Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances. The ability to focus a laser beam tightly, to the diffraction limit, can be effectively utilized to trap microscopic dielectric particles; this was established some decades ago in pioneering experiments conducted by Ashkin and co-workers opt, is the dimensionless parameter that becomes useful in comparing the performance of different objectives. The standard multi-element microscope objective has a rear aperture that limits the size of the incident laser beam. The effect that such an aperture has on the trap stiffness remains to be investigated even though, in general, the effects of hard apertures on Gaussian beam propagation are reasonably well established For a specified laser power, the parameters that affect the trap stiffness are incident beam size and the objective's numerical aperture (NA). On the basis of ray optics calculations, prevailing wisdom is that the objective should be overfilled in order to optimize trap stiffness Here, we investigate and quantify how truncation of the incident laser beam in our optical trap affects its strength (trap stiffness). In our experiments beam truncation is achieved by utilizing apertures of various sizes to alter the laser beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. We also quantitatively probe the trap stiffness using apertures that have square cross section. Further, we measure the post-objective laser power as well as the profile of the focused beam. Our truncation method of enhancing trap stiffness is simple to implement and inexpensive; it may be of interest to many in the increasing community of users of optical traps in diverse areas of the biological and biomedical sciences.4 laser  with a 2-mm beam that was expanded to 8 mm. This beam was coupled to an inverted microscope (Nikon TE 2000U). Optical trapping was achieved by focusing 1064 nm wavelength, linearly-polarized light from the laser through a 100×oil-immersed objective  onto either a microscope slide material to be trapped or to a liquid flow cell. Details of our apparatus have been published in earlier reports pertaining to studies with red blood cells Our experimental set-up, in brief, consists of a 1 W Nd:YVO4 laser (Suwtech 2500) possessing a 2 mm beam diameter. The beam is expanded using lenses (L1 and L2) to 7.6 mm diameter . This expanded beam is then passed through a 1∶1 telescopic arrangement (L3 and L4) and is routed into an inverted microscope (Nikon TE2000-U) by reflecting through a set of mirrors . The telescope permits the laser focus plane to coincide with the microscope image plane.The experimental set-up used in the present series of measurements that we report in the following is shown schematically in Optical trapping was achieved by tightly focusing the incoming laser beam by means of a 100×oil immersed objective with a NA value of 1.3. A circular aperture (made by drilling a hole in a stainless steel plate of ∼5 mm thickness) of fixed diameter (not an iris or a diaphragm) is placed in the path of the expanded beam for truncation. In order to quantitatively probe the efficacy of the thus spatially-truncated beam vis-à-vis optical trapping, we trap polystyrene beads that have been dispersed in water and placed on a #1 cover slip mounted on a sample holder (S) fixed onto a translation stage.5 (f = 15 cm) onto a QPD (UDT SC-4D). The photodiode signals are amplified  and acquired on a 2 GHz bandwidth digital oscilloscope (Lecroy WavePro 725Zi).Trapping efficacy is quantified in terms of trap stiffness. To determine the trap stiffness we measure the power spectrum of the thermal motion of a trapped bead. For this measurement we make use of a quadrant photodiode (QPD) −1.To calibrate the QPD, a 2 µm polystyrene bead (Polysciences Inc.) is initially fixed to the cover slip and is subjected to a known linear displacement  using the translation stage (Peizosystem Jena PXY 101 CAP). The corresponding QPD signal is recorded at each position; the QPD signal is plotted as a function of linear displacement and the resulting slope yields a calibration factor. In our set-up, we obtain a calibration factor of 6 mV nmcf. Determination of cf is straightforward cf by fitting to the plotted data the function The QPD signals allow us to measure the Brownian fluctuations of a trapped bead  as a function of time. S denotes the power spectral density  are dispersed.It is pertinent to draw attention to a possible experimental constrain in such measurements. This constrain concerns the electronic instrumentation that is used. The bandwidth of the amplifier used in our power-spectrum measurements imposed a maximum sampling rate of 7 kHz. Consequently, the Nyquist theorem limits the frequency range that we can quantitatively determine to 3.5 kHz. However, this limiting frequency is much higher than typical values of corner frequency measured in our experiments; the power spectrum shown in The laser power at the trap was directly measured by an integrating sphere attached to a calibrated photodiode.The results that we present in the following were all obtained under conditions wherein polystyrene beads, of 2 µm diameter, were trapped at a height of 10 µm from the bottom of a microscope cover slip. The inset to We measured the beam profile of incident beam when it is truncated by various apertures using a beam profiler (Spiricon LBA-SCOR-20); typical profiles are plotted in 5 by a beam profiler. Initially, a 2 µm polystyrene bead was imaged to calibrate the imaging system.In order to determine the size of the beam after 100×magnification, we introduced an aperture and replaced the QPD and lens LThe transmission through the aperture was measured to be 72.8% for a 5.3 mm aperture, 68.2% for a 5.0 mm aperture, 58.7% for a 4.3 mm aperture, and 53.2% for a 3.9 mm aperture. In each case the aperture was placed in the beam path and the size of the focus spot was measured see . The ins2 value along the x axis) and 1.03  may be compared with the expected theoretical value of 1.04 µm for a Gaussian beam focused by an objective . The power within the beam's central lobe is ∼87% for a truncation factor of 0.68 et al.P = T A, where A and P are, respectively, the coefficient matrices of the incident and scattered beam.We have also carried out theoretical studies that have a bearing on the truncation-based enhancement in trap strength that has been measured by us. To this end we have utilized the computational toolbox developed by the Nieminen The optical forces result from transfer of momentum to the trapped bead in the course of scattering. By considering momentum transfer to different parts of our spherical bead, the Nieminen toolbox In our computations, a Gaussian beam with y-polarization was assumed. We followed the modification to the Nieminen toolbox described in –1; the trap stiffness at the zero-order location was 0.0038 pN nm–1. In the present series of experiments, we used two different meshes to measure the trap stiffness for the zero-order diffracted spot as a function of incident laser power. The transmission of the incident laser power was 58% for Mesh 1 (a square mesh of dimensions 178 µm×178 µm) and was 35% for Mesh 2 . The zero-order diffracted spot size measured for Mesh 1 (with a 100×objective) was 2.0 µm and an image is shown in the inset of Having established that enhancement of stiffness of a single trap is achieved simply by introducing an aperture  using a circular aperture compared to the case when no aperture is inserted. The corresponding enhancement in axial stiffness is as much as 16%. With increase in truncation the trap stiffness reduces. The important role played by effective NA in determining trap stiffness is highlighted by our results obtained when the laser beam is truncated using circular and square apertures.Our method is very simple to implement compared to other methods that use variable beam expanders. It is also the most inexpensive of possible methods.From an optics perspective our results open opportunities for further work on determining the extent and magnitude of aberrations that may be introduced in our trapping apparatus that would contribute to the enhancement of stiffness that we report here. The challenge would be to overcome the difficulties associated with identifying and quantitatively determining such aberrations. We note that the use of apertures of different sizes and shapes has started to find utility in altering lateral and axial stiffness in trap, which is likely to be of critical importance in the development of holographic tweezers


