 (code 250) and the 10th revision for years 1996-2007 (codes E10-E14).th year: 1985, 1990, ..., 2005. These were used for the mortality analyses in the years 1988-1990, 1991-1995, ..., 2006-2007 respectively. Four groups were used: (1) upper non-manual, (2) lower non-manual and (3) manual workers and (4) farmers or other entrepreneurs. Those working at home were classified according to the occupation of the head of the household [Information on occupation-based socioeconomic position was available for every 5ousehold ; in the ousehold . A smallFor the trend analysis between years 1988-2007 we calculated age-specific diabetes death rates by gender and age-standardized diabetes death rates by gender and socioeconomic position. Standardization was done by using the direct method and using the sum of all person-years in the total population in 5-year age groups as the standard.The analysis of potential years of life lost (PYLL) between the ages 35 and 80 due to diabetes related deaths by socioeconomic position was performed for the period 2004-2007. PYLL was calculated as the sum of deaths weighted by the difference between age at death and age 80. We calcAltogether 346 878 deaths occurred among men and 235 466 deaths among women during the follow-up period 1988-2007. Numbers for diabetes related deaths were 27 224 and 26 113, corresponding to eight percent of deaths among men and 11 percent among women. Figure At the beginning of the study period in 1988-1989 those in lower socioeconomic position had higher diabetes mortality than those in higher positions and the differences remained throughout the study period  among men and six percent among women (Table This study examined the total burden of diabetes mortality in the Finnish population in 1988-2007. Both underlying and contributory causes were included. A systematic age pattern was detected in diabetes related deaths for both genders: the older the age-group the higher the mortality. Diabetes related mortality declined during the follow-up period especially among women, and the decline was especially large in the oldest age-groups. More research is needed to evaluate the causes of these trends. In line with earlier studies of total mortality among persons with diabetes and studies of diabetes related mortality ,13,20 a The longitudinal nature of our individual level data enabled us to analyze trends in diabetes mortality. The Finnish Causes of death register is considered valid and reliable by international standards . FurtherIt is possible however that our data may still underestimate the number of diabetes deaths. We thus compared the diabetes deaths in our data to those in earlier studies examining mortality among diabetes patients ,23. We oAccording to our results diabetes related mortality declined especially among older age groups in Finland suggesting improvements in diabetes care in the 1990s and 2000s particularly among women. Since diabetes incidence has increased in Finland and elsewhere [The authors declare that they have no competing interests.KM contributed to the conception and design of the study, planning of analyses, and drafted the manuscript. RP contributed to the conception and design of the study, performed the statistical analyses and took part of the revision of the manuscript for important intellectual content. SK contributed to the conception and design of the study, planning of analyses and took part of the revision of the manuscript for important intellectual content. PM contributed to the conception and design of the study, planning of analyses, the collection of the data and took part of the revision of the manuscript for important intellectual content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/747/prepub


Dissolved Fe (dFe) concentrations in the ocean are of the order of a few nM, and Fe is often a factor limiting primary production. Bioavailability of Fe in aquatic environments is believed to be primarily controlled through chelation by Fe-binding ligands. Marine microbes have evolved different mechanisms to cope with the scarcity of bioavailable dFe. Gradients in dFe concentrations and diversity of the Fe-ligand pool from coastal to open ocean waters have presumably imposed selection pressures that should be reflected in the genomes of microbial communities inhabiting the pelagic realm. We applied a hidden Markov model (HMM)-based search for proteins related to cellular iron metabolism, and in particular those involved in Fe uptake mechanisms in 164 microbial genomes belonging to diverse taxa and occupying different aquatic niches. A multivariate statistical approach demonstrated that in phototrophic organisms, there is a clear influence of the ecological niche on the diversity of Fe uptake systems. Extending the analyses to the metagenome database from the Global Ocean Sampling expedition, we demonstrated that the Fe uptake and homeostasis mechanisms differed significantly across marine niches defined by temperatures and dFe concentrations, and that this difference was linked to the distribution of microbial taxa in these niches. Using the dN/dS ratios (which signify the rate of non-synonymous mutations) of the nucleotide sequences, we identified that genes encoding for TonB, Ferritin, Ferric reductase, IdiA, ZupT, and Fe They identified that the TBDTs were less common in the metagenomes than in the genomes, an