Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress. Normal cardiovascular function depends on the biomechanical properties of blood vessels, and changes in these properties are characteristic of disease. Increased aortic stiffness precedes and predicts for the development of hypertension and subsequent negative cardiovascular outcomes, including myocardial infarction, stroke, and heart disease The mechanical properties of a blood vessel, like any material or tissue, are determined by its components and their organization. Accordingly, the stiffness of vascular tissue can be derived from the composition of the extracellular matrix (ECM) and the cells in the tissue, the organization of the matrix and cells, and the connections that link these components (cell-matrix and cell-cell contacts). The prevailing view is that the ECM is the principal determinant of vascular stiffness Vascular smooth muscle cells (VSMCs), the predominant cell type in the wall of large blood vessels, influence tissue stiffness when modulating contractile tone or when depositing matrix or matrix-degrading enzymes In the present study, we examine the mechanical properties of blood vessels across multiple length scales and identify, for the first time, the Src-FAK signaling complex of FAs of the non-migratory, non-proliferating VSMCs embedded in the blood vessel wall as a significant regulator of aortic stiffness. Finally, we show that FA size, cortical stiffness, and VSMC contractility influence the FA-regulated component of aortic stiffness.All procedures were performed in accordance with protocols approved by the Boston University Institutional Animal Care and Use Committee (Permit Number: A3316-01). The animals were maintained according to guidelines in the NIH Guide for the Care and Use of Laboratory Animals and were obtained and used in compliance with federal, state, and local laws. Tissue removal was quickly performed after isoflurane euthanasia.A7r5 vascular smooth muscle cells, originally derived from rat aorta , were maintained as described previously For magnetic tweezer studies, an arginine-glycine-aspartic acid (RGD) peptide  was covalently coupled to amine-modified superparamagnetic Dynabeads M-270  according to a protocol from Bangs Laboratories . Briefly, amine-modified Dynabeads were suspended in phosphate buffered saline (10 mg/ml), activated with 10% glutaraldehyde, reacted with RGD peptide (0.5 mg/ml), quenched with 40 mM glycine and blocking agent, and stored with blocking agent and preservative in phosphate buffered saline.5β1 and αvβ3 integrins, which are present in vascular smooth muscle tissues and recognize fibronectin and vitronectin, respectively RGD is an ECM protein motif that is a minimal sequence required for binding by transmembrane integrin receptors 5 beads were added to the 35-mm glass bottom dish containing cells. The beads were incubated with the cells for 30 min at 37°C with 5% CO2 to facilitate their attachment to the cells. Following this incubation, the serum-free medium was exchanged twice to remove unbound beads. Measurements of cortical stiffness were performed at room temperature. Measuring time was limited to 30 minutes per dish.Before making measurements with the magnetic tweezers, the beads were washed and sonicated, and then 6.7×10The magnetic tweezers (MT) apparatus is modeled after a system designed by the Fabry group 2O (viscosity η = 0.052 Pa s at room temperature) were used to compute forces on the beads for different solenoid currents using Stokes’ formula for viscous drag, F = 6πηrv, where η is the viscosity, r is the radius of the bead, and v is the velocity of the bead through the mixture. The calibration data for all beads were then fit to a simple empirical equation relating the force on a bead to both the bead’s distance from the magnetic tweezers tip and the current through the solenoid Calibration of the magnetic tweezers was performed similarly to what has been done by others To measure cortical stiffness, the MT tip is positioned approximately 150–200 µm from a cell-adherent bead using a micromanipulator. This distance was chosen because the force-distance relationship is highly nonlinear at short bead-tip distances but flattens out at distances greater than 100 µm. Thus, at an operating distance of 150–200 µm, a pull on a bead will displace it towards the tip and only negligibly increase the force exerted by the MT on the bead . An operating current of 1.5 A was chosen for experiments because it produces low forces appropriate for cortical probing Cortical stiffness, which we define as the stiffness of the bead-integrin attachment and the underlying FA and cortical cytoskeleton, was measured by employing a priming protocol described by others that consists of a series of five identical square-wave cycles that are 4 sec in duration and 4 sec apart 2, Photometrics) at 10 frames per second using the Nikon NIS Elements imaging software (Nikon Instruments). Positions of beads were tracked with an intensity-weighted center-of-mass algorithm using the MTrackJ plug-in for ImageJ Experiments were performed at 20× magnification (NA 0.5) with a Nikon Plan Fluor objective under bright-field illumination on an Eclipse TE2000-E inverted microscope (Nikon Instruments). Videos of beads and the MT tip were recorded in real time with a charge-coupled device camera  was added for 15 minutes, PP2 (10 µM) was added for 30 minutes, or PP2 was added for 30 minutes followed by LPA for 15 minutes. Experiments were performed at room temperature, and measurement time with the MT for each sample was limited to 30 minutes to ensure cell viability.2, Photometrics), and filters optimized for double-label experiments. NIS-Element Advanced Research software (Nikon Instruments) was used to capture images and for removal of out-of-focus fluorescent blur by deconvolution of Z-stacks  as previously described A7r5 cells were fixed and stained as previously described Areas of individual FAs were determined using NIS-Elements Advanced Research software. FAs were measured for 5 to 13 A7r5 cells per condition  per experiment . In each cell that was selected for analysis, all peripheral FAs were examined for analysis because these adhesions are most relevant to the question of force transmission, as numerous studies have reported that the largest traction forces are found in the cell periphery Mus musculus)   were euthanized with an overdose of isoflurane by inhalation. The aorta was promptly excised, rinsed six times to remove blood within or around the vessel, which can harm the smooth muscle cells and induce vasoconstriction 2–5% CO2) physiological salt solution (PSS) . Following careful dissection in oxygenated PSS to remove connective tissue and adventitia, aortic rings were cut (4 mm long) then suspended in vitro in tissue baths containing oxygenated PSS at 37°C. Smaller rings immediately proximal and distal to these suspended segments were also cut for determining ring thickness. Following the measurements made in the tissue baths, aortic rings were quick-frozen at −78.5°C in a slurry of dry ice and liquid acetone containing 10 mM dithiothreitol and 10% TCA for subsequent biochemical analysis C57bl/6 Mice , a vital nuclear stain, for 30 minutes. Each ring was placed in a small PSS-filled chamber on a slide with the major axis of the vessel perpendicular to the slide for examination in cross-section. Images were taken under the blue channel (cell nuclei) and the green channel  at 20x, moving around the ring to acquire a total of six images for thickness measurements. For each of the six images, four line segments were arbitrarily drawn to measure stiffness at different positions on the vessel wall. All twenty-four measurements per ring were averaged. Then, the thicknesses of the two bordering thin rings were averaged to determine the mean thickness of the aorta ring segment (∼60 µm).in vitro force and stiffness measurements, thin triangular shaped wire clasps (d = 0.005 in) were threaded through the lumen of the aorta rings then attached at one end to a fixed hook and at the other end to a computer-controlled motorized lever arm  capable of setting tissue length while simultaneously measuring force. The rings were stretched uniaxially in the circumferential direction, as vascular smooth muscle cells in the aorta wall are oriented primarily in this direction, and this is the primary direction of strain induced by pulsatile flow in vivo.For LO  for 30 minutes to allow adequate time for stress relaxation . The optimal length for maximal contraction was predetermined by length-tension curves (data not shown); for mouse aorta, OL is near the upper end of the tissue’s physiologic strain range during systolic/diastolic pressure transients LO were collected continuously during ten minutes of stimulation with the alpha-agonist phenylephrine  at a maximally effective concentration.Rings were stretched to optimal length E = E’+iE”, where E’ is the elastic or storage modulus (the in-phase component of the stiffness), E” is the viscous or loss modulus (the out-of-phase component of the stiffness), and i is the unit imaginary number. As reported previously E is calculated as the ratio of the stress σ to the strain ε, E = (ΔF/A)/(ΔL/LO), where ΔF is the amplitude of the force response to the cyclic stretches, A is the cross-sectional area, and ΔL is the amplitude of the cyclic stretches. When stretched to optimal length, the walls of the ring collapse, allowing the cross-sectional area A to be calculated as twice the product of the wall thickness h and the axial length of the ring l, A = 2hl.Tissue stiffness was measured by small-amplitude (1%), high-frequency (40 Hz) sinusoidal length perturbations as previously described 3VO4, 1 mM NaF, 100 µM ZnCl2, 20 µM AEBSF, and 5 mM EGTA. Western blot densitometry was performed using Odyssey Infrared Imaging System . Glycerol-urea gel electrophoresis was performed to separate nonphosphorylated, monophosphorylated, and diphosphorylated light chains as previously described Quick-frozen tissues were homogenized and processed at 4°C as previously described Agonists used were LPA from Cayman Chemical  and phenylephrine from Sigma-Aldrich. PP2 was purchased from EMD Biosciences ; FAK inhibitor 14 was purchased from Santa Cruz Biotechnology ; and ML-9 was purchased from Calbiochem . General laboratory reagents were of analytical grade or better and purchased from Sigma-Aldrich and Bio-Rad Laboratories . The following primary antibodies were used: phospho-tyrosine  from BD Biosciences ; phospho-FAK Y925 , phospho-paxillin Y118 , and phospho-CAS Y165  from Cell Signaling Technology ; vinculin  from Sigma-Aldrich; and a-tubulin . For immunofluorescence experiments, goat anti-rabbit and goat anti-mouse Alexa Fluor 488 and Alexa Fluor 568  were used as secondary antibodies. For Western blot experiments, Goat Oregon Green 488 or Alexa Fluor 568 labeled anti-rabbit or anti-mouse IgGs were used as secondary antibodies .Data are reported as means +/− standard error. Differences between individual means were determined by two-tailed Student’s t-test. Differences were considered significant at p<0.05.To test the role of FAs in regulating cell stiffness, minimizing the contribution of the ECM, we measured cortical stiffness of serum-starved A7r5 VSMCs with MT . For theMean cortical stiffness  was measAs a control, we performed experiments with bovine serum albumin (BSA)-coated beads, which adhere nonspecifically to cell membranes A larger FA would have greater stiffness as measured by the MT. We hypothesized that changes in FA dimensions might underlie our cortical stiffness observations since FAs of migrating cells grow in a force-dependent manner 2 versus 3.24±0.14 µm2 in control cells, a 55% increase) (p<0.001). This is consistent with effects reported in migrating cells upon myosin activation and suggests that a tension-induced increase in FA size in the presence of LPA increases cortical stiffness. Additionally, the growth in FA size with LPA was prevented by PP2, which is also consistent with the known effect of PP2 to prevent FA remodeling and growth in other cell types −2) in any of the conditions examined.Mean FA size did not change with PP2 treatment alone. FAs were significantly larger in cells stimulated with LPA -dependent myosin phosphorylation To test the role of FA remodeling in regulating stiffness of the intact vessel wall, we measured the stiffness of aortic rings pre-treated with PP2 before PE activation. PP2 significantly decreased PE-induced stiffening (71–84%) (p<0.001–0.05) .These results are consistent with the concept that Src-dependent FA recycling is a significant regulator of aortic stiffness.4/mol LC; PE: 0.375+/−0.049 mol PO4/mol LC, p<0.05; PP2+PE: 0.362+/−0.039 mol PO4/mol LC, n.s., n = 5). Src is also known in some systems to regulate actin polymerization Unexpectedly, PP2 pre-treatment also significantly decreased PE-induced stress generation (78–88%) (p<0.001–0.05) . At firsWe also measured stress and stiffness for aortic rings pre-treated with the specific small molecule inhibitor FAK inhibitor 14 before PE activation To confirm that PP2 inhibits Src-dependent FA signaling in aortic tissue, we performed phosphotyrosine screens of aorta homogenates. As seen in The p125 band co-stained with an antibody against 125-kDa FAK, a major FA signaling protein and Src binding protein To further explore agonist-induced FA signaling, we examined phosphotyrosine levels in the presence of FAK inhibitor 14. Phospho-specific antibodies against p130 CAS Y165 and p68 paxillin Y118, both substrates of the FAK-Src signaling complex Thus, taken together, these results are consistent with the concept that, at the tissue level, vasoconstrictor activation initiates Src- and FAK-dependent FA signaling, which, in turn, regulates aortic stiffness and contractility.The main finding of this study is that the FA, as a cell-matrix linker, via the FAK-Src signaling network, is a significant and regulated component of aortic stiffness and tone. As a first step towards determining whether the cytoskeleton-FA linkage modulates vascular tissue stiffness, we measured cortical stiffness on the surface of aortic smooth muscle cells. To the best of our knowledge, our use of MT to measure cortical stiffness in VSMCs is novel but complementary to other techniques employing larger forces to sample whole cell stiffness The MT experiments revealed that cortical stiffness is increased by contractile stimulation with LPA in a Src-dependent manner. Cell immunofluorescence imaging was employed to explain the effects of LPA and PP2 on cortical stiffness. The imaging data demonstrate that contractile activation with LPA induces FA growth, similar to what has been observed in migrating and spreading cells in vitro stiffness measurements of rodent aorta. We found that smooth muscle contraction accounts for roughly 20% of stiffness, with the remaining 80% likely being due to extracellular matrix. Others have published that the ECM plays an important role in determining tissue stiffness and that changes in the ECM that occur with age or disease can affect tissue stiffness After demonstrating that FAs and especially the FAK-Src signaling network are important to the cortical stiffening of a VSM cell model during contractile stimulation, we scaled up to Taking these findings together, it appears that agonist-induced FA growth and remodeling entails strengthening of cytoskeleton-matrix linkages in VSM, permitting adequate force transmission from contracting cells to the vascular wall (see Model, In summary, our results point to the FA of the VSMC, specifically through interactions of FAK and Src, as a regulator of aortic vessel stiffness. We report that the FAs of these nonmigrating cells embedded in the wall of the aorta are regulated by vasoconstrictors and coordinate changes in force and stiffness development in the tissue. Given that increases in aortic stiffness are linked to cardiovascular disease, these results also point to the FA as a potential novel therapeutic target.