Background. H-Y antigen incompatibility adversely impacts bone marrow transplants however, the relevance of these antigens in kidney transplantation is uncertain. Three previous retrospective studies of kidney transplant databases have produced conflicting results. Methods. This study analyzed the Organ Procurement and Transplantation Network database between 1997 and 2009 using male deceased donor kidney transplant pairs in which the recipient genders were discordant. Death censored graft survival at six months, five, and ten years, treated acute rejection at six months and one year, and rates of graft failure by cause were the primary endpoints analyzed. Results. Death censored graft survival at six months was significantly worse for female recipients. Analysis of the causes of graft failure at six months revealed that the difference in death censored graft survival was due primarily to nonimmunologic graft failures. The adjusted and unadjusted death censored graft survivals at five and ten years were similar between the two genders as were the rates of immunologic graft failure. No difference in the rates of treated acute rejection at six months and one year was seen between the two genders. Conclusions. Male donor to female recipient discordance had no discernable effect on immunologically mediated kidney graft outcomes in the era of modern immunosuppression. Identifying candidate minor histocompatibility antigens that impact outcomes after transplantation is difficult, and antigens that have been identified often have disparate effects depending on the organ or tissue transplanted. One minor histocompatibility antigen system that has been identified is the H-Y antigen system. The H-Y antigen system is produced by a series of genes found on the Y chromosome of males which produce proteins with distinct amino acid sequences from their X chromosome homologs and have significant tissue expression. Seven genes have been identified on the Y chromosome that posses these characteristics, and these antigens constitute the H-Y antigen system –4. OrganThe evidence for an effect of the H-Y antigen system in renal transplantation is less certain. The expression of these antigens on the surface of human kidney tissue is not well studied, so it is not clear if target H-Y alloantigens are expressed on kidney tissue. Antibody formation to H-Y antigens has been demonstrated, and in one small study female recipients of male kidneys who had detectable antibodies to H-Y antigens had a higher rate of acute rejection . RegistrSince donor quality is an important factor determining both short- and long-term outcomes in kidney transplantation, and the covariates used in modeling do not always capture all the differences, a paired cohort analysis of the OPTN database was undertaken in order to better analyze the impact of the donor recipient gender discordance on kidney transplant outcomes. Because death censored graft failure as an endpoint includes graft failures due to both immunologic and nonimmunologic causes, this paired study includes a more detailed analysis of the causes of graft failure and also an analysis of rejection rates reported to the registry. The primary graft outcomes analyzed were death censored graft survival at six months, five years, and ten years, treated acute rejection rate at six months and one year, and rates of graft failure at six months and from six months onward by putative etiology coded in the database. All male deceased donor kidneys from January 1, 1997 to November 27, 2009 in whom the recipient of each pair of kidneys was discordant for recipient gender, were selected from the OPTN database . Only reThe etiology of graft failure was determined from two data fields. One was the numerically coded cause of graft failure which included the following categories: hyperacute rejection, acute rejection, primary nonfunction, thrombosis, infection, surgical complication, urologic complication, recurrent disease, chronic rejection, or other. The second field used was a write-in field. For those graft failures coded as other, the write-in field was queried, and based on the documentation two additional causes were included: noncompliance and polyoma-virus-associated nephropathy. Write-in causes of graft failure not included in the list were coded as other. Also, if the query matched one of the numerically coded groups, the cause of graft loss was changed to that category. Graft losses without coding in either field were considered unknown. Recipients who died during followup and whose graft survival time was equal to their patient survival time were classified as death with graft function.Cox proportional hazard ratio survival analysis was carried out to determine the independent association of graft failure with donor recipient gender match . The following covariates were included in the model : recipient age, race/ethnicity , and body mass index (BMI), donor: recipient cytomegalovirus (CMV) serologic status  delayed graft function (DGF) , duration of dialysis prior to transplant, history of previous transplant, cold ischemia time, human leukocyte antigen mismatch, type of induction , maintenance immunosuppression at discharge, and panel reactive antibodies (PRA). Statistical significance for categorical variables was determined using the Pearson's chi-squared test and Fisher's exact test. ANOVA testing was used to determine statistical significance between scalar variables. Log rank testing was performed to determine statistical significance for Kaplan Meier survival analysis. Cox analysis included correlation due to the match pairs using the robust sandwich estimate of the covariance matrix specifying the linked recipient-donor pairs . A probaBetween January 1, 1997 and November 27, 2009, 12,479 male donors were identified in which both kidneys were transplanted as single kidney transplants in two recipients who were discordant for gender. The characteristics of the pairs are shown in P = 0.013). The survival curves show that the decrement in survival occurs in the first 30 days. The slopes of the curves after 30 days are nearly identical. Adjusting for the differences in the two populations, female recipients of male kidney had a statistically significant worse outcome when compared to their paired male recipient (Hazard Ratio 1.28 95% C.I. 1.12–1.46). This analysis suggests that the male donor to female recipient gender discordance does not have a significant impact on immunologic graft outcomes in the current era of immunosuppression. Our analysis is consistent with the finding from the previous analysis of the OPTN database by Kim and Gill that short-term graft outcomes were worse among female recipients of male kidneys when compared to male recipients while the long-term graft outcomes were not. Our analysis goes further to show that the difference in short-term outcomes occurs very early after transplant and is driven not by immunologically mediated graft failures but by a higher rate of graft thrombosis and graft failure due to other nonimmunologic causes. Similarly, our analysis did not show a difference in the rates in early acute rejection or late graft failures due to acute or chronic rejection consistent with the conclusion that for the vast majority of female recipients of male kidneys receiving modern immunosuppression the H-Y antigen incompatibility did not increase the risk of rejection or graft failure. The strength of this study is that it better controls for donor quality and assessed not only death censored graft survival but also the rates of rejection and graft failure by cause. The study has a number of caveats regarding the conclusions. First, the study is limited by the accuracy of the registry data regarding the coding of the cause of graft failure and episodes of rejection. Over 95% of case did have a specified code for cause of graft failure, and the number of cases coded as unknown was similar between the two groups. Determining causality of graft failure can be difficult and depends on medical sophistication of the person entering the data. That being said, there is no reason to believe that the gender of the recipient would bias the presumptive cause of graft failure entered even if the cause of graft failure was incorrect. Second, 28% and 35% of cases, respectively, lacked coding for treated acute rejection at six months and one year. For each time point, the percentage of missing data for acute rejection was similar between the two genders. Although the rates of rejection were similar in the patients in whom coding was present, it is possible that the rejection rates could be different if the missing data were present, and as a result the strength of the conclusion from the acute rejection data must be interpreted in that context. On the other hand, the data from the acute rejection rates is concordant with the findings regarding the rate of immunologic graft loss in this population. This study also demonstrates the shortcomings of using death censored graft failure as a surrogate marker for immunologic failure of the graft. Short-term differences in outcomes previously demonstrated in the analysis of the OPTN database appear by our analysis to be due to nonimmunologic graft failures, mainly due to vascular thrombosis and other nonimmunologic causes of renal failure like acute tubular necrosis due to sepsis. In fact, in the present era of immunosuppression, early graft failure in the first six months due to rejection is a rare event occurring in approximately 1% of transplants in this analysis. In contrast, death with functioning graft occurred in approximately 3% of recipients and vascular thrombosis or primary nonfunction in approximately 1%, respectively. The finding of a higher rate of thrombosis among female recipients was unexpected but is biologically plausible given that estrogen is a prothrombotic hormone and females are more likely to have conditions such as antiphospholipid syndrome or lupus erythematous which are associated coagulation abnormalities that increase the risk of thrombosis.Why H-Y incompatibility did not affect outcomes remains speculative. There are a number of possibilities why one tissue or organ may be more or less susceptible to immunologic attack. One potential explanation for the lack of effect is that H-Y antigen expression on human kidney tissue is absent or weak. There are no published studies of H-Y antigen expression on human kidney tissue. Second, even if the H-Y antigens are expressed on kidneys, the use of potent immunosuppression may prevent kidney rejection in the majority of cases limiting its impact on outcomes. Finally, it is possible that sensitization through previous exposure to the H-Y antigens by pregnancy or previous transplant is necessary before an effect would be seen. One study indicated that the presence of antibodies to H-Y antigens predicted a higher rate of rejection . In our In summary, this analysis of the OPTN database indicates that the male donor to female recipient gender discordance has minimal if any impact on immunologic kidney graft outcomes in the modern era of immunosuppression. Female recipients of male donor kidneys had neither an increased rates of acute rejection in the first year after transplant nor increased risk of graft failure related to rejection.