observation that reflects the numerical dominance of Pelagibacter and Prochlorococcus . However the GOS samples are distributed along a wide range of latitudes possibly confounding the effect of temperature, which varies widely within both Open Ocean and Coastal niches. In addition, prior studies did not directly investigate the exact nature of the link between taxonomic diversity and Fe-pathway prevalence within the metagenomes.The availability of a large number of marine prokaryotic and eukaryotic microbial genomes and the Global Ocean Sampling (GOS) metagenomes, have greatly enhanced our understanding of the Fe-acquisition strategies used by various groups of microorganisms. Recent studies by Hopkinson and Barbeau  and ToulHere, we build on the results presented in the studies of Hopkinson and Barbeau  and ToulThe set of non-redundant Uniref 100) protein sequences for the genes listed in Table 00 proteihmmbuild from the HMMER2 package  sets were combined to form the train set and used to build an HMM. The sequences in t were scored using this HMM by hmmsearch program to get a True Positive (TP) score distribution. False positives (FP) were identified from the negative training set (in this case the entire UniProt database excluding the training sequences for the gene in question). The sensitivity, specificity, and Matthews Correlation Coefficient (MCC) distribution for each of n sets was calculated  , a pelagophyte (Aureococcus anophagefferens), green algae (Ostreococcus lucimarinus and Ostreococcus tauri), and a prymnesiophyte (Emiliania huxleyii) were also downloaded from NCBI as FASTA files of protein sequences. We also analyzed 12 Freshwater microbial genomes mentioned in . This matrix was used for making the non-parametric multidimensional scaling (NMDS) plots using the Primer-E v6 software  test for statistically significant differences between prior groupings of the samples made according to taxonomy or location and the Similarity Percentages (SIMPER) analysis comparing relative abundances of genes in the said prior groupings to identify discriminating genes were carried out using Primer-E. Principal Components Analysis (PCA) of the GOS samples using the environmental matrix  was also performed using Primer-E. Following is a brief description of the non-parametric statistical methods implemented in Primer-E Clarke,  that we Whereas the abundance matrices were log-transformed, for the environmental matrix, the variables were individually transformed to reduce the collinearity as much as possible. So, Latitude and Longitude were square-root transformed, “Water column depth” was log-transformed and “dFe deposition” was exponential-transformed.j and k the Bray–Curtis similarity is described byThe first step in the analysis of multivariate data was the calculation of a similarity measure between the samples. The similarities between all pairs of samples (the similarity matrix) were then used for a number of analyses. The Bray–Curtis similarity coefficient is the most common measure for comparing ecological samples with species abundance data. The Bray–Curtis measure is independent of scale of measurements  and joint absences of variables in a pair of samples have no effect on the similarity between them. For two samples ijy and iky are the abundance of the ith variable in the jth sample and kth sample respectively and p is the total number of variables. The Bray–Curtis dissimilarity is then simply represented as 100 − jkS.where Ordination plots visually display the similarity between ecological samples by mapping the high-dimensional community structure to two or three dimensions such that the physical distance between samples on the plot reflects the similarity between their communities.In an NMDS ordination plot, the distances between the samples (in this case genomes or metagenomes) are first calculated using complete profiles of occurrence of the variables (in our case the iron metabolism genes). The sample objects are then placed randomly in a 2-d space and the Euclidean or physical distance between the objects in 2-d is calculated. This distance matrix is then non-parametrically regressed on to the original distance matrix to calculate a stress value (goodness-of-fit of the regression) that gives an indication of the best fit between the two matrices. The samples are then iteratively rearranged such that the stress value is minimized. The NMDS plot thus is a 2-d representation of the distances between the samples in a high-dimensional space. The distance between two genomes in such an ordination diagram gives an indication of the similarity of their gene profiles. A stress value less than 0.2 combined with an overlay of pre-defined group names provides reliable inferences about the clustering of the samples.n samples having replicates for two or more categories  a test statistic R is calculated as followsThe ANOSIM test is the non-parametric multivariate analog of the Analysis of Variance tests for univariate, normally distributed data. Instead of the group means as in the univariate case, here only the rank similarities between the samples in the underlying similarity matrix are considered. For M = n(n − 1)/2 and n is the total number of samples. The statistical significance of the observed R value is evaluated using the null hypothesis H0 that there are no differences between the groups of