Cardiovascular Journal of Africa is pleased to announce an increase in its 2011 impact factor to 0.767, as provided by Thompson Reuters (ISI). Currently, we rank 161 out of 273 indexed journals in the field of cardiology and cardiovascular medicine throughout the world, according to the Scopus journalrating system. The Cardiovascular Journal of Africa is truly entrenched in Africa and worldwideThe Cardiovascular Journal of Africa has developed the capability of publishing your article to PubMed Central (PMC), which is the free electronic archive of the Biomedical and Life Sciences Journal literature at the US National Institutes of Health National Library of Medicine (NIH/NLM). Currently your article is available on PubMed as a PDF from Sabinet e-publications. PubMed Central is therefore an additional repository of your full-text article. This ensures a long existence for your research and availability for generations to come.In order to improve visibility for our authors, the The further value of PMC lies in its capacity to store and cross reference data from diverse sources using a common format within a single repository. With PMC, a user can quickly search the entire collection of full-text articles and locate all relevant material. PMC also allows for the integration of its literature with a variety of other information resources that can enhance the research and knowledge fields of scientists, clinicians and others. We are offering authors from our 2012 published articles first opportunity to use this service at an initial fee of 80 US dollars.Cardiovascular Journal of Africa, 590 peer-reviewed articles have been published and are freely available as linked full text.Our authors will also be pleased to note that PubMed will now index our electronic journal. This allows us to publish more articles as we are not restricted by print dynamics. Since listing in Index Medicus, PubMed in 2007 as the Cardiovascular Journal of Africa receives articles from across the world, but gives priority to articles from Africa.Authors and reviewers deserve our gratitude as their efforts help to record the development of cardiovascular research across our continent. The www.cvja.co.za and register as a reviewer.In order to assist Francophone Africa, we are enlisting French reviewers with competency in cardiovascular medicine. We need to expand this list, so if you are able to assist in reviewing, please contact our development editor, Glenda Hardy on Glenda@clinicscardive.com or go online to Cardiovascular Journal of Africa will be present at the forthcoming PASCAR conference in Dakar, Senegal, 15–20 May 2013. We look forward to seeing you there, and of course, also at the 6th World Congress of Paediatric Cardiology and Cardiac Surgery, 17–22 February 2013 in Cape Town.The


The majority of hCGβ epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of β-sheet loops 1 and 3  and the second around the cystine knot . Four mAbs recognized epitopes on hCGβcf-only  and six mAbs epitopes on the remote hCGβ-carboxyl-terminal peptide . For routine diagnostic measurements, methods are used that either detect hCG-only, hCGβ-only, or hCG together with hCGβ or hCG together with hCGβ and hCGβcf. Sandwich assays that measure hCG plus hCGβ and eventually hCGβcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGβcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope β1  in combination with epitopes β2 or β4 located at the top of β-sheet loops 1 + 3 of hCGβ involving aa hCGβ20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGβ, hCGβn, hCGβcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGβ-, 8 hCGα-, and 13 αβ-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no  contains supplementary material, which is available to authorized users. Improving between method comparability for measurement of the heterogeneous glycoprotein hCG requires harmonization of epitopes of the Abs used and broad consensus about assay specificity. To address this, the Second International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) Workshop (WS) by a three-step algorithm characterized and epitope typed 69 Abs directed against hCG and variants submitted by diagnostic companies and research groups. The results of this WS in combination with those of the First WS enable recommendations to be made regarding epitope combinations to be used for the design of immunoassays for hCG and its variants .The glycoprotein hormone hCG is essential for maintaining pregnancy. Physiologically, it is produced and secreted by the placental trophoblast and pathophysiologically by trophoblastic cancers and by germ cell tumors of the testis and ovary .hCG is a protein heterodimer consisting of hCGα noncovalently linked to the hCGβ subunit. As all glycoprotein hormone (GPH) subunits, hCGα and hCGβ share structural homology with members of the cystine knot growth factor superfamily that includes nerve growth factor, platelet-derived growth factor and transforming growth factor beta . The comThe subunits are noncovalently linked in antiparallel, i.e., a head-to-toe fashion, such that loops 1 + 3 of one subunit are adjacent to loop 2 of the other subunit , hLH and synthetic hCGβCTP peptides to enable grouping of the mAbs according to their main specificities , and tentative assignment of epitopes by comparing specificity profiles to those of reference mAbs with known epitope recognitionConfirmation of epitope recognition and spatial arrangement of epitopes using sandwich assays with mutual antigen recognition or inhibition by pairs of mAbs to provide information about epitope disparity or identity/vicinityCross-referencing of the ISOBM-Abs’ reaction profiles in specificity and sandwich assays to those of reference mAbs recognizing previously defined epitopes.For the Second ISOBM TD-7 WS on hCG, a previously reported epitope mapping algorithm , which wThis approach frequently enabled definitive assignment of epitopes at the molecular level. In rare cases where no exact molecular localization could be determined due to the lack of appropriate reference mAbs, additional circumstantial evidence, e.g., mutual steric inhibition in simultaneous antigen recognition with mAbs of known epitope localization or recognition of breakdown products like hCGβcf or inter-species cross-reactivity, was used to elucidate the epitope’s antigenic domain .Sixty-nine ISOBM-TD-7 Abs were submitted by eight participants to Dr. Kjell Nustad at the Central Laboratory, Norwegian Radium Hospital (NRH), Oslo, Norway , and six of these shared by hCGβ . Two Abs recognized epitopes on hCGβ plus hCGβn plus hCGβcf (β6 and β7). Three reference mAbs against epitopes β10, β11, and β13 recognized exclusively hCGβcf. MAb FB12 recognizing hCGβCTP epitope β9 ., 50.1–11β113–116 , 24 was When immunizing with the glycoprotein hCG, the vast majority of antibodies will be generated against composite epitopes on hCGα or the core region of hCGβ (aa 1–112) but only rarely against linear peptide sequences of low structural order like the hCGβCTP. MAbs against hCGβCTP are generally of fairly low affinity. Nevertheless, they are used in diagnostic sandwich-type immunoassays as they do not cross-react with hLH Fig. .In the panel of ISOBM-mAbs, 8 of 69 recognize hCGα epitopes. One of these mAbs, ISOBM-404, seems to be specific for free hCGα, and it is speculated that it might recognize the sequence hCGα33-42 on the single loop 2. As no reference hCGα-mAbs (Table 1–c4) are present only on hCG ± hCGn but not on either free subunit or hCGβcf [1 (reference mAb INN-hCG-10) and c2 (reference mAbs INN-hCG-40 and INN-hCG-53) are (1) dependent on intact hCG and thus sensitive to nicking of assembled hCGβ loop 2, (2) not compatible in sandwich-type assays with the cystine knot-related hCGβ epitope β1 (aa hCGβArg10 + Arg60 and possibly Gln89) [1, α2, and α4 [1-mAbs show identical reactivity patterns as α1- and α2-mAbs reflecting sterical epitope relatedness [At least four epitopes  , and 3), and α4  on loop , and α4 atedness .3 are sterically related to epitope c2, highly specific versus hLH as well as non-combined intact and modified subunits (<1 % cross-reactivity), not influenced by nicking of assembled hCGβ loop 2, and thus recognize hCGn and hCG equally [MAbs against epitope c equally Fig. 4)..3 are st4 has not been resolved yet. It is present on hCGn and hLH, remote from and thus sterically compatible with all other c-epitopes and to a minor extent determined by hCGβ as shown by low cross-reactivity [4-epitope represented by ISOBM-424  that is not shared with hLH (cross-reactivity <0.1 %) seems to exist. A presumably fifth highly specific c-epitope has been observed in sandwich assays wherein mAb ISOBM-433 is compatible with mAbs to c1–c4 .The exact molecular localization of epitope cactivity , 26, 38.–c4 Fig. . Its molSandwich-type assays measuring hCG alone or in combination with free hCGβ and metabolites are used for detection of pregnancy, pregnancy-related disorders, trophoblastic disease, and various other female and male tumors . DetaileWhile assays measuring hCG and all hCGβ-related variants are useful as first line methods, for diagnosis of pregnancy and cancer, it is often advantageous to specifically measure only selected variants . Thus, sElevated plasma concentrations of hCGβ are reflected by high levels of hCGβcf in urine , and spe2 or β4 (the hCGβloops 1 and 3 domain) combined with a mAb-recognizing epitope β1 , a mAb against epitope c/4 Table . Alternainations  or β2/4 possible . These dommended  (Table 6A unique mAb coded B152 is used for the measurement of hCG-h that carries a core-2 glycan on Ser132 located on hCGβCTP . HoweverConsiderable reduction in between-method and between-laboratory variability in results can be achieved by a number of measures: 1) the establishment and usage of a clear nomenclature of hCG and its variants  the esta; (2) endESM 1(PDF 20194 kb)Below is the link to the electronic supplementary material.