Dr. Murthy J. R. KollurithOctober, 1939 to 25thFebruary, 2011)(From 19The eminent chest physician, Dr. K. J. R. Murthy, was a doyen of respiratory medicine and a pillar of the Indian Chest Society. He has made a huge contribution in the field of tuberculosis.He obtained his MBBS from Gandhi Medical College, Hyderabad, in 1961 and did his Post Graduation in Internal Medicine from Guntur Medical College, Guntur, in 1967. After acquiring the postgraduate degree, he immediately joined as Assistant Professor of Medicine at Osmania Medical College, Hyderabad. After serving there for 12 years, he moved to Bhagwan Mahaveer Hospital and Research Centre, Hyderabad, as a Consultant Chest Physician and subsequently became its Medical Director in 2005. He worked at the same institution as Director, Mahaveer PPM DOTS, till his last days.“Lung India” and was one of the founding members of Indian Chest Society.He distinguished himself as a popular teacher, good administrator and an excellent clinician. He was associated with several operational research studies for the control of tuberculosis. Dr. Murthy was member of A.P.I., A.C.C.P., and TB Association of India. He was in the Editorial Board of He was recipient of several awards including Mahaveer Mahatma Award 2005, and conducted research projects funded by WHO, Overseas Development Agency UK, University of Newcastle UK, and Government of India. He was involved in many community welfare activities.Dr. Murthy has to his credit 126 publications in national and international journals, 115 presentations and many a contributions to books and updates. He was invited as a guest speaker to almost all countries of the world and acted as Panelist and Session Chairman in many world congresses.In view of his tremendous contributions to various organizations and to Indian Chest Society, he was highly respected as an elder statesman and his invaluable advice was sought on various occasions. His knowledge regarding constitutional intricacies was exemplary. May God Almighty give eternal peace to the departed soul!Lung India Editorial Board deeply regrets this irremediable loss and expresses sincere regards to grieving family, relatives and colleagues.We are grateful to Prof. S. R. Kamat, Former Head of Respiratory Medicine at the King Edward Memorial Hospital, Mumbai, for providing material and inspiring us to get this document prepared. The obituary was prepared with courtesy of Prof. U. S. Mathur, Former Head of Division of Allergy and Pulmonary Medicine, SMS Medical College, Jaipur, and Prof. Sundaram Challa, Head of Department of Pathology, Nizams’ Institute of Medical Sciences, Hyderabad.


The Association of Medical Physicists of India (AMPI), now 30 years young, was founded in the year 1976 with the objective of promoting the application of physics in medicine and biology. It provides a forum for medical physicists in the form of annual conferences  and publication of a quarterly Journal of Medical Physics for the exchange of ideas and dissemination of knowledge. The host of the AMPI Conference is decided by the general body of the association on receipt of the formal request from the head of the host institution. The association and the host institution are jointly organizing the conference.th annual conference of the association is being organised at Bhubaneswar, Orissa during 9-12 November 2006. This is also the seventh International Conference on Medical Physics (ICMP) in India. The host of this conference, the Hemalata Hospitals and Research Centre, Bhubaneswar is equipped with all the adequate facilities for the treatment of cancer. It is pleasure to know that this hospital has come to this stage by the relentless efforts made by one of our medical physics colleagues, Dr. A.K. Rath who is also the Chairman of the organising committee of the conference.The 27This issue of the journal is exclusively dedicated to AMPICON 2006. About 200 abstracts were received by the organisers, which were evaluated by the scientific committee for presentation at the conference. Extended abstracts of proffered papers -both oral (63) and poster (127) - are included in this issue. The scientific programme consists of invited talks including Ramaiah Naidu Memorial oration. Ramaiah Naidu Memorial Oration (RNMO) Award was instituted by the association in 1992 in the honour of pioneer Indian medical physicist Dr. Ramaiah Naidu. The oration award consists of a citation and a silver plaque. As per the norms of AMPI, the recipient of RNMO award shall be a person of national/ international repute in the field of medical physics. The recipient of this award is selected by the Executive Committee of AMPI. There are 14 scientific sessions, including 19 invited talks, 2 guest lectures and 3 poster sessions. For the first time the scientific programme includes a debate on a topic that I am sure that all the medical physicists in radiation oncology will find it interesting. Keeping in line with the recent trends in radiation oncology three invited talks are being organised on image guided radiotherapy (IGRT) by experts from India and abroad. It is also decided to keep early morning lectures on various topics for the benefit of our young colleagues in the specialty.While going through the abstracts, the scientific committee felt that there is scope for improvements in the methodology for abstract submission, their speedy evaluation and informing the authors on decision taken. We will certainly pass on our suggestions to the association for consideration. Couple of years down the line I hope that complete process of submission to publication of abstracts should be made on-line.As a guest editor I take this opportunity to request all the authors of proffered papers to prepare the full manuscript of their good work and submit to the editorial office for publication in this journal.I also thank the members of scientific and publication committees for their full cooperation and hard work in bringing out this issue in time.