samples. This is accomplished by a permutation test where all the group labels are sequentially applied to all the samples and the R statistic recalculated for each permutation. The null hypothesis H0 is rejected if the observed R value lies outside of the distribution of R values from the permutation test. For instance if tR values of the T total permutations are greater than or equal to the observed R value then we can reject H0 at a significance level of (t + 1)/(T + 1). This is what is referred to as the Global R test, i.e., between all the groupings of the samples. Pairs of the groups were also similarly compared to each other in terms of the R statistic and its significance value. Following convention, we tolerated a significance value of up to 5%  as being small enough to rule out the possibility of H0 being true.where j and k SIMPER calculates the contribution for the ith species as follows:This method disaggregates the Bray–Curtis similarity matrix in order to identify the species that contribute most to the differences (average dissimilarities) between the prior groupings of the samples. For two samples ijy and iky are defined as before for Eq.  for all pairs  such that j is from the first group and k is from the second group. If i) of the δjk (i) values is low, it implies that this species i has a significant contribution to the overall dissimilarity in a majority of pairwise comparisons between the two groups. A high i is a good discriminator.The terms  for Eq. . The avePrincipal components analysis is an ordination where the high-dimensional data is represented in terms of two or three orthogonal axes . The procedure involves finding a linear combination of the original variables (first PC) such that the variance of the sample points projected perpendicularly on this new axis is maximized. The second PC is restricted to be perpendicular to the first PC and again chosen in the direction that maximizes the variance of the sample points and so on. The percentage of the variance explained by the first three PCs gives an idea about the loss of information resulting from reducing the dimensions. The variable vectors can be plotted on top of the PCA ordination to visualize the directions of the variable gradients.E. coli 16S rRNA sequence. These were aligned using MUSCLE and imported into the ARB software  is the set of pairwise phylogenetic distances between all pairs of genomes where gene g occurs.where For the genes discriminating between taxa or locations, the nucleotide sequences were retrieved from GenBank and the Maximum Likelihood estimations of average pairwise non-synonymous by synonymous mutation (dN/dS) ratios were calculated using CodeML (runmode = −2) from the PAML package Yang, .Cyanobacteria, eukaryotic phytoplankton, Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria  as compared to Alphaproteobacteria , Flavobacteria , and Cyanobacteria . Fe2+ or divalent cation transporters were abundant in all the taxa but were most abundant in the eukaryotic phytoplankton genomes (39%). Ferric reductase was characteristic of the eukaryotic phytoplankton group (71%), but was also present in Cyanobacteria (2%), Alphaproteobacteria (16%), and Gammaproteobacteria (10%). Fe3+ transporters occurred in Cyanobacteria (37%), Alphaproteobacteria (33%), and Gammaproteobacteria (27%), but were uncommon in Flavobacteria (2%) and absent from eukaryotic phytoplankton. NRPS and NIS components involved in siderophore biosynthesis were present in Alphaproteobacteria (14 and 16%), Gammaproteobacteria (14 and 39%), Flavobacteria (14 and 22%), Cyanobacteria (32 and 11%), and also in eukaryotic phytoplankton (24 and 11% respectively).A set of proteins involved in iron metabolism Table  was recoa Figure A using Ha Figure . The hema Figure  were preP. marinus MIT9303 and NRPS along with the NIS component RhbB (a PLP dependent decarboxylase) were detected in P. marinus MIT 9303 and MIT 9313. It is possible that the high specificity of our HMM-ModE models led to a slight drop in sensitivity. To confirm whether the other components of this pathway were indeed present in the phototrophic genomes and were being missed due to this lowered sensitivity of HMM-ModE, we used the Search Tool for Interacting Genes/Proteins (STRING) database  in P. marinus CCMP1375, NATL1A, CCMP1986, MIT9211, MIT9515, MIT9215, MIT9312, NATL2A, AS9601, and MIT9301. A corresponding siderophore uptake gene was not detected in the Prochlorococcus genomes. Our profiles detected a putative gene for NRPS in eukaryotic phytoplankton A. anophagefferens, E. huxleyi, O. tauri, P. tricornutum, and T. pseudonana, and the NIS component RhbB in E. huxleyi, F. cylindrus, and O. lucimarinus. Using the STRING database we detected genes similar to rhizobactin biosynthesis components RhbA and RhbB in O. tauri, O. lucimarinus, T. pseudonana, and P. tricornutum along with RhtX, a special permease involved in the uptake of rhizobactin 1021. The sequences identified as RhbA, RhbB, and RhtX from these genomes shared 48.18, 35.48, and 30% identity at the protein level within each group, respectively. A neighbor-joining tree calculated from the multiple sequence alignments of these sequences showed a higher similarity of the Freshwater organisms with the Sinorhizobium genes while the eukaryotic sequences clustered with the Prochlorococcus sequences  and IucA_IucC (siderophore