International Journal of Integrated Care (IJIC) it gives me great pleasure to welcome you to the Republic of San Marino for the 12th Annual Conference on Integrated Care.On behalf of the Editorial Board of the May you live in interesting times” is a well-known quote that has been used and interpreted both as an old Chinese curse or as a dynamic reality in International Law. This conference today is an opportunity to shape this reality by learning from each other’s experiences, both practical and academic, and to get a deeper understanding of the dynamic multi-dimensional field that is integrated care.“IJIC’s annual conference supplement. We cordially invite you to write-up your policy analyses, academic research and/or innovative case studies and submit them formally or informally to the Journal.This year’s conference programme in San Marino is rich in interesting presentations and poster papers and we look forward to publishing the abstracts in IJIC has consolidated itself as the leading international scientific journal in its field. IJIC remains a pioneer in providing an open access and e-based publication that is widely-read. IJIC, as a result, is also highly responsive enabling one of the shortest periods from article submission to publication that there is—25 weeks in 2010. In 2011, IJIC’s unofficial Impact Factor rose to 1.75—a good number for a specialist journal—and an application for an official impact factor is in progress.During the last 12 years of existence, IJIC and its related annual conferences have delighted us with important developments in the knowledge and practice of integrated care. This collective learning experience has been possible because of the leading role of Prof. Guus Schrijvers, our sponsors at the Julius Centre, and our helpful publishers—IGITUR—at the University Library Utrecht. It has also been steered over the years by a highly committed and experienced Board of Editors, all of whom have given their time voluntarily, and supported by a range of excellent authors and reviewers. But, especially, it has been a very rich experience due to the on-going support of our more than 1500 monthly readers from more than 25 countries.Since its innovative start, International Foundation of Integrated Care (IFIC) will replace the Julius Centre as the journal sponsor. IFIC is an ambitious and exciting initiative with a growing portfolio of initiatives including: a new World Congress; an Integrated Care Knowledge Centre; a new ‘high tech’ website; a range of educational courses; and a core partner in international research projects.By the end of 2012, the new IFIC is an evolving organization and you are welcome to help contribute to growing portfolio of work and also its planning, decision-making and development processes. To learn more about IJIC and IFIC, please attend our workshop session (4.3) on day 2. It is indeed a very interesting time for all of us who wish to join this initiative and contribute to shape the work of IJIC and IFIC in the future.Finally, we are extremely grateful to the conference organizers and the scientific committee for their hard work in putting together such a fine event. Special thanks go to Paolo Pasini and his colleagues in San Marino for agreeing to be such welcoming hosts.We wish all of you an interesting and effective conference.


Whereof what's past is prologue; what to come,In yours and my discharge.Shakespeare The Tempest, Act 2, scene 1, 253-254Croatian Medical Journal (CMJ) is ready for a new future. The team that made the Journal what it is today is leaving – its two Co-editors in Chief and almost the whole Editorial Board. The two Co-editors in Chief have been elected as editors emeriti, the title that had already been given to the founder and creator of the CMJ, Prof. Matko Marušić, who stepped down as the editor when he became the dean of the School of Medicine at the University of Split , we have probably reached the peak of achievement for a small general medical journal from a small country. We always closely followed the journal in its childhood and adolescent years (Journal in the mainstream science. On CMJ’s fifteenth birthday in 2006, we were about to get our first official impact factor and predicted that it would be over 0.8 (Over the first 20 years (and we count from the first publication of the nt years -25 and wover 0.8 . It was over 0.8 . This waCMJ was not to increase its impact factor but to increase its activities as a busy two-direction bridge between the small and large scientific communities. Instead of racing for the impact factor, we opened “new doors and windows into areas such as research integrity, education in scientific writing, and excellence in publishing clinical research” (In 2006, we also reaffirmed our position that the goal of the esearch” .CMJ with all of its cover pages up to this issue (CMJ’s adult life.With this editorial we do not say goodbye to our colleague editors and authors – all of us will go back to our busy academic lives and hopefully make up for our absence. As a picture is worth a thousand words, we tell the history of the is issue . In wordCMJ as the door for the small scientific communities to the mainstream science and the window for the world to see good research in small and developing countries (Our strategy to develop the ountries -25 has bountries .CMJ – to become a publishing outlet for the wealth of research in molecular medicine around the world. Statistics of the cross-citations between the CMJ and other journals provides solid evidence base for this proposal. As in previous years, the relatedness statistics (CMJ are forensic science journals: Forensic Science International: Genetics, International Journal of Legal Medicine, and Forensic Science International. Since 2001, the CMJ has been publishing a theme issue dedicated to forensic DNA research every two years, and the articles have always been our most-cited publications (We believe that the latter topics are the future of the atistics  of the 2ications . In 2006ications .CMJ further along this exciting and challenging path. It is better that the past does not restrain them. All advice we can give was already voiced many years ago by Mark Twain (Now that we have left , we hoperk Twain :“Twenty years from now you will be more disappointed by the things that you didn't do than by the ones you did do. So throw off the bowlines. Sail away from the safe harbor. Catch the trade winds in your sails. Explore. Dream. Discover.”


The International Meeting on Computational Intelligence Methods for Bioinformatics and Biostatistics (CIBB) is a small but active conference where a group of researchers share their ideas and visions. The proceedings of the conference are usually published in a book and a group of selected papers are collected in a post proceedings book published by Springer.th CIBB conference was hosted in Palermo at Palazzo Comitini during September 16-18. We found several of the papers to merit further development and publication as full journal articles to achieve the broader diffusion that they deserve. The result is this BMC Bioinformatics supplement. All of the papers published in this supplement are completely new and a further advancement of the research presented in the CIBB meetings. Moreover the goal of this supplement is not only to present the research of the participants of the CIBB meeting but to promote the CIBB conference to the world bioinformatics community.In 2010 the 7The CIBB meeting series is organized by the Special Interest Group on Bioinformatics of the International Neural Networks Society (INNS) to provide a forum open to researchers from different disciplines to present and discuss problems concerning computational techniques in bioinformatics, systems biology and medical informatics with a particular focus on neural networks, machine learning, fuzzy logic, and evolutionary computational methods. From 2004 to 2007, CIBB meetings were held with an increasing number of participants in the format of a special session of bigger conferences, namely WIRN 2004 in Perugia, WILF 2005 in Crema, FLINS 2006 in Genoa and WILF 2007 in Camogli. With the great success of the special session at WILF 2007 that included 26 strongly rated papers, we launched the first autonomous CIBB conference edit starting with the 2008 conference in Vietri.Riccardo Rizzo and Paulo JG LisboaThe authors declare that they have no competing interests.