Administration of a recombinant peptide of Periostin (rPN) has recently been shown to stimulate cardiomyocyte proliferation and angiogensis after myocardial infarction (MI) in vitro and in vivo release of rPN. Administration into the pericardial cavity of pigs, following a complete occlusion of the left anterior descending artery, leads to greater induction of cardiomyocyte mitosis, increased cardiomyocyte cell cycle activity, and enhanced angiogenesis compared to direct injection of rPN alone.We investigated the ability of intrapericardial injections of drug-eluting hydrogels to deliver and prolong the release of rPN to the myocardium in a large animal model of myocardial infarction. Gelfoam is an FDA-approved hemostatic material commonly used in surgery, and is known to stimulate fibrin clot formation. We show that Gelfoam disks loaded with rPN, when implanted within the pericardium or peritoneum of mammals becomes encapsulated within a non-fibrotic fibrin-rich hydrogel, prolonging the The results of this study suggest that intrapericardial drug delivery of Gelfoam, enhanced by triggered clot formation, can be used to effectively deliver rPN to the myocardium in a clinically relevant model of myocardial infarction. The work presented here should enhance the translational potential of pharmaceutical-based strategies that must be targeted to the myocardium. Myocardial infarction (MI) results in the irreversible damage of cardiac muscle, which ultimately leads to heart failure. Current clinical strategies are mainly focused on attenuating the progressive deterioration of the remaining viable myocardium We have recently demonstrated that administration of a recombinant truncated peptide of periostin (referred to as rPN throughout this manuscript) in rats after MI increased cardiomyocyte mitosis, improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis Use of rPN for the treatment of heart disease in humans would require a system that permits the effective and targeted delivery to the myocardium. The most commonly employed strategies for delivering factors to the heart, which includes systemic, intracoronary, and intramyocardial injections, are of limited utility due to the low solubility of rPN at therapeutically relevant concentrations. In addition, without a tailored delivery vehicle, these approaches have very poor specificity for the myocardium, necessitating relatively large doses and increasing the potential for adverse reactions in neighboring normal tissue.Alternate, clinically relevant strategies that enable targeted delivery to the heart and that minimize systemic circulation are drastically needed. One such approach is the implantation of drug-eluting devices into the pericardial cavity . Previous studies in chronically ischemic pigs have shown that direct intrapericardial injection of growth factors resulted in increased myocardial deposition and retention and decreased systemic recirculation, compared to intracoronary or intravenous delivery in vivo fibrin-hydrogel formation and enable the prolonged release of rPN to the myocardium following MI.Gelfoam is an FDA-approved hemostatic medical device prepared from purified pork skin gelatin USP granules and crosslinked by a thermal treatment method in vitro and in vivo drug delivery of rPN from Gelfoam disks. We demonstrate that Gelfoam disks become encased within fibrin-rich hydrogels when implanted in the pericardial or peritoneal fluid of pigs or mice, respectively. We show that Gelfoam-induced fibrin hydrogels enable the prolonged release of rPN in vitro and within the peritoneal cavity of mice, in vivo. We also demonstrate that administration of Gelfoam disks loaded with rPN into the pericardial cavity of pigs, following a complete occlusion of the left anterior descending artery, leads to increased cardiomyocyte cell cycle activity and angiogenesis, compared to direct injection of rPN alone.In the present paper, we characterize the in vivo clot formation would enable the prolonged release of rPN when administered intrapericardially. To test whether Gelfoam disks become encapsulated within fibrin hydrogels in the absence of severe bleeding, we implanted pre-swollen Gelfoam disks (in PBS) into the peritoneum of mice. Animals were sacrificed after 2 hrs and 7 days, the devices were recovered, and histological analysis was performed . The release profile of fluorescently-labeled rPN (rPN-AF488) from Gelfoam was assessed in vitro (50t and 90t) is ∼0.5 and ∼2 hrs, respectively , compared to its diffusion through cryo-injured cardiac tissue . To further demonstrate that Gelfoam-induced fibrin hydrogels were critical for the sustained release of rPN to the myocardium, rPN alone was injected into the pericardial space of pigs after myocardial infarction. Quantification of H3P-positive cardiomyocytes on sections revealed that Gelfoam disks loaded with rPN had a 2.2-fold increase in the number of H3P-positive cardiomyocytes compared to pigs injected with rPN alone . Collectively, these data demonstrate that Gelfoam delivered to the pericardial space are useful vehicles for the sustained release of rPN to the myocardium after MI.The degree of vascularization, twelve weeks after MI, was visualized by staining tissue sections with antibodies against CD31 to identify the endothelial layer of blood vessels and with myomesin to demarcate cardiomyocytes . The degree of fibrosis was quantified using two independent techniques. First, we imaged the sections using light microscopy followed by thresholding of the fibrotic areas (stained blue) relative to the myocardial area (stained deep red), which showed no difference between the groups . Second, a pathologist (R.P.) scored the degree of fibrosis on AFOG-stained sections in a blinded manner, using a semi-quantitative scale and found it to be similar between groups . To further investigate the possibility that rPN administration may have an effect on fibroblast proliferation, we quantified the number of non-myocytes undergoing mitosis in the border zone after one week of treatment . rPN did not affect mitotic activity of non-myocytes, consistent with recent findings in periostin knockout mice The periostin gene plays a role in the recruitment of fibroblasts to the MI region and in the formation of scar tissue Figure S3D).The periostin gene has also been reported to influence reactive inflammation Identification of novel factors that regulate myocardial regeneration is an exciting new paradigm in cardiac biology In vivo implantation of Gelfoam is known to induce the formation of fibrin-rich matrices without triggering significant inflammatory or fibrotic responses. We hypothesized that this spontaneous in vivo deposition of fibrin within the interstitial sites of Gelfoam could be used as a natural retardant to prolong rPN release, increase rPN bioactivity, and decrease non-specific uptake of rPN by non-target organs.We chose to encapsulate rPN within Gelfoam disks for several reasons. First, these devices are FDA approved, which greatly enhances the clinical applicability of the proposed method. In fact, a recent pre-clinical study has shown that intrapericardial administration of Gelfoam into the pericardial space of pigs is both feasible and safe in vitro and in vivo drug release experiments. The in vitro release profile of rPN from Gelfoam disks alone was very rapid compared to rPN release from Gelfoam-fibrin composite hydrogels. These results suggest that fibrin deposition within the interstitial sites of Gelfoam disks is able to effectively modulate rPN release. We performed similar in vivo release experiments within the peritoneum of mice. Peritoneal and pericardial fluids have similar compositions, as both are ultra filtrates of the plasma in vivo release profile of rPN from Gelfoam was much slower than its in vitro release from Gelfoam alone. To determine if fibrin deposition played a critical role in modulating the release kinetics, we implanted Gelfoam disks loaded with heparin and rPN. Heparin is known to inhibit fibrin formation by activation of antithrombin and factor Xa in vivo administration of Gelfoam is critical for the extended release of rPN from these devices.To examine the role of fibrin deposition on the release kinetics of rPN from Gelfoam disks, we performed both in vivo applicability of our approach was tested by injecting our delivery systems into the pericardial space of pigs two days after MI. Histological analysis one week after injection revealed minimal inflammation, no evidence of a fibrotic capsule, and extensive fibrin(ogen) and fibrin deposition on the surface of the Gelfoam as well as within the interstitial sites, respectively. These results are similar to those observed from Gelfoam disks explanted from the peritoneal cavity of mice, and suggests that Gelfoam-induced fibrin hydrogels are present after intrapericardial administration of Gelfoam disks. We quantified PCNA and H3P-positive cardiomyocytes as well as CD31-positive vessels in order to characterize the bioactivity and the in vivo release of rPN from Gelfoam-induced fibrin hydrogels. Our results clearly indicate that Gelfoam-induced fibrin hydrogels provide a sustained release of rPN for an extended period of time. This correlates with our in vivo release profile, in which rPN was shown to be released from the Gelfoam-induced fibrin hydrogels over a period of seven days. This data also suggests that Gelfoam disks injected into the pericardial space are not degraded at an accelerated rate (i.e. they are present 7 days after implantation). It is also important to note that the therapeutic effect of rPN encapsulated within Gelfoam-induced fibrin hydrogels is superior to rPN alone. Injection of rPN into the pericardial space is most likely eliminated by either lymphatic drainage and/or proteolytic cleavage; whereas encapsulation within the Gelfoam hydrogels retains the drug within the cavity and may preserve its bioactivity The Previous studies have shown that the effects of intrapericardial administration are limited to the epicardial surface of the heart in vivo. This strategy may serve as a platform for rapid testing of new pharmaceuticals that have to be targeted to the heart. In addition, our findings may inspire the future development of advanced biomaterials that enable, for example, the spatio-temporal delivery of multiple factors over various time scales to mediate cardiac injury and to induce myocardial regeneration.Intrapericardial administration of drug eluting biomaterials is a novel, clinically attractive, and effective method for the targeted delivery of therapeutics to the myocardium after MI. We utilized the body's innate immune response (i.e. thrombus formation) to Gelfoam implantation to facilitate the prolonged release of rPN to the myocardium. The approach presented here utilizes established clinical procedures and FDA approved medical devices to