synthetase for Aerobactin) suggest that none of the eukaryotic phytoplankton and only around 4% of marine picocyanobacteria possess this system  in the type and frequencies of occurrence of Fe uptake systems, the SIMPER method was used to identify the proteins that contributed the most to this difference , other-Cyanobacteria (Cyanobacteria excluding picocyanobacteria), and the eukaryotic phytoplankton. The genomes were grouped into niches based on only the isolation location of the source organism, e.g., Open Ocean, Coastal, or Freshwater .We generated occurrence matrices showing the abundance of Fe-metabolism proteins (variables) in each genome or metagenome (samples) and used NMDS plots to visualize the clustering of the samples based on similarities of their gene occurrence profiles. The distance between the sample points on a NMDS plot is indicative of the extent to which samples share species (or proteins in this case). We grouped the individual genomes according to either their taxonomic class or to their ecological niches and applied a multivariate non-parametric test (ANOSIM) to check for differences in distribution of the Fe uptake systems across these groupings. For groups that showed a significant difference , and FbpA (Fe3+ transporter component) as well as regulatory elements Fur and RirA. The ferric citrate uptake protein FecA, the FeoAB proteins (Fe2+ uptake), and Ferritin were amongst the most abundant in Flavobacteria and infrequent in the other two groups of heterotrophic bacteria. The regulatory element DtxR was only present in Flavobacteria and absent in Alphaproteobacteria and Gammaproteobacteria.The NMDS plot of 110 heterotrophic genomes showed three distinct clusters corresponding to the three taxa Figure A. The diia Table . The AlpCyanobacteria and eukaryotic genomes formed separate clusters. Within the cyanobacterial cluster the picocyanobacteria, which had the highest number of representative genomes, and other-Cyanobacteria formed sub-clusters. The phototrophic genomes showed a statistically significant difference between the eukaryotic phytoplankton, picocyanobacteria, and other-Cyanobacteria groups , ferritin, HO1 (heme oxygenase), and NRPS were abundant in Freshwater niche. The Fe3+ transporter components (IdiA and HitB) were evenly present in all the three niches. The NIS biosynthesis protein RhbB was most abundant in the Coastal niche. The Open Ocean niche had a greater abundance of IsiA and IsiB than both other niches. The niche groups in the heterotrophic genomes were not distinguishable from each other in terms of the frequencies of iron uptake genes . There was a marked difference in the surface dFe concentration in samples collected from the Atlantic and the Pacific basins. However, apart from the dFe differences in the niches  defined in Toulza et al. , and their median temperature difference was also significantly different . The most noticeable difference of Fe concentrations was seen in the Atlantic Open Ocean samples and Pacific  samples  whereas the corresponding temperature difference between these groups was not significant . Conversely, the Temperate, Coastal samples were primarily separated from the Tropical  samples by a temperature difference  with no difference in the corresponding average Fe concentrations . Consequently, only these three pairs of groups  were further analyzed for the differences in their taxonomic profiles and their associated Fe-metabolism systems.To further understand the interaction between dFe and temperature and to define meaningful niche groups with contrasting parameters, we compared the median and range of the dFe concentrations as well as the temperatures (suitably transformed as described in the methods) for different group of GOS samples  and IsiA had a higher representation in the Atlantic Open Ocean (compared to the Pacific), Tropical (compared to Temperate), and Open Ocean  groups. The Fe2+ uptake components (FeoAB) and Ferric reductase were more abundant in Atlantic Open Ocean, Temperate, and Coastal groups. The Flavodoxin protein IsiB and heme oxygenase HO1 had a higher average abundance in the Pacific, Tropical, and Open Ocean groups. While ZupT (zinc uptake protein) was more abundant in Pacific, Temperate, and Coastal groups, the storage protein ferritin had a higher abundance in Atlantic Open Ocean, Temperate, and Open Ocean groups.The taxonomic profiles (the occurrence of sequences from various taxonomic groups) of the aforementioned GOS metagenome groups were obtained from MG-RAST . The dN/dS ratio therefore provides a measure of the selection pressure operating on a gene. The dN/dS ratio for some of the genes was plotted Figure  along wis Figure . The remHidden Markov models of protein families or folds have been routinely used in genome wide studies of protein functions such as metal binding capabilities  that suggest the presence of some of the components for rhizobactin siderophore biosynthesis in Cyanobacteria and eukaryotic phytoplankton genomes . However, the confirmation of siderophore biosynthesis pathways in these organisms will depend in part on the positive identification of the remaining genes for the many necessary components that could not be detected in these genomes using the HMMs and the STRING database as input for the search. Also, the homologs of RhtX detected in the eukaryotic genomes from