It is critical to effectively use every available organ to meet the increasing demands for liver transplantation. Situs inversus is a rare congenital anomaly caused by obstruction of viscus rotation during embryonic development. Situs inversus was once regarded as a contraindication to liver transplantation because of the technical difficulties associated with the unique vascular anatomy and concern about achieving accurate graft positioning. Here, we present a successful case of liver transplantation using a graft from a donor with situs inversus totalis. The related experience will contribute to opening up new realms for the use of such rare organ resources. Situs inversus is a rare congenital anomaly caused by obstruction of viscus rotation during embryonic development. Its incidence is about 0.01% worldwide. The viscus distribution in the situs inversus individuals is opposite to ordinary people. All thoracoabdominal organs and retroperitoneal organs  are symmetrically positioned to the midline, which is often called mirror-image reversal , 3. For A 23-year-old male was referred to our hospital in May 2008 due to severe traumatic brain injury caused by a traffic accident and was diagnosed as brain dead shortly after admission. Dextrocardia was found during physical examination and situs inversus totalis was verified by further X-ray and ultrasonic inspection. During the retrieval of donor organs, it was found that the liver, pancreas, and stomach were located in the opposite positions. The descending aorta was located directly in front of the spine and the inferior vena cava was found in the front left of the spine. Organ procurement was smoothly performed without great difficulty even though the procedure took a little longer due to the operator's habitual thinking about the anatomy. During preparation of the donated liver, the anatomical relations were clearly identified, that is, liver artery locating in right side and common bile duct in the left, and portal vein still being behind common bile duct and liver artery. The hepatic superior and inferior vena cava was cut to angle well in preparation for vascular anastomosis.A 58-year-old female recipient was diagnosed with end-stage liver disease caused by type B hepatitis. Preoperative condition was evaluated with Child-Pugh (class A) and MELD (4.3). The liver transplantation was performed in May 2008. The incision was made double subcostally. Following isolation of porta hepatis and hepatic ligament, the retrohepatic inferior vena cava was partially clamped and then the diseased liver was removed preserving the inferior vena cava. The right hepatic vein was suture ligated while preserving the left and middle hepatic veins. At the point of left hepatic vein entering the hepatic superior and inferior vena cava, a transection was taken on an anterior wall of the interval between the left and middle hepatic veins, and meanwhile, the anterior wall of inferior vena cava was cut downward 2 cm, thereby forming a “T” type anastomotic rim in preparation for hepatic venous outflow reconstruction.Liver transplantation was performed with a modified piggyback technique. The donor liver was directly placed into the recipient's right upper quadrant without rotating adjustment, that is, the right hepatic lobe was positioned in the recipient's left side and the smaller lobe was positioned in the recipient's liver fossa. The hepatic superior and inferior vena cava was directly anastomosed to recipient's inferior vena cava with 3-0 vascular suture. Portal veins were end-to-end anastomosed with 6-0 vascular suture followed by albumin solution perfusion. The residual UW solution (University of Wisconsin solution) in the donated liver was flushed away with albumin solution. Then, the intrahepatic vena cava was completely ligated and the clamps were removed from portal vein and vena cava, thereby ending an hepatic phase. Cold and warm ischemia times were 7 hr and 2 min, respectively. For artery reconstruction, the cut off at the junction of the proper hepatic artery and gastroduodenal artery (recipient) was anastomosed with the cut off at the junction of common hepatic artery and splenic artery (donor) with 7-0 vascular suture. Biliary reconstruction was performed by duct-to-duct anastomosis between recipient's common hepatic duct and donor's common bile duct with 6-0 vascular suture without placing a “T” type drainage tube. The total operation time was 6.5 hr and life support was withdrawn at 6 hr after operation. The shortage of donated organs has been a critical issue with regard to the increasing demand. In order to enhance usable organ sources, people try to expand the criteria for donor evaluation and attempt the maximum possible use of every donor resources. Situs inversus totalis is a very rare anatomical abnormality, which is characterized by dextrocardia and “mirror-image” distribution of abdominal organs. It may coexist with other malformations such as underdevelopment of portal vein and inferior vena cava, polysplenia syndrome, and congenital biliary atresia . Situs iIn 1995, Asfar et al. , 13. TheIn the present report, the donor with situs inversus totalis died from severe traumatic brain injury caused by-traffic accident. The distribution of viscera was confirmed with X-ray and type-B ultrasonic checks, and a planned procedure was laid out before the organ retrieval. In consideration of the recipient being at the end-stage of liver disease without cancerization, a modified piggyback technique was used for the orthotopic liver transplantation. During operation, the liver graft was slightly rightward rotated and a water bag was temporarily placed in the liver fossa supporting the graft in order to avoid its self rotating. After the removal of the diseased liver, there was enough space in right upper quadrant to hold the liver graft, and vascular bypass was not required. The inferior vena cava involved a “T” type anastomotic rim which ensured the hepatic venous outflow to be unobstructed. The hepatic artery was closely related to bile duct, which was adjusted by slightly changing liver position during anastomosis of vascular and biliary systems. End-to-end anastomosis was applied for the reconstruction of hepatic artery and biliary duct. Since the left hepatic lobe was relatively large, the liver graft was laid up in situ placement. The remaining space in the right upper quadrant was filled with omentum majus and the liver was mounted to the diaphragm with falciform ligament. This manipulation could protect the liver from graft self-rotation without repressing the stomach and the surrounding intestinal tract. The achievement of long-term survival proved the rationality of the planned procedure in this particular case.In the face of critical shortage of donor livers, we treasured every valuable donor resource to save as many lives as possible. All donors received routine auxiliary examinations and the condition of the anticipated organ was predetermined prior to organ procurement. Especially to liver graft with situs inversus, timely awareness of the exceptional situation and a detailed procedure in advance are prerequisites to the success of transplantation. During donor liver retrieval, the location and relationship among various vessels need to be carefully distinguished to prevent operation damage to the organ. During implantation of the liver graft, the following two aspects should be seriously considered, the reconstruction of hepatic venous outflow which is not to be obstructed and fixation of liver graft to prevent self-rotation. Thus to our experience, the success of transplantation using such infrequently donated organs could be achieved through careful preoperative examination and an elaborately planned operation procedure.


From thSources: McElhinny T. A mammalian lexicon. [cited 2005 Oct 13]. Available from http://www.msu.edu/~mcelhinn/zoology/mammalwords.htm; Webster's Third New International Dictionary (unabridged). Springfield (MA), 1993; and wikipedia.org.


In Reply: The Croatian Medical Journal strictly follows the rule that manuscripts based on unregistered clinical trials are immediately rejected (rejected -3.