enhance factor retention and bioactivity within the pericardial cavity and to prolong the biological effect All experiments in this study were conducted in accordance with the guidelines established by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine and Children's Hospital Boston and the American Association for Accreditation of Laboratory Animal Care and were approved by the Institutional Animal Care and Use Committee at MSSM and CHB. All efforts were made to minimize any pain and suffering felt by the animals.ex = 494 nm, λem = 519 nm), and the amount (in µg) of rPN-AF488 released was calculated by interpolation from a standard fluorescent calibration curve. At the end of the experiment, all Gelfoam disks were degraded and the amount of retained rPN-AF488 was determined. The cumulative release was calculated by summing the amount of rPN-AF488 released over the various time intervals. The percentage of cumulative rPN-AF488 released is reported as the ratio of the cumulative release of rPN-AF488 (in µg) at any point in time to the initial amount of rPN-AF488 (in µg) that had been loaded.Gelfoam (Pfizer) was cut into disks using a standard 6 mm biopsy punch (6 mm×2 mm). Loading was accomplished by application of an aliquot of rPN-AF488 (∼2 µg) to each Gelfoam disk followed by incubation at 4°C for 12 hrs. The volume added to each Gelfoam disk was much less than the maximal absorbable volume to ensure that the applied dose was completely loaded within each gel. Loading was determined by quantifying the amount of fluorescence in solution after degradation of at least 3 individual disks by enzymatic treatment (trypsin). For release studies, the Gelfoam disks, loaded with or without rPN-AF488, were placed within the wells of a 96-well plate containing 50 µL of phosphate-buffered saline (PBS). The plate was covered and placed at 4°C with intermittent agitation. To determine the fraction of rPN-AF488 released from the Gelfoam gels, the PBS within each well was removed at various time points and replaced with an equivalent volume of fresh buffer. The collected buffer was then analyzed on a fluorescent plate reader (λGelfoam disks encapsulated within fibrin gels were prepared as follows. Gelfoam disks (6 mm×2 mm) were pre-swollen in a stock solution of thrombin (25 U/mL) for 30 mins at room temperature. The disks were subsequently allowed to dry at 4°C for 3 days. rPN-AF488 (∼2 µg) was loaded into individual thrombin-loaded Gelfoam disks by incubation at RT for 30 min. Again, the volumes added were much smaller than the maximum swelling volume, which ensured that the applied dose was completely loaded within each gel. The thrombin/rPN-AF488 loaded Gelfoam disks were then placed into a solution of fibrinogen  for 20 mins at 37°C. The amount of rPN-AF488 loaded was determined by enzymatic degradation of at least 3 Gelfoam-fibrin hydrogels. The percentage release of rPN-AF488 was determined as described above.fX is the amount of rPN-AF488 remaining in the Gelfoam disk at time t and iX is the amount of rPN-AF488 initially loaded.Gelfoam disks (6 mm×2 mm) were pre-swollen in a stock solution of heparin (5000 U/mL) for 30 min at room temperature. The disks were subsequently allowed to dry at 4°C for 3 days. rPN-AF488 (∼2 µg) was allowed to absorb into Gelfoam disks, loaded with or without heparin, for 30 min at room temperature prior to implantation. The Gelfoam disks were subsequently implanted into the peritoneal cavity of healthy adult mice . Gelfoam disks loaded with heparin and rPN-AF488 were extracted after 30 mins, 1 hr, and 2 hrs; whereas Gelfoam disks loaded with rPN-AF488 only were explanted after 30 mins, 1 hr, 20 hrs, 48 hrs, 96 hrs, and 168 hrs. The animals were euthanized, the disks retrieved, and they were degraded in Eppendorf vials via treatment with trypsin . Vials were spun down at 14,000×g, the supernatant was added to a black, 96-well flat bottom plate, and the fluorescence was quantified using a standard fluorescent plate reader. The amount of rPN-AF488 loaded was determined by enzymatic degradation of at least 3 Gelfoam-heparin disks (not implanted). The amount of rPN-AF488 retained within the disk (µg) was determined as described above. The percent rPN-AF488 released was calculated according to the following equation:Diffusion of rPN into the myocardium was evaluated ex vivo using fluorescently-labeled rPN. Adult mouse hearts (CB57/SL6) were isolated using standard techniques and immediately placed in ice cold PBS to flush out the ventricles. Hearts were glued to the bottom of Petri dishes (35 mm) with the left ventricle pointing up. Hearts then received no injury/Gelfoam/rPN-AF488 (control), cryo injury/Gelfoam/rPN-AF488 (3 independent 5 second exposures over the same region using a 3 mm probe) or cryo injury only (same exposure). Gelfoam sponges loaded with rPN-AF488 (0.84 µg) were glued to the surface of the heart. In the case of the cryo-injured hearts, care was taken to ensure that the Gelfoam was directly over the injured area. Culture media (containing 10% FBS) was then added to the Petri dishes and the plates incubated at 37°C in 5% CO2 for 2 hours. The hearts were then carefully released from the dishes using a razor blade, rinsed in ice-cold PBS, and immediately embedded in OCT. Detailed information on the histological analysis and quantification of rPN-488 diffusion is provided in the File S1.All manipulations were approved by the IACUC and performed at Mount Sinai School of Medicine (MSSM). In order to enhance the clinical applicability of this delivery system, Gelfoam disks were milled using a standard bone rasp, which enabled administration via injection through standard catheters Heart samples were embedded in optimal cutting temperature compound (OCT) and placed at −80°C. We prepared cryosections (14 µm thick), fixed them in 10% buffered formalin, permeabilized with 0.5% NP-40 dissolved in phosphate buffered saline (PBS), and blocked with goat serum . Sections were subsequently treated with primary antibodies against PCNA (Abcam), Ki67 (Abcam) and phosphorylated histone-3  to visualize cardiomyocytes in the cell cycle, troponin T, tropomyosin, and myomesin (Abcam) to visualize cardiomyocyte-specific proteins in the contractile apparatus, CD31 (Abcam) to visualize the presence of blood vessels, and fibrinogen to visualize the presence of fibrin/fibrinogen  within Gelfoam disks. Sections were subsequently treated with secondary antibodies conjugated to either Alexa 488 or 594. Nuclei were stained with DAPI. To quantify the number of PCNA and H3P positive cardiomyocyte nuclei per millimeter squared of myocardium we analyzed 3 different hearts per group. For each heart we randomly selected and counted the number of PCNA or H3P positive cardiomyocyte events from 6 slides containing 3–4 sections per slide (only 1 section/slide was quantified). The area of each tissue section was determined using the method of point counts Gelfoam disks explanted from the peritoneal cavity of mice were harvested and immediately fixed in 3.7% formaldehyde overnight. Samples were either frozen at −80°C or embedded in optimal cutting temperature. Gelfoam disks were cut into 10 µm sections and re-fixed in 10% buffered formalin. Gelfoam disks explanted from the pericardial cavity of pigs were harvested and either embedded in OCT compound or snap frozen in liquid nitrogen. Samples were cut into 14 µm sections and fixed in 10% buffered formalin. Sections were stained with hematoxylin and eosin to visualize inflammation and acid fuchsin orange-G (AFOG) to visualize fibrin deposition. Images were acquired using a Zeiss Axioplan 2 epifluorescence microscope with Plan-Neofluar 10× and 40× lenses equipped with a Zeiss Axiocam CCD camera.n = 8 hearts.Frozen samples of myocardial tissue were brought to room temperature and immediately fixed in 10% buffered formalin. The tissue was subsequently incubated with collagenase D  and B  for 24 hours at 37°C with constant agitation. The supernatant was collected and spun down to yield the isolated cardiomyocytes. This procedure was repeated until no more cardiomyocytes were dissociated from the tissue. The cardiomyocytes were stained for the presence of cell cycle markers as described in the Immunofluorescence section. The efficiency of the isolation procedure was 91.3±3% (by mass) and the percent intactness was 92.4±0.4% (determined by cadherin staining). The percents are given as mean ± SEM, One-way ANOVA was used to analyze myocardial fibrosis and cell cycle data. Two-tailed Student's t-test was used for vascular density and non-cardiomyocyte proliferation analyses. All measurements were reported as the mean ± SEM. Significance was accepted at p<0.05. Number of animals in pig experiments: 6 control (Gelfoam only) and 7 treatment (Gelfoam/rPN). Number of animals in mouse experiments: 3 control (Gelfoam only) and 3 treatment (Gelfoam/rPN-AF488).File S1Supplemental information is available for the following: synthesis and characterization of rPN-AF488, quantification of non-myocyte cell cycle activity, and quantification of myocardial fibrosis and inflammation.(DOCX)Click here for additional data file.Figure S1Synthesis and characterization of rPN-AF488. (A) Schematic comparing the protein structure of the type 1 variant of human periostin with the recombinant truncated peptide used in this study. The recombinant PN peptide is truncated from amino acid 1–22 and from 672–836. (B) Schematic representation of the bio-conjugation technique used to fluorescently-label rPN with Alexa Fluor 488. (C) SDS-PAGE analysis of rPN-AF488 and rPN (control).(TIF)Click here for additional data file.Figure S2Administration of Gelfoam loaded with rPN does not stimulate non-cardiomyocyte cell cycle activity (A) Photomicrograph of a H3P positive non-cardiomyocyte within the infarct border zone. (B) Quantification of non-cardiomyocyte mitosis after 1 wk of treatment. Ctr., control hearts receiving Gelfoam with buffer; rPN, hearts receiving Gelfoam with periostin peptide; n is the number of animals per group. Scale bars, 100 µm.(TIF)Click here for additional data file.Figure S3Delivery of Gelfoam loaded with rPN does not alter myocardial fibrosis or inflammation in the infarct border zone. (A–C) Visualization and quantification of myocardial fibrosis from tissue sections stained with acid fuchsin orange G (AFOG). Fibrosis was determined both quantitatively using an image analysis software (B) and semi-quantitatively by a pathologist in a blinded manner (C). (D) Visualization of myocardial inflammation using Hematoxylin and eosin staining (H&E). (E) Quantification of myocardial inflammation was performed in a blinded manner by a pathologist. Ctr., control hearts receiving Gelfoam with buffer; rPN, hearts receiving Gelfoam with periostin peptide; n is the number of animals per group. Scale bars, 100 µm.(TIF)Click here for additional data file.