the STRING database, were mostly annotated as Acetyl CoA transporters and had only a weak similarity with the S. meliloti RhtX protein , making their identification tenuous without supportive evidence. Siderophore biosynthesis has been reported in some Synechococcus species  and RhbB (The increased specificity (and the related sensitivity drop) of the HMM search implies that we might miss identifying Fe-metabolism components in the genomes and metagenomes and our bottom-up approach (starting from known sequences) would prevent us from discovering novel or highly diverged forms of these Fe uptake systems. Also, for the metagenomes, since we sampled equal numbers of sequences, the reduced search space led to the under-representation of some Fe-metabolism systems. For example, even though the hydroxamate TBD uptake systems turned up as discriminating between the genome groups, they were not detected in sufficient numbers or discriminating between metagenome groupings Table . To see a priori groupings of the samples. Our multivariate analysis detected distinct patterns of co-occurrence in the groups (both genomes and metagenomes) including the co-occurrence of multiple components of the same system. For example, in most of the comparisons of the groups that we performed, all components of the heme uptake machinery HmuTUV, Fe3+ transporters, or the FeoAB system were reported as significant. Concomitantly, the characteristic occurrence patterns for each genome group  and picocyanobacteria (11.55%). These taxa were notably under-represented in the Temperate metagenomes (16.6 and 0.28% respectively). On the other hand the Temperate metagenomes were mainly composed of Flavobacteria  and Gammaproteobacteria  samples whereas ps Table , i.e., mps Table .Cyanobacteria are well documented. For example, siderophore production and its associated receptor mediated uptake is more common in freshwater and coastal cyanobacteria but is not the preferred iron acquisition strategy in open ocean Cyanobacteria . The Flavobacteria and picocyanobacteria were both over-represented in the Coastal group  were almost six times more abundant in the Coastal group than in Open Ocean (data not shown). The over-representation of Fe3+transporters, IsiA, and IsiB in the Open Ocean could be explained by the abundance of Prochlorococcus while the FeoAB and Ferric reductase in the Coastal group could be due to the abundant Flavobacteria. In conclusion for each metagenomic group the Fe-metabolism component was representative of the taxonomic groups dominant in those metagenomic groups. The environmental niche defined the taxonomic dominance which in turn led to the enrichment of particular Fe uptake components in that particular niche.Niche specific adaptations and diversity in Fe uptake mechanisms among idiA and fbpA bind Fe3+, but only idiA was under positive selection pressure while fbpA was under purifying selection pressure. However the phylogenetic spread of FbpA was high and occurrence of idiA was more or less confined to the Cyanobacteria. It has been postulated that Fe3+ transporters such as idiA interact with Fe bound organic complexes rather than with free Fe3+(Hopkinson and Barbeau, 3+ transporter idiA or fbpA the Ópen Ocean cyanobacteria could make a variety of siderophores available to themselves. Similarly the non-specific nature of Ferric reductase mechanism (Schroder et al., Synechococcus spp. reported that around 98% of the genes evolved under purifying selection (Tai et al., The genes for some of the proteins which were discriminatory between the niches also showed evidence of positive selection pressure at the sequence level Figure . Most ofIn this time of rapid change in global oceanic conditions, the selection mechanisms operating on the evolution of genes conferring adaptation to a particular oceanic habitat are continuously shaping the genetic composition of microbial communities. We defined aquatic ecological niches for the GOS metagenomes in terms of dFe concentrations and temperature and investigated the differences in distribution of the taxonomic groups as well as the Fe-metabolism systems between these niches. The distribution of the Fe uptake proteins correlated with the taxonomic distribution of the organisms that possessed these systems, suggesting a role for temperature and Fe in shaping the microbial community in these niches. The biological availability of Fe is complicated by the presence of diverse organic ligands that bind to it. The high demand and low bioavailability of Fe mean that it is an abiotic stressor driving the evolution of microbial Fe-metabolism. We calculated rates of non-synonymous mutations for a set of genes that were discriminating between the above mentioned niches which were distinct with respect to temperature, dFe concentrations, or Coastal and Open Ocean location, and inferred that genes that exhibited higher rates of non-synonymous mutations were the ones involved in non-specific uptake of Fe bound to diverse ligands. This indicated that in the highly diffusive, oligotrophic open ocean marine environment possession of Fe uptake strategies with broad specificities provides a competitive edge to the microorganisms.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Microbiological_Chemistry/10.3389/fmicb.2012.00362/abstractThe Supplementary Material for this article can be found online at