Plants deal with resource management during all their life. During the day they feed on photosynthetic carbon, sucrose, while storing a part into starch for night use. Careful control of carbon partitioning, starch degradation, and sucrose export rates is crucial to avoid carbon starvation, insuring optimal growth whatever the photoperiod. Efficient regulation of these key metabolic rates can give an evolutionary advantage to plants. Here we propose a model of adaptive starch metabolism in response to various photoperiods. We assume the three key metabolic rates to be circadian regulated in leaves and that their phases of oscillations are shifted in response to sucrose starvation. We performed gradient descents for various photoperiod conditions to find the corresponding optimal sets of phase shifts that minimize starvation. Results at convergence were all consistent with experimental data: (1) diurnal starch profile showed linear increase during the day and linear decrease at night; (2) shorter photoperiod tended to increase starch synthesis speed while decreasing its degradation speed during the longer night; (3) sudden early dusk showed slower starch degradation during the longer night. Profiles that best explained observations corresponded to circadian regulation of all rates. This theoretical study would establish a framework for future research on feedback between starch metabolism and circadian clock as well as plant productivity. Arabidopsis thaliana, about 50% of the carbon assimilated during the day accumulates as starch in the leaves  cycle. Starch degradation speed immediately decreases while its accumulation speed increases . Further evidence for the involvement of the circadian clock in the control of starch degradation comes from the study of mutants in which the period of the clock is altered. The cca1 lhy double mutant is known to have a fast-running circadian clock with a period of about 17 h when placed in constant light. When mutant plants are grown in a 17 h period L/D cycle starch degradation pattern is similar to that of wild-type plants grown in 24 h L/D cycles, while starch is degraded too fast when the mutants are grown under normal 24 h L/D cycles  acts as part of the sucrose-signaling network , where g is the constant magnitude rate of photosyntates production and L(t) is the light availability function taking value 1 for light and 0 for dark. During light period a fraction γ(t) of carbon assimilated by photosynthesis is partitioned into starch (C)—which accumulates in the leaf through the day—and a fraction 1−γ(t) is partitioned into sucrose (S)—immediately available for growth. No partitioning occurs at night as there is no photosynthesis. Starch is degraded into sucrose with rate β(t) which is the only source of sucrose at night to support leaf respiration and growth. Sucrose is exported with rate η(t) to non-photosynthetic tissues such as roots and immature leaves via the phloem. The three rates γ(t), β(t), and η(t) are aggregate parameters of multiple processes  and are treated here as equivalent to the activity of carbon partitioning, activity of starch degradation, and activity of sucrose export, respectively.We build a mechanistic model for sucrose and starch regulation in plant photosynthetic leaves Figure . The groS, C, and the three rates γ, β, and η are functions of the phase of circadian oscillation. Details will be explained later.Taken together, temporal dynamics of sucrose and starch concentrations in photosynthesizing leaves are represented by the following equations Figure :(1)dSdtS) in Equation (1) is composed of three terms; the first is equal to the fraction of photosyntate flux that is distributed into sucrose pathway, the second is the flux of sucrose that comes from starch breakdown, and the third is the loss due to leaf maintenance respiration/growth (with constant rate h) and export to non-photosynthetic tissues (with rate η(t)). Similarly the rate of change in starch concentration (C) in Equation (2) is equal to the complementary fraction of the photosyntate flux distributed into starch pathway, minus the loss due to starch breakdown into sucrose for remobilization.The rate of change in sucrose concentration (t) to be governed by the internal circadian clock with period τ and formalize it as:aβ and bβ represent the maximum and minimum values of oscillations with 0 ≤ bβ ≤ aβ; ϕβ represents the shift of internal oscillator relative to the external L/D cycle  is completely synchronized with the initiation of light period. When ϕβ is positive, oscillations of β(t) are delayed by an amount ϕβ compared to the external L/D cycle, that is, oscillations of β(t) are shifted to the right along the time axis. On the contrary, when ϕβ is negative, oscillations of β(t) are advanced by an amount ϕβ and oscillations of β(t) are shifted to the left along the time axis.There is increasing evidence that the rate of starch degradation is under circadian control  and sucrose export η(t) are governed by the internal circadian clock with period τ, which is formalized as:aγ and aη represent the maximum values while bγ and bη represent minimum values of oscillations for γ(t) and η(t), respectively. Note that 0 ≤ bγ ≤ aγ ≤ 1 and 0 ≤ bη ≤ aη are satisfied; ϕγ and ϕη represent the relative shift of each oscillation compared to the external L/D cycle. When ai = bi, , there is no circadian oscillation for process i.We also assume that the rate of starch synthesis γ, as in Equation (1), is the sucrose concentration at time t for given shift values ϕβ, ϕγ, and ϕη. In Equation (6), [x]+ = x when x > 0 and 0 otherwise. When Sγ, ϕβ, ϕηϕ (t) is less than S*, the plant is assumed to react to carbon starvation by producing a signal accumulated proportionally to starvation intensity during the time interval t1 to t2 assumed to be 24 h counted from dawn.We first sought for a set of phase shifts for the three rates that minimizes carbon starvation using gradient descent method. If sucrose drops below the level of requirement s Figure . We assuSγ(0),ϕβ(0),ϕη(0)ϕ (0) and Cγ(0),ϕβ(0)ϕ (0) and obtain a stable rather than transient profile for these two variables. Then we calculated the level of carbon starvation cn)( [defined in Equation (6)] during the 24 h of the 10th day to obtain the starvation corresponding to the stabilized dynamics. The updated values of the three phase shifts were calculated as follows:cn)( − cn + 1)( ≤ 10−6). The set of phase shifts that satisfies this criterion is the optimal set that minimizes the starvation level.To find the optimal set of phase shifts that minimizes carbon starvation, we performed a gradient descent of the cost function (6) in phase shift space. Initial shift values were set to 0 for each rate:Parameter values of the model, minimum, and maximum for the rate of starch partitioning, starch degradation and sucrose export, gross production rate of photoassimilates and respiration rate were estimated from published data of Gibon et al.  and are We first sought for a set of phase shifts that minimizes the level of carbon starvation in 12 h light/12 h dark condition, starting with an initial phase shift value of 0 for each oscillation and initial concentrations for sucrose and starch equal to 0. The initial diurnal pattern of starch was very different from the observed pyramid shape ) was increasing during the night C constant, and gives a linear decrease. During the day starch degradation rate was decreasing until dusk in order to replenish the starch pool for the next night. Optimal starch partitioning (γ(t)) was low in the morning ) showed the lowest level during the night , β(t), and η(t)) in minimizing carbon starvation, we investigated the situation where either or all of these rates were no longer circadian regulated. We examined the eight different models built up with each combination of constant/oscillating  rates (Table To examine the role of circadian regulation on the three rates (γ(es Table  for eaches Table . Model 1t), β(t)) and sucrose export (η(t)). Starch partitioning (γ(t)) has no effect on starch dynamics at night. Similarly, sucrose export (η(t)) has no direct effect on the starch dynamics (Equation 2). Thus, starch profile at night only depends on starch degradation rate β(t). When starch degradation rate was no longer circadian regulated , starch decayed exponentially at night  oscillated  starch showed features of linear decay for a part of the night, similar to the data in Gibon et al. (t) with a minimum at dusk.Starch profiles illustrated in Figure t Figure . Howevern et al. . To obtat)  (model 7). This shows that autonomous rhythm in starch degradation has greater importance than in starch partitioning and sucrose export in order to minimize carbon starvation .Models with only one parameter with circadian regulation  did not perform well to minimize carbon starvation level, but among the three worst models the one performing the best was generated by oscillating β(t)) plays an important role to realize a linear increase in starch level during the day (compare models 1 and 2). However, circadian regulation of γ(t) only was not enough to reduce starvation  and β(t) (model 5) could further reduce the level of carbon starvation  has no effect at night we obtained the same feature of parallel slopes of starch degradation as in model 3.The circadian regulation of starch partitioning ) is required to reduce this discrepancy by allowing a decrease in the slope of starch degradation along with a decrease of day length. η(t) is involved only in sucrose dynamics. However, it still affects starch profiles since sucrose dynamics modified by η(t) affects the value of β(t) during convergence of the gradient descent  on slopes of starch degradation is clearly illustrated in starch profile of model 8, which was the best model minimizing the level of carbon starvation. This model had all of the features of observations, especially different slope values for starch degradation depending on L/D cycles. Indeed, in model 5 the relative phase shift between γ(t) and β(t) was conserved through the various L/D cycles  and the two other rates were different through the various L/D cycles and slopes of starch degradation became different through the various L/D cycles   Figure  in Equat) Figure , furtherArabidopsis leaves is rather unusual. Nevertheless, by using a sinusoidal degradation (partitioning) rate for starch we could obtain such linear decrease (increase). Furthermore, shifting of the internal phase of oscillations of the key metabolic activities was enough to reproduce observations, namely a faster starch accumulation in shorter light period followed by a slower degradation during longer dark period, while keeping linear features in both cases. As a result of this behavior sucrose starvation was decreased.The presented model, with simple assumptions of circadian regulated rates and starvation avoidance by shifting of oscillation phase, could reproduce most of the features observed in experiments. While most biological quantities follow exponential behavior, the linear decrease (increase) of starch concentration at night (in the light) in The model could also explain the reduced degradation speed of starch in case of sudden early dusk. In fact, this behavior may not be the result of a sophisticated regulation from the plant but simply the result of a lower starch level at the time of early dusk for a similar value of the degradation rate. Although this behavior is not the result of any regulation from the plant it offers the advantage of saving starch.t) should follow an accelerating increase. Starch decay will diverge from linear otherwise  we used a minimum number of assumptions making it time (clock)-dependent only. However, it can include more detailed dynamics such as saturation by high starch level. If the surface of a starch granule can host all the enzymes degrading it into sucrose, the degradation would occur at a constant rate until the moment the granule surface is too small to host all the enzymes. In this case β(t) would be proportional to the enzyme level and inverse proportional to C.There are many possibilities to extending our model. Regarding the aggregate parameter β(t) obtained in our simulations.In our results we noticed that circadian regulation of all rates gave the lowest sucrose starvation. However, it is possible that other parameters of the model change with time or with other variables independently from the clock. For example, export rate of sucrose from the leaf may be inhibited by low sucrose level to prevent starvation in the exporting organ. However, with such regulation the profile of sucrose export should be close to the profile of η(In the optimization procedure we considered an objective function including only cost of sucrose starvation. It is likely that plants may try to maximize growth at the same time by supplying growing organs with a constant flux of sucrose. To do so, one can modify the objective function in the present model by including other terms such as sucrose export. Thus, plants would maximize sucrose export to growing organs while avoiding depleting sucrose level in exporting organs.However, it is interesting to notice that minimization of sucrose starvation, an intuitively critical quantity for plant growth and survival, could recreate seemingly complicated behaviors observed in starch profiles under various photoperiods. This choice of modeling offers valuable information regarding how the metabolic network of plants may have been shaped by natural selection to optimize resource management. That is the type of metabolisms that remain nowadays.Nevertheless, because of its simplicity, the present model helps understanding the mechanism of adaptive starch management behind the seeming complicated starch and sucrose behaviors. Proper management of starch allows suitable export of sucrose to other organs, which significantly influences the productivity of plants. Thus, a better understanding of starch management would have important consequences for crop performance and plant evolution. In this direction, further interaction between empirical and theoretical studies is essential.Akiko Satake proposed the model. François G. Feugier and Akiko Satake discussed and improved the model together. François G. Feugier wrote the programs, fitted the data, and ran the simulations to produce the results. Both authors discussed the results and were involved in the manuscript preparation.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.


IJMS[1 and H2 in the previous article were reversed. These errors have been amended in an amended version of the manuscript, which is available from the International Journal of Molecular Sciences website. The authors and publisher apologize for the inconvenience.The authors wish to change Figure 2 of the paper published in IJMS. The poshttp://www.mdpi.com/1422-0067/13/12/17295/s1.The corrected version can be accessed at:


P = 0.005). Adjustment for multiplecovariates did not affect this result . CXCR4rs2228014 polymorphisms from donor or recipient did not affect the incidence ofacute rejection. SDF1 was differentially expressed in renal tubular epitheliumwith acute rejection according to genetic variations of donor rs1801157 showinghigher expressions in the grafts from GG donors. Contrary to the development ofBPAR, the presence of minor allele rs1801157 A, especially homozygocity,predisposed poor graft survival(P = 0.001). This association wassignificant after adjusting for several risk factors . The allelic variation ofrecipients, however, was not associated with graft loss. A donor-derived geneticpolymorphism of SDF1 has influenced the graft outcome. Thus, the geneticpredisposition of donor should be carefully considered in transplantation.Genetic interaction between donor and recipient may dictate the impendingresponses after transplantation. In this study, we evaluated the role of thegenetic predispositions of stromal-derived factor-1 (SDF1) [rs1801157(G>A)] and CXC receptor 4 (CXCR4) [rs2228014 (C>T)] onrenal allograft outcomes. A total of 335 pairs of recipients and donors wereenrolled. Biopsy-proven acute rejection (BPAR) and long-term graft survival weretraced. Despite similar allele frequencies between donors and recipients, minorallele of SDF1 rs1801157 (GA+AA) from donor, not from recipients, has aprotective effect on the development of BPAR compared to wild type donor (GG)( A variety of recently discovered potent immunosuppressive agents have significantlyimproved short-term renal allograft survival in the last two decades. However,long-term allograft survival has not changed significantly. Although variousimmunological and non-immunological factors may affect the outcome of an allograft,the genetic interactions between donors and recipients may also be an importantaspect for the impending responses after organ transplantation. Recently, wedemonstrated genetic interactions of the CCR5 cytokine between donors and recipientsand its effects on allograft rejection and allograft survival Stromal-derived factor-1 (SDF1) is the ligand for chemokine CXC receptor 4 (CXCR4).SDF1 is up-regulated in various organs that respond to tissue damage fromirradiation or hypoxia We assume that the graft outcome is dependent not only on the recipient's immuneresponses but also on the responses of the graft as an active participant.Therefore, we hypothesized that genetic variations in SDF1 and CXCR4 of donors aswell as recipients might alter leukocyte trafficking in the short-term and thetissue repair process in the long-term, and would thereby be associated with anincreased risk of AR and long-term graft survival. To evaluate this hypothesis, weanalyzed the genetic variations of SDF1 and CXCR4 with regard to donor-recipientgenetic interactions in kidney transplantation.All variables except for the recipient's gender were not significantlydifferent between the BPAR (+) and BPAR (−) groups . Allele P = 0.005] (P fortrend  = 0.003). Adjustment for multiple covariates did notsignificantly affect this result  experienced BPAR after renaltransplantation. = 0.005] . In the = 0.006] . Allelic= 0.006] , 3When = 0.006] .P = 0.028, The most prominent staining of SDF1 was detected in tubular epithelium. When wecompared the expression of SDF1 according to donor SDF1 genotypes in 21recipients who experienced BPAR, we found that SDF1 expression was significantlyhigher in the graft from donors with the wild-type genotype (GG) compared withthe GA or AA genotype . The median graft survival of recipients who had grafts from AAdonors was significantly shorter than those of grafts from GGor GA donors (9.8 vs. 17.2 years). Again, recipients'polymorphisms did not affect graft survival.When we performed a survival analysis using the Kaplan-Meier method, recipientsreceiving the homozygous variant allele (AA) from donors showed poor graftsurvival compared to recipients who received kidneys with the wild-type (GG) orheterozygous genotype (GA) in the log-rank test, contrary to the association ofBPAR and SDF1 polymorphism (P<0.001). Inrecipients experiencing BPAR, the number of recipients with AAgrafts was too small. No one had lost an AA graft .Next, we performed a subgroup analysis according to the presence of BPAR. Inrecipients who had not experienced BPAR, subjects with AA grafts hadsignificantly poorer allograft survival , P<0.001P = 0.003). This association wasstatistically significant after adjusting for several risk factors   .Our analysis identified a statistically significant association between the rs1801157genetic variation at 3' UTR of SDF1 and graft outcome in terms of BPAR andgraft loss after kidney transplantation. Specifically, the presence of the rs1801157polymorphism variant allele of donors was associated with a significantly lower riskof BPAR, but it increased the risk of graft loss irrespective of BPAR.Kidney tubular epithelial cells express cytokines, chemokines, or many of theirreceptors. They may play different roles in allograft rejection and chronicallograft dysfunction. Therefore, polymorphisms of cytokine and chemokine geneexpression have been recognized as significant variables affecting allograftoutcome. Recently, we reported that a CCR5 polymorphism in donors and recipientscontribute together to provoke subsequent acute rejection in the early period andinfluence graft outcome in the later phase This study showed that the presence of the rs1801157 variant allele (A) wasassociated with a significantly lower risk of BPAR, but it increased the risk ofgraft loss. It is unusual that one cytokine or chemokine performs in a different wayin acute rejection than in chronic allograft nephropathy. In accordance with ourresults, IFN-γ prevented necrosis during a rejection process, but it potentiatedchronic allograft dysfunction Our results are informative, but this study has some limitations. First, the numberof recipients transplanted from AA donor, who had a significant low graft survival,was small. There may be a likelihood of type 1 error. Study dealing with more largenumber of population is necessary to confirm our finding that polymorphism of SDF1affect graft survival. Second, the protocol biopsy and works for the functionalrelationship of the SDF1 rs1801157 sequence variation of donor with BPAR or renalgraft loss will be necessary.In conclusion, a donor-derived, not recipient-derived, genetic SDF1 polymorphism hasdifferent effects on graft outcome. Thus, the genetic influence from donors shouldbe carefully considered for the proper management of allografts after kidneytransplantation.A total of 335 pairs of Korean recipients and donors who were followed-up for atleast 1 year were recruited for this study. They had received kidney transplantsat Seoul National University Hospital between 1982 and 2008. We collected wholeblood samples from the patients and their donors as follows: 307 samples fromdonors and recipients; 22 from recipients only; 6 from a donor only. Theresearch protocol used for this study was approved by the Internal Review Boardof Seoul National University Hospital (IRB No.H-0911-011-299). Written informedconsents have been obtained and all clinical investigations have been conductedaccording to the principles expressed in the Declaration of Helsinki. Weconducted medical record reviews of recipients based on the electronic medicalrecord system. Clinical parameters that could have influenced graft outcome werecollected, i.e. recipient's gender and age at transplantation, history ofhypertension, diabetes mellitus, and donor type. The primary outcome of thisstudy was biopsy-proven acute rejection (BPAR) within 1 year of the transplant.The secondary outcome was graft loss which was defined as graft dysfunction thatnecessitated new renal replacement therapy after transplantation.5′-GTGAAGGCTTCTCTCTGTGGGA-3′and 5′-TCTGCCCTGGCCTCACAC-3′; CXCR4 rs22280145′-CAACCTCTACAGCAGTGTCCTCAT-3′ and5′-CAGCTTCCTTGGCCTCTGAC-3′.DNA was extracted from whole blood, and genotyping for SDF1 [rs1801157(G>A)] and CXCR4 [rs2228014 (C>T)] was carried out by theTaqMan® method .Primers used for SDF1 and CXCR4 were as follows: SDF1 rs1801157 GGGTCTGCC-3′(TAMRA) and 5′(TET)-CATGGGAGCCAGGTCTGCCTCTT-3′(TAMRA); CXCR4 rs2228014 5′(FAM)-CGCTACCTGGCCATCGTCCA-3′(TAMRA) and 5′(TET)-CTGGCCATTGTCCACGCCAC-3′ (TAMRA).Reaction mixtures consisted of a 1.0 µL 10× AmpliTaq buffer, 1.0µL deoxynucleotide triphosphates (2.5 mM each), 0.2 µL forwardprimer (20 pmol/µL), 0.2 µL reverse primer (20 pmol/µL), 1.0µL genomic DNA (50 ng/µL) and 0.15 µL iMax II Taq polymerase.PCR reactions were carried out under the following conditions: 5 min 94°C(one cycle); 30 s 94°C; 30 s 56°C (35 cycles); 50 s 72°C, 7 min72°C (one cycle). PCR products were analysed on 2% agarose gels.Confirmation of genotypes was done by repeated PCR and DNA sequencing using theABI Prism BigDye Terminator Kit  of10% of the study population samples.A different fluorescence label was used to label the 5′ segment of allelic probes. Probe sequences asfollows: SDF1 rs1801157 5′(FAM)-CATGGGAGCC2O2). To examine the expression of human SDF1,deparaffinized sections were stained with 1∶200 diluted anti-SDF1 antibody and incubated at 4°C overnight. Sectionswere then incubated with biotin-conjugated anti-mouse IgG biotinylated secondaryantibody . Streptavidin and 3,3′-diaminobenzidine tetrahydrochloride  were used for immunohistochemical detection. Sections were thencounterstained with Mayer's hematoxylin and examined by light microscopy.The SDF1 score was graded in a blinded fashion by a renal pathologist. The scorewas expressed semi-quantitatively as 1 to 4 as follows. Score 1 indicatedbasically no staining or faint staining in a few tubules, score 2 indicated weakstaining, score 3 corresponded to moderate staining, and score 4 signifiedstrong staining . Values ofP<0.05 were considered as statistically significant.We used SPSS for Windows package 12.0K  for all analysesand calculations. Student's Table S1Association between donor SDF-1 rs1801157 (G>A) genetic variationsand severity of acute cellular rejection.(DOC)Click here for additional data file.


The editors of Journal of Physiological Anthropology would like to thank all our reviewers who have contributed to the journal in Volume 31 (2012). Tatsuro AmanoJapanSumiko AnnoJapanKiyoshi AoyagiJapanDouglas E. CrewsUnited States of AmericaJanet DufekUnited States of AmericaKatsuo FujiwaraJapanYoshiyuki FukubaJapanYoshiyuki FukuokaJapanElena GodinaRussian FederationHanns-Christian GungaGermanyAntonio GutierrezUnited States of AmericaHajime HaradaJapanShinya HayasakaJapanShigekazu HiguchiJapanMasahiro HoriuchiJapanYuhei IchimaruJapanKaoru InoueJapanYoshimitsu InoueJapanKeita IshibashiJapanKoichi IwanagaJapanMasatoshi KatagiriJapanYoshiaki KikuchiJapanYeon-Kyu KimJapanShingo KitamuraJapanHiromitsu KobayashiJapanShunsaku KogaJapanSatoru KojimaJapanTakaaki KondoJapanKatsuyasu KoudaJapanTomoaki KozakiJapanSusumu KudoJapanSoomin LeeJapanAkira MaedaJapanTakafumi MaedaJapanYoshifumi MiyazakiJapanJordan MoonUnited States of AmericaTakeshi MoritaJapanSatoshi MurakiJapanYumiko NagaiJapanHarunobu NakamuraJapanMasashi NakamuraJapanKyuichi NiizekiJapanJun-Ya OhashiJapanKazue Okamoto-MizunoJapanAnna OoueJapanMelany RichterGermanyDiane Riddiford-HarlandAustraliaKiyoshi SanadaJapanYayoi SatsumotoJapanKenichi ShibuyaJapanYoshihiro ShimomuraJapanWon Sop ShinKorea, SouthNami SomeyaJapanYoshika TakahashiJapanMotoharu TakaoJapanYuji TakasakiJapanNigel TaylorAustraliaKumpei TokuyamaJapanSei-Ichi TsujimuraJapanKazuyo TsuzukiJapanHitoshi WakabayashiJapanJim WaterhouseUnited Kingdom