PLOS Medicine's Chief Editor looks back at some highlights in the way the journal has sought to constructively disrupt medical publishing over its history.Please see later in the article for the Editors' Summary PLOS Medicine is from a tremendously skilled lawyer who has held our hands through some difficult times:The phrase that best sums up my time at “I'm always happy to help provide guidance on how to stir up trouble without getting into too much yourself.”Is that too disruptive? On the contrary—the history of PLOS is of an innovative organization started by people with a vision of changing publishing, who employed staff who shared that vision, who themselves also understood the need for disruption as well as the pragmatic realities of publishing  to make that vision a reality. Many times over the following years we have debated our purpose and strategy and concluded with certainty that the best way to innovate in publishing was to challenge assumptions head on, to take the debate to the field.We have published many excellent research and magazine articles since the journal began, but some of the articles I am most proud of exemplify that disruption and are not the obvious fare of medical journals. Among these are papers on: why lethal injection is not a humane method of killing, as it probably asphyxiates prisoners—which led to lethal injection being ruled unconstitutional in the state of Tennessee 10.1371/journal.pmed.0040156.Zimmers TA, et al. (2007) Lethal Injection for Execution: Chemical Asphyxiation? PLoS Med 4(4): e156. doi:10.1371/journal.pmed.0050243.Hicks MH-R, et al. (2008) The Dirty War Index: A Public Health and Human Rights Tool for Examining and Monitoring Armed Conflict Outcomes. PLoS Med 5(12): e243. doi:Fugh-Berman AJ (2010) The Haunting of Medical Journals: How Ghostwriting Sold “HRT”. PLoS Med 7(9): e1000335. doi:10.1371/journal.pmed.1000335.http://www.plosmedicine.org/static/ghostwritingThe Wyeth Ghostwriting Archive: Stuckler D, et al. (2012) Big Food, Food Systems, and Global Health. PLoS Med 9(6): e1001242. doi:10.1371/journal.pmed.1001242PLOS Medicine Series on Big Food: http://www.ploscollections.org/article/browse/issue/info%3Adoi%2F10.1371%2Fissue.pcol.v07.i17The 10.1371/journal.pmed.1000431Kesselheim AS, Mello MM, Studdert DM (2011) Strategies and Practices in Off-Label Marketing of Pharmaceuticals: A Retrospective Analysis of Whistleblower Complaints. PLoS Med 8(4): e1000431. doi:10.1371/journal.pmed.1001431McCambridge J, Hawkins B, Holden C (2013) Industry Use of Evidence to Influence Alcohol Policy: A Case Study of Submissions to the 2008 Scottish Government Consultation. PLoS Med 10(4): e1001431. doi:10.1371/journal.pmed.1001249Weishaar H, Collin J, Smith K, Grüning T, Mandal S, et al. (2012) Global Health Governance and the Commercial Sector: A Documentary Analysis of Tobacco Company Strategies to Influence the WHO Framework Convention on Tobacco Control. PLoS Med 9(6): e1001249. doi:PLOS Medicine, PLOS Neglected Tropical Diseases, and PLOS Pathogens) but no day-to-day input into content. I very much look forward to engaging with medical publishing in a new part of the world and will continue to blog, tweet, and agitate from there. I'm delighted to say that my successor will be Larry Peiperl, who is currently a Clinical Professor at UCSF School of Medicine. He previously worked at PLOS Medicine and was the journal's first Senior Research Editor.I'm now moving to Australia and as a result felt that the time was right to step down as Chief Editor. However, I am remaining with PLOS, in the other post I also hold as Medicine Editorial Director. In this position, I will have oversight of the medical journals (PLOS Medicine is in rude (translation: excellent) health as I hand it on. This is undoubtedly due to the tremendously talented editors and editorial staff at the journal along with the staff working on other teams within PLOS, as well as our many academic editors, reviewers, and authors. Looking back, I'm gratified to remember how many of these individuals grasped our purpose immediately, supported us enthusiastically, and berated us  when we fell short. I hope they continue to do all three.Have we succeeded overall? I like to think so.But this isn't the time to be complacent. Medical publishing, like publishing in general, still has many issues to address, and how these play out over next few years will, I believe, determine the place journals have in the wider dissemination of medical knowledge and, more importantly, its translation into practice and policy.PLOS Medicine was founded; its acceptance, although not without opposition, has gained substantial and sustainable traction. Undoubtedly in some cases there remain practical obstacles to implementation, but the remaining opposition in the industry to the principle of OA to the medical literature is a throwback to the dark days when restricting access was the norm.First, Open Access (OA) to the medical literature has to become universal. By OA I mean, of course, free, immediate access that also allows human and machine reuse without restriction, ensuring that authors are credited. This was the principle on which Second, the tying of the medical literature to online formats that essentially replicate the limitations of print medium has to change. Journals need to learn from the rest of the Web, where communication has been revolutionized. Articles need to be allowed to evolve, to have new versions, to reflect the dynamic web within which that they now exist. Articles need to be evaluated on their own merit by the most relevant standards. The paper on lethal injection PLOS Medicine's stance of not taking ads for drugs or devices has allowed us extraordinary freedom. Other journals that continue to rely on funding from these industries cannot lead the debate on what is best for the public's health, such as the imperative to register and report all clinical trials Third, medical journals need to address the inherent conflicts in their way of doing business with the pharmaceutical and associated industries. PLOS Medicine have been a rollercoaster ride, and my only prediction is that my successor will have an equally exciting time. I'm looking forward to watching the ride.I don't think there's ever been an uninteresting or predictable time in medical publishing. The almost 10 years that I have been at


Shear wave elastography is a new ultrasound technique which shows promise in the differential diagnosis of solid breast masses. This study compares the mean tumour stiffness with mammographic findings and mammographic density in women with invasive breast cancer.Mammographic morphological features and BIRADS density assessment of the contralateral breast were documented in 96 consecutive patients with operable invasive breast cancer who had undergone shear wave elastography. Mammographic assessment was performed blinded to the elastography and pathological findings. Mean stiffness values of individual tumours were classified as either above or below the average mean stiffness of the cancers. Chi-squared tests for trend were used to compare mean stiffness with mammographic features and density.There was no statistically significant difference in the mean stiffness of tumours according to their mammographic features. Mean tumoural stiffness increased with breast density , with a statistically significant trend of stiffness being above average for a cancer in a denser breast. No statistically significant correlation was shown between tumour size or grade (factors known to be associated with increased stiffness) and mammographic density.Tissue stiffness of breast cancers is greater in women with mammographically denser breasts. Assuming elastographic stiffness reflects characteristics of stroma and tumour, our findings suggest that tumour-stroma interactions may vary with mammographic density.


Journal of Radiation Research (JRR) has served as an official journal for the scientific communication of the Japan Radiation Research Society (JRRS) for more than 50 years. Partnered with the Japanese Society for Therapeutic Radiology and Oncology (JASTRO) since 2009, JRR has entered into a new phase of growth over multidisciplinary radiation research fields including biology, oncology, chemistry, physics, epidemiology and environmental sciences. Since 2012, JRR has shifted to a new partnership with Oxford University Press, which has the potential to be successful in the coming years. JRR ended 2013 with more than 340 new submissions compared to previous years.The The quality and impact of JRR are recognized throughout the multidisciplinary fields in radiation research. We have been providing an international forum for radiation scientists, but this trend has been changing in recent years in some statistics. JRR reached its highest impact factor of 2.034 in 2009, but it declined to 1.447 in 2012.As I step in as a new Editor-in-Chief from 2014, I would firstly like to aim to further international influence. The continuous high rate of papers from outside of Japan is indeed pleasure for us, but we would also like to pursue increased number of high quality papers. In order to achieve that, we hope to keep working on smoother review process to make prompt decisions to benefit authors and to keep up with the international standard for this discipline.Journal of Radiation Research.Finally, I would like to thank previous EIC, Yoshiya Furusawa, who has contributed immensely for the development of the journal. I would hereby like to express my gratitude to all personnel for their continuous support and would like to ask for your further cooperation to improve the quality and expand the international prominence of the


To compare the transmural extent of hyperintensity (“edema”) on T2-weighted (T2W) CMR to the transmural extent of infarction by delayed-enhancement(DE)-CMR.It has been reported that hyperintensity on T2W-CMR represents the area-at-risk. This conclusion is based upon the observation that the region of T2-hyperintensity is always larger than region of infarction. However, differences in measured size may be due to many factors, including the choice of different image intensity thresholds :547-9.). Additionally, basic physiology studies have established that the area-at-risk is not simply larger than the region of infarction, but is nearly always transmural regardless of the transmurality of infarct size. The relationship between the transmural extent of T2-hyperintensity and the transmural extent of infarction is unknown.34 canines underwent coronary occlusion of the LAD or the LCx followed by reperfusion. Occlusion time was varied between 45-120 minutes to produce a variable TEI. T2W-TSE and DE-CMR were performed at median of 3 days post MI. A standard T2W-TSE pulse sequence was used with vender supplied coil normalization  to obtain short-axis slices of the LV. T2W-TSE and DE-CMR images were separately scored on a per-infarct basis (blinded to the other technique) for the presence/absence of transmural T2-hyperintensity or transmural hyperenhancement, respectively, at any location within the heart. Additionally, the T2W and DE images were compared using a 6-point scale  to determine the relative transmural extents.On a per-infarct basis, 59% (20/34) subjects were scored to have transmural T2-hyperintensity, which was similar to the proportion with transmural hyperenhancement 56%, 19/34). On a per-slice basis (n=244), transmural T2-hyperintensity at any location was found in 30.3% of slices, which was similar to the proportion of transmural hyperenhancement (32.0%, p=0.53. Table 9/34. On T2-hyperintensity is frequently not transmural and is highly associated with the transmural extent of infarction. This is problematic for the use of T2W-TSE as a representation of the area-at-risk.


AtISA1 and AtISA2) with the monocotyledonous rice (Oryza sativa) isoforms. We demonstrate that the ISA1 and ISA2 are sufficiently well conserved between these species to form heteromultimeric chimeric Arabidopsis/rice isoamylase enzymes. Furthermore, we were able to reconstitute the endosperm-specific rice OsISA1 homomultimeric complex in Arabidopsis isa1isa2 mutants. This homomultimer was able to facilitate normal rates of starch synthesis. The resulting amylopectin structure had small but significant differences in comparison to wild-type Arabidopsis amylopectin. This suggests that ISA1 and ISA2 have a conserved function between plant species with a major role in facilitating the crystallization of pre-amylopectin synthesized by starch synthases and branching enzymes, but also influencing the final structure of amylopectin.This study tested the interchangeability of enzymes in starch metabolism between dicotyledonous and monocotyledonous plant species. Amylopectin - a branched glucose polymer - is the major component of starch and is responsible for its semi-crystalline property. Plants synthesize starch with distinct amylopectin structures, varying between species and tissues. The structure determines starch properties, an important characteristic for cooking and nutrition, and for the industrial uses of starch. Amylopectin synthesis involves at least three enzyme classes: starch synthases, branching enzymes and debranching enzymes. For all three classes, several enzyme isoforms have been identified. However, it is not clear which enzyme(s) are responsible for the large diversity of amylopectin structures. Here, we tested whether the specificities of the debranching enzymes (ISA1 and ISA2) are major determinants of species-dependent differences in amylopectin structure by replacing the dicotyledonous Arabidopsis isoamylases ( Starch is the major storage carbohydrate in plants and an important renewable resource for both the food and non-food industry sectors. Starch is comprised of two glucose polymers (amylopectin and amylose) and accumulates in plant tissues as semi-crystalline granules. Amylopectin accounts for the majority of the granule mass . It is a branched molecule in which α-1,4-linked glucan chains are connected via α-1,6-bonds The higher-order structures adopted by amylopectin are thought to occur in all wild-type starches and underlie the water-insoluble granular characteristics of starch. Nevertheless, there is considerable variation in starch granule morphology, structure and composition between plant sources. These factors are important due to the impact they have on starch properties, which are relevant for downstream functional applications. A detailed understanding of how different biosynthetic enzymes influence amylopectin structure has been hindered by the fact that it is currently not possible to determine the exact structure of amylopectin. Also, we can only partially relate the physical properties of starch to its structure, and hence it is difficult to predictably control these characteristics through manipulation of enzyme abundance and/or specificities.Chlamydomonas reinhardtii: C. reinhardtiiStarch synthesis is mediated by three enzyme classes. Starch synthases extend α-1,4-linked chains by transferring new glucose units from ADP-glucose to the non-reducing end. Branch points are introduced by branching enzymes, which cleave an existing α-1,4-bond of a linear chain and transfer the cut end to another chain, creating an α-1,6-bond. Both starch synthases and branching enzymes exist as multiple isoforms, thought to have different specificities ISA1 or ISA2 gene expression has been reduced or abolished. The loss of ISA1 results in a reduction of granular starch in endosperms of maize C. reinhardtii cells ISA2 is more variable. In endosperms of maize and rice, loss of ISA2 had no measurable effect, explained by the fact that these tissues contain still the active ISA1 homomultimeric complexes The role of these isoamylase complexes in starch biosynthesis has been determined through phenotypic analysis in a range of plant species and tissues in which AtISA1 and AtISA2) with the respective monocot rice isoforms (OsISA1 and OsISA2). Our data show that active heteromultimeric complexes can be formed between AtISA1 and OsISA2 as well as AtISA2 