Croatian Medical Journal :942-9) was previously published as a letter to the editor in the International Journal of Cardiology :398-400, published online March 22, 2005). The letter published in the International Journal of Cardiology includes the description of the first case described in the article published in the Croatian Medical Journal, while the other cases presented in the Croatian Medical Journal are original. The authors have apologized.It has come to our attention that a part of an article published in the


Acinetobacter are Gram-negative cocobacilli that are frequently found in the environment but also in the hospital setting where they have been associated with outbreaks of nosocomial infections. Among them, Acinetobacter baumannii has emerged as the most common pathogenic species involved in hospital-acquired infections. One reason for this emergence may be its persistence in the hospital wards, in particular in the intensive care unit; this persistence could be partially explained by the capacity of these microorganisms to form biofilm. Therefore, our main objective was to study the prevalence of the two main types of biofilm formed by the most relevant Acinetobacter species, comparing biofilm formation between the different species.The members of the genus Acinetobacter spp. and it appeared to be generally more important at 25°C than at 37°C. The biofilm formation at the solid-liquid interface by the members of the ACB-complex was at least 3 times higher than the other species (80-91% versus 5-24%). In addition, only the isolates belonging to this complex were able to form biofilm at the air-liquid interface; between 9% and 36% of the tested isolates formed this type of pellicle. Finally, within the ACB-complex, the biofilm formed at the air-liquid interface was almost 4 times higher for A. baumannii and Acinetobacter G13TU than for Acinetobacter G3 .Biofilm formation at the air-liquid and solid-liquid interfaces was investigated in different Acinetobacter spp to form two different types of biofilm: solid-liquid and air-liquid interfaces. This ability was generally higher at 25°C which might contribute to their persistence in the inanimate hospital environment. Our work has also demonstrated for the first time the ability of the members of the ACB-complex to form biofilm at the air-liquid interface, a feature that was not observed in other Acinetobacter species.Overall, this study has shown the capacity of the  Pseudomonas aeruginosa or Salmonella spp. [The observation of natural habitats has shown that bacteria generally aggregate in biofilm structures to persist . Biofilmlla spp. ,5.Acinetobacter are ubiquitous Gram-negative cocobacilli that are frequently found in the environment but also in the hospital setting where they have been associated with outbreaks of nosocomial infections [Acinetobacter calcoaceticus - Acinetobacter baumannii (ACB) complex contains phenotypically closely related species, i.e. the clinically most important species, A. baumannii, Acinetobacter Genospecies 3 and Acinetobacter Genospecies 13TU, also referred to as the A. baumanni group, and the environmental species A. calcoaceticus [A. baumannii and Acinetobacter Genospecies 13TU are responsible for most of the infections while Acinetobacter Genospecies 3 is less often associated with disease. On the other hand, A. calcoaceticus is mainly an environmental microorganism rarely involved in human infections [A. baumannii has emerged as the most common pathogenic species involved in hospital-acquired infections [Acinetobacter species. Overall, this study has shown the capacity of the members of the ACB-complex to form two different types of biofilm (SLI and ALI), and it revealed that biofilm formation increased at 25°C, a condition that might contribute to their persistence in the hospital environment.The members of the genus fections . The so-aceticus . Althougfections . Among tfections ,8-10; thfections . One reaAcinetobacter spp.: 64 clonally unrelated A. baumannii clinical isolates collected during the GEIH-Ab2000 project [Acinetobacter johnsonii (n = 34); Acinetobacter lwoffii (n = 26); Acinetobacter radioresistens (n = 20); Acinetobacter Genospecies 3 (n = 46); Acinetobacter Genospecies 13TU (n = 60); A. calcoaceticus (n = 10); Acinetobacter junii (n = 5). The Acinetobacter spp. other than A. baumannii were mainly recovered from catheter-related bloodstream infections and from the skin of patients outside the ICU and of healthy controls [This study has investigated the ALI and SLI-biofilm formation in different  project ; Acinetocontrols . Both bi600 of 0.01 and incubated at 25°C or 37°C for 48 h without shaking. Two wells were left uninoculated and used as negative controls. After checking that all the isolates had grown at a similar rate, the culture media was removed by inversion and the wells were washed twice with distilled water. The biofilm was stained with 0.5% crystal violet (w/v) for 20 minutes at room temperature and the wells were washed again to remove the unbound crystal violet. Biofilm formation was finally quantified at 550 nm after solubilisation with 95% ethanol. The bacterial isolates were considered to be positive for SLI-biofilm formation when the readings obtained were at least 3 times greater than the negative control.SLI-biofilm formation was performed in 96-well plates; biofilm formation was determined in Mueller Hinton broth  using an initial OD600 of 0.01 and incubated at 25°C or 37°C for 72 h without shaking. Positive ALI-biofilm samples were identified visually  using an initial ODAcinetobacter spp. producing biofilm is illustrated in Table All the experiments were performed in duplicate at two independent time-points. The percentages of isolates producing biofilm were compared by the chi squared test, or the Fisher exact test, as appropriate. A 2- tailed P value < 0.05 was considered significant. The percentage of isolates of the various Acinetobacter Genospecies 3, Acinetobacter Genospecies 13TU, and A. baumannii than in A. johnsonii, A. lwoffii, and A. radioresistens . Thus, these results might explain, at least in part, the marked persistence of the three former species in hospitals and their involvement in nosocomial infections. Moreover, in a previous study , Rodriguez-Baño et al. [A. baumannii clinical isolates formed SLI-biofilm at 37°C. Based on these results, our study revealed that the rates of SLI-biofilm formation obtained for Acinetobacter Genospecies 13TU and A. baumannii were similar, as well as their variation related to the temperature (higher at 25°C than at 37°C). Likewise, ALI-biofilm was significantly more frequent at both 25°C and 37°C in Acinetobacter Genospecies 13TU and A. baumannii than in A. johnsonii, A. lwoffii, A. radioresistens, and Acinetobacter Genospecies 3 (p < 0.05). Overall, this similar behaviour in biofilm formation between A. baumannii and Acinetobacter G13TU is coherent with the fact that these two species are the most commonly found in the hospital.As shown in Table o et al.  showed tA. junii had some capacity to form this characteristic biofilm, the number of isolates studied was too small to draw conclusions. By contrast, as a member of the ACB-complex, A. calcoaceticus has shown a different pattern at 37°C with a complete absence of ALI-biofilm formation together with a reduced ability to form SLI-biofilm, both of which could be explained by the fact that this species' natural habitat is the environment where lower temperatures usually prevail.Our results showed , and probably contributing to the increased risk of clinical infection.In summary, the members of the Acinetobacter calcoaceticus - Acinetobacter baumannii complexSLI: Solid-Liquid Interface; ALI: Air-Liquid Interface; ACB-Complex: The authors declare that they have no competing interests.SM carried out the biofilm studies, participated in the design of the study and drafted the manuscript; JRB performed the statistical analysis and helped to draft the manuscript; MCF helped to perform the biofilm studies; TJ participated in the design of the study; JV participated in the design of the study and helped to draft the manuscript; HS participated in the design of the study and helped to draft the manuscript; ED participated in the design and coordination of the study and helped to draft the manuscript. All authors have read and approved the final manuscript.Due to the observational and retrospective design of the study, the Ethic Committee of the participating centres waived the need for obtaining written informed consent.


Natural polysaccharides such as starch are becoming increasingly interesting as renewable starting materials for the synthesis of biodegradable polymers using chemical or enzymatic methods. Given the complexity of polysaccharides, the analysis of reaction products is challenging.Thermomyces lanuginosus. The reaction showed a pronounced optimal water content of 1.25 mL per g starch, where a degree of substitution (DS) of 0.018 was obtained. Incomplete gelatinization probably accounts for lower conversion with less water.Esterification of starch with fatty acids has traditionally been monitored by saponification and back-titration, but in our experience this method is unreliable. Here we report a novel GC-based method for the fast and reliable quantitative determination of esterification. The method was used to monitor the enzymatic esterification of different starches with decanoic acid, using lipase from Lipase-catalysed esterification of starch is feasible in aqueous gel systems, but attention to analytical methods is important to obtain correct DS values. Naturally abundant polysaccharides, in particular starch and cellulose, are increasingly being considered as renewable and potentially biodegradable starting materials in many traditional industries ,2. In maBiocatalysis is an attractive option for chemical modification of starch, and has been extensively used for partial hydrolysis to generate oligomers. However, the enormous range and diversity of available enzymes offers the possibility to obtain completely novel products with new or improved functionalities that were impossible to produce through traditional means. The use of enzymes to acylate the starch molecule, and generate compounds with commercial importance, is a relatively unexplored field -7. Even Starch fatty acid esters have traditionally been dominated by starch acetates which presented more desirable properties for the intended uses. In recent years however starch acylates of medium or longer chain acids have emerged as candidates for novel uses, mainly in the biodegradable and renewable materials industries. One of the main advantages of these materials is the ability of longer chain esters to work as internal plasticizers of starch's glucan matrix . The extWhen starch has been esterified, whether by conventional chemical or enzymatic routes, product analysis is important. The analysis of esterified starch samples has been approached in a few different ways. The oldest and most common method however has been a titrimetric one, first proposed by Genung and Mallatt . The priOther more recent methods include the quantification based on the absorption band assigned to the vibration of the carbonyl in the ester group, with FTIR  and a meThe published saponification method Figure  indicateFigure Another problem is the nature of the titration curve for the starch/fatty acid mixture. When sodium decanoate was titrated alone, there was a clear phase of significant acid consumption between pH of around 8 to 6. This titration occurs well above the true pK value, because the neutral decanoic acid formed comes out of solution, pulling the equilibrium over. However, after the saponification treatment in a mixture with starch, there is no indication of this titration of sodium decanoate Figure . The curThese same problems were found when using the titration method with a range of different starch types (data not shown). Overall, our observations suggest that the titration method is unreliable, at least for starch esters of medium chain fatty acids. It might be acceptable for high DS acetyl derivatives, for example, where blanks may be smaller relative to the sample titrations.Transesterification of acetyl groups from starch to methanol has been employed, where the resulting methyl acetate was distilled, then analysed with alkaline saponification and titration ,21. In tIn initial trials the quantity of methyl ester found declined again after longer transesterification times, probably because of its hydrolysis. The rate of disappearance was faster at higher temperatures, but could be reduced by the use of special grade anhydrous methanol. A transesterification reaction at 50°C gave maximum methyl ester at just under one hour. The hydrolysis of the methyl ester was not detectable under these conditions.Once the FAME was quantified, the average mol of acyl groups per anhydroglucose unit was calculated to give the DS of the modified starch. As there are three hydroxyl groups per unit present, the maximum value the DS can reach is 3. It should be pointed out that when used for systems with fatty acid substrates, any residual free fatty acid in the final product will not esterify and thus will not affect the resulting DS. This is expected for alkaline methanolysis, because the fatty acid is converted to the unreactive carboxylate anion. Nevertheless, because we were measuring small DS values, a control was performed where decanoic acid was added to the mixture prior to transesterification. No detectable methyl ester was found, unless fatty acid esters were also present. This is a very important advantage of this method, as, in our experience, it is not uncommon for unreacted fatty acid to remain non-covalently attached to the starch, even after several washes. The titration analysis, will not distinguish between covalently and non-covalently bound residues.The reproducibility of the method was tested by replicate analysis of samples taken from the same reaction product, showing a standard deviation of less than 3% (e.g. DS of 0.0042 ± 0.0001).Systems using lipase-catalysed esterification of gelatinized starch with a low water concentration, surfactant and a free fatty acid have been described in literature ,7 Figur. In our The analysis of these enzymatic reaction systems with the GC method described earlier showed clear peaks of methyl decanoate, whilst controls (no enzyme) gave very small peaks, or no peaks at all. Quantification of these peaks showed that starch decanoate with a DS ranging from 0.004-0.007 was obtained Table . Such DSAnother point is that in general, starches with higher amylopectin fractions seem to result in higher DS values Table . Rather It is worth noting that a control where the enzymatic reaction mixture is analysed without incubation (yield for time zero), did not result in the generation of methyl ester. This control was run in case any methyl ester was generated from residual fatty acid, catalysed by residual lipase, during the work-up and analysis process.The reaction progress with time was monitored for the starch type that resulted in the higher DS. From Figure Because water plays an important role in both stages of the process, as a gelatinization medium and as an enzymatic reaction solvent, the effect of water concentration in the gelatinization mixture was also investigated. It seems that although the enzymatic reaction as expected is favored by reduced water concentration (or increased starch concentration), a minimum amount of water is necessary for the progression of the gelatinization. This seems like a reasonable interpretation of the optimum observed in Figure 6 medium, showing perfect agreement of chemical shifts with peaks a, c and e. We further analyzed a sample of acylated starch precipitated with acetone instead of ethanol. The peaks attributed to ethanol were indeed now missing, with instead a large peak appearing at 2.08 ppm, as expected for acetone protons. Small low-shift peaks were revealed, which might be attributable to methyl and methylene protons in the decanoyl chains. Their small size made accurate quantitation difficult, but estimated DS values were about twice those from the GC method. However, fatty acid non-covalently attached to the carbohydrate would also contribute to these peaks. No 1H-NMR peaks were detectable that could be assigned exclusively to covalently  bound fatty acid. Hence we conclude that this NMR approach is not a reliable method for estimation of DS in the low range encountered with our samples. It may well be accurate for much higher DS starch derivatives, especially when purified  with decanoate was achieved to substitution yields of close to 0.02 DS with good reproducibility.We have developed a new analysis scheme for determining degrees of substitution in starches acylated with fatty acids. This analysis method has been used to evaluate a number of enzymatic acylation reactions and to measure the DS for a number of acylated starch types. We found an optimum water concentration in the gelatinization mixture, which can be attributed to its role in the disruption of starch's granular structure while inhibiting the synthetic reaction. Thus, acylation of Tapioca derived starch using lipase from Thermomyces lanuginosus (lipolase), Triton X-100 and decanoic acid were obtained from Sigma-Aldrich(UK).Hylon VII (70% amylose), Tapioca Starch and Amioca Powder TF (near 100% amylopectin) were provided by National Starch (Germany). Amylose, lipase from T. lanuginosus, approximately 50 kU for Sigma assay), per gram of starch were added and mixed thoroughly. Finally 0.5 g of liquid decanoic acid per gram of starch (0.46 mol per mol anhydroglucose units) was placed in the vessel. The reaction solution was mixed with an overhead stirrer , for 1 hour. Smaller scale reactions were carried out in 2 mL screw-top centrifuge tubes and mixed in a Vortex Disruptor Genie. The reaction solution was then quenched with 10 times its volume of acetone or absolute ethanol, to precipitate the starch. Ethanol is most commonly used in published methods to precipitate starch, but we found that its use could lead to formation of small but detectable amounts of ethyl decanoate, probably through catalysis by the enzyme still present. The amounts found  corresponded to less than 0.0005 DS, but it is recommended to avoid ethanol, especially when low DS values are to be measured. After centrifugation (4000 rpm or 2690 g) the supernatant was removed and the product was washed twice with the same volume of acetone or ethanol. After washing, the product was dried in a desiccator under vacuum. A control used an equal volume of buffer instead of enzyme solution.Starch (dried in a convection oven to constant weight) was mixed with sodium phosphate buffer , at a ratio of typically 40% w/v in a 50 mL round bottomed flask and gelatinised while mixed at 90°C, for approximately 1 hour, followed by cooling to 50°C. 0.5 mL of lipolase solution (lipase from 2O (3.6 mL) at 30°C for one hour, followed by measuring the pH, and further treating with 1.4 mL 1.0 M NaOH at 50°C for 48 hours [Potentiometric titration analysis was performed in an automatic titrator . The substituted starch (300 mg) was treated with H48 hours . The solAnhydrous methanol and sodium methoxide in methanol solution were obtained from Sigma-Aldrich (UK). A small sample (5-30 mg) of esterified starch dissolved in 0.5 mL DMSO was mixed with 1 mL of sodium methoxide 0.07 M in methanol solution and a known amount (10 μL of a 100 mg/mL solution in n-heptane) of internal standard (n-tetradecane was suitable in the case of decanoic acid). Reagents were of anhydrous grade to minimise hydrolysis of the methyl ester as a side-reaction. This mixture was then heated (50°C) under reflux for 60 min, while shaken, then cooled and 1 mL of deionised water and 1 mL of n-heptane were added. The mixture was shaken for 1 min and left to settle. The top organic phase contained the methyl ester and could be removed and injected into the GC-FID .1H and 2D-COSY experiments were conducted at 50°C on an AVANCE/DRX500 spectrometer at 500 MHz. The starch samples were dissolved at 50°C in DMSO-d6 or DMSO-d6 : methanol-d4 (4:1) at a concentration of 10 mg/mL (DMSO-d6 and methanol-d4 were obtained from Sigma-Aldrich). The samples were incubated at 50°C for 5 min in the spectrometer, prior to analysis.AA performed all experiments, participated in all data analysis, and drafted much of the manuscript. NB and BH provided regular advice as the study progressed. SLF and PJH conceived of the study, participated in all data analysis, and drafted parts of the manuscript. All authors read and approved the final manuscript.


Introduction: Graft monitoring following vascularized composite allotransplantation (VCA) relies primarily on serial skin biopsy. However, given that VCA comprised multiple tissue types, skin biopsy may not accurately reflect rejection in other transplanted tissue.Methods: A review of the literature on episodes of both acute and chronic rejection following VCA was completed. Special attention was given to how these patients were monitored for rejection and whether skin biopsy accurately reflected the state of other tissue types within VCA.Results: Following VCA, skin biopsies accurately reflected episodes of acute rejection, but chronic rejection, resulting in both muscle fibrosis and graft vasculopathy, did not present with any ostensible skin changes.Conclusion: Various tissue types within VCA can reject at different times and rates. We define this phenomenon as “split rejection.” Split rejection has significant implications on flap monitoring, as it suggests that skin biopsy alone may not be sufficient in monitoring long-term graft rejection. Given each tissue has differing antigenicity and has different presentation mechanisms, varying tissue in VCA may elicit nonsynchronized and varying immune responses.18-24There exist only a few examples of chronic rejection following VCA. The first report is of a patient having undergone hand transplantation in 1998 who later discontinued his immunosuppressive medications.21-Petruzzo et al26In contrast, the University of Louisville has performed 6 hand transplants, with follow-up from 9 months to 12 years, all of which demonstrate some degree of graft vasculopathy not initially inferred on the basis of appearance of the graft or skin biopsy.“Split rejection” suggests that various tissues in VCA will reject at different times and rates. Evidence from previous animal models of VCA as well as recent hand VCAs performed at the University of Louisville substantiates this finding and therefore suggests a need for improved graft surveillance.-32Deep tissue biopsy may be the most reliable way to diagnose and evaluate chronic rejection, but its morbidity makes it impractical for monitoring. Serial MRI may be a way to monitor structural tissues such as bone, tendon, and muscle. Methods to evaluate graft vasculopathy may be more difficult, however, as magnetic resonance angiography fails to diagnose nascent intimal thickening in donor vessels.29