and OsISA1. Additionally, we were able to reconstitute the OsISA1 homomultimeric complex as found in rice endosperm. The resulting transgenic lines synthesize comparable amounts of starch to wild-type Arabidopsis plants. This illustrates that rice isoforms can replace the endogenous Arabidopsis proteins. Furthermore, in addition to playing a major role in facilitating starch biosynthesis, differences in the final structure of amylopectin between the transgenic lines suggests that the isoamylases enzymes composed of ISA1 and ISA2 subunits also contribute to the species-specific differences in amylopectin structure.In this study, we wanted to address three hypotheses arising from our current knowledge. First, are differences in starch structure between different plant species due to different catalytic specificities of ISA1 and ISA2 protein complexes? Second, are the functions between the ISA complexes from different plant species conserved? Third, are the subunits of the ISA complexes adequately conserved such that they are interchangeable between species? To answer these questions we replaced the endogenous ISA proteins in the dicot Arabidopsis , the reported amylopectin structures differ considerably between different plant species and tissues In the CLDs of leaf starch from all four species, chains with a degree of polymerisation (d.p.) of 12 were most abundant, but the profiles differed in other ways. The CLD of potato, had a prominent peak of chains of d.p. 6 and d.p. 7 , which wISA1 and ISA2 have been characterised, together with the double mutant We investigated the role of the isoamylases (ISA1 and ISA2) in determining the starch structure between plants using a gene-swap experiment. We chose the rice, which had the largest difference in starch structure compared to Arabidopsis . The latOsISA1 and OsISA2 into the pB7WG2 plant Gateway-compatible expression vector OsISA2 was expressed in the Arabidopsis isa2 single mutant under the cauliflower mosaic virus (CaMV) 35S promoter. The OsISA1 construct was transformed into the Arabidopsis isa1 single mutant and the isa1isa2 double mutants. In contrast to OsISA2, which can only form a heteromultimeric complex with OsISA1, the OsISA1 protein is reported to also form a homomultimeric complex OsISA1 might be able to complement both of the missing Arabidopsis proteins.We cloned the protein-coding region of 1 generation. We then screened these lines by iodine staining to search for those in which the loss of the endogenous isoamylase activity was complemented by the expression of the rice gene. Half or more of the T1 transformants stained like the wild type, suggesting complementation. We confirmed that this phenotype was stable in the T2 generation of the three most promising candidate lines per genotype . Comparable results were also recently described for these isa mutants transformed with the Maize ISA1 gene Iodine staining is suitable for rapid screening but is largely qualitative. Therefore we measured the starch and phytoglycogen in all of the transformed lines at the end of a 12-h photoperiod. As expected, wild-type plants had around 8 mg starch per gram plant fresh weight and contained only trace amounts of soluble glucan. In contrast, the ild type . All thrstaining  as they isa2-35S::OsISA2 lines showed a marked change in phenotype compared with the parental line, but complementation was incomplete. Although all three lines contained only starch, with little or no phytoglycogen, the starch content varied significantly between the lines , whereas isa2-35S::OsISA2 C contained almost wild-type amounts of starch.The three he lines . The linisa1, isa2 and isa1isa2 lacked the debranching activity found in wild-type plants  while the third ran at the same height as the endogenous Arabidopsis ISA1/ISA2 complex  were the same, having more very short chains (d.p. 4 to 7) but less medium-length chains (d.p. 10 to 16) compared to the wild type (only isa1isa2 is shown). We compared the amylopectin CLDs of the starch synthesized in our Arabidopsis mutants complemented with the rice isoamylases to the wild type, to the isa1isa2 double mutant, and to the rice starches. For each transformation, the three independent lines had very similar CLD profiles . Consistent with our observations, early studies in which the homomultimer was purified to homogeneity from rice endosperm also yielded an enzyme which migrated in several positions on a native gel and in two positions during isoelectric focusing In this work, we demonstrated the interchangeability of enzymes in starch metabolism between monocotyledonous and dicotyledonous plants. Arabidopsis mutants lacking the ISA1/ISA2 complex have reduced capacity to synthesise crystalline starch - instead they accumulate water-soluble phytoglycogen. Here, we show that the rice isoamylase proteins are able to rescue this phenotype. All the transgenic lines analysed contained exclusively starch , even thomplexes . In thesisa1 lines expressing OsISA1, we observed an additional enzymatic activity besides the two homomultimeric OsISA1 complexes . However, there is residual AtISA1 protein in the isa2 mutants, which was comparable in lines overexpressing OsISA2. This might mean that there is sufficient protein to partner OsISA2. It is also possible that a chimeric complex is formed which has an altered migration such that it co-migrates with another starch degrading activity and is therefore masked. Alternatively, the chimeric enzyme may not resolve well under our experimental conditions or may be very labile during extraction.In the  mutants . It is pAtISA1/OsISA2 isoamylase complex may be present at a very low abundance in our isa2-35S::OsISA2 plants would suggest that extremely low levels of a functional isoamylase complex are sufficient to facilitate starch synthesis. Although all three lines had a similar starch structure it is worth noting that the starch level was not the same as in the wild type (StISA1 or StISA2 were silenced. Transgenic lines were identified which showed no detectable isoamylase activity on native PAGE (as used here) but still accumulated almost exclusively starch and only tiny amounts of phytoglycogen The idea that a chimeric ild type . The facThe fact that the rice proteins were able to functionally replace the Arabidopsis proteins does not necessarily mean that the converse is true, and that the Arabidopsis proteins would be able to complement the phenotypes of the corresponding rice mutants. This may be especially the case for ISA1, because Arabidopsis ISA1 seems unable to form an active homomultimer. This may be important as it is specifically the ISA1 homomultimer that is thought to facilitate starch synthesis in the rice endosperm isa1isa2 mutant CLD (compared to wild-type Arabidopsis amylopectin) was lost. Instead an increased abundance of longer chains between d.p. 6 and d.p. 14 was seen. This structure is somewhat intermediate between Arabidopsis and rice transient starch. In the isa1-35S::OsISA1 lines the amylopectin CLD was much more similar to wild-type Arabidopsis amylopectin, but the same tendencies towards increased number of chains between d.p. 6 and d.p. 14 was seen. In this line, the homomultimeric OsISA1 is accompanied by a chimeric Arabidopsis/rice isoamylase complex - OsISA1/AtISA2. This chimeric enzyme presumably functions in a similar way to the endogenous Arabidopsis enzyme. Plants putatively containing the other chimeric Arabidopsis/rice isoamylase complex - AtISA1/OsISA2 also had starch with modest increases in the numbers of chains between d.p. 6 and d.p. 14. It has been proposed that the catalytically inactive subunit ISA2 has a regulatory function, provides substrate specificity, or increased stability to the catalytic ISA1 subunit AtISA2 with OsISA2 causes a change in substrate specificity, but this conclusion needs to be treated with caution since no activity was directly measured. Further analysis, such as X-ray scattering, scanning electron microscopy and differential scanning calorimetry could be used to study in more depth the structure and properties of starch produced in plants with different chimeric isoamylase complexes.The replacement of Arabidopsis ISA1 and ISA2 with the rice orthologs enabled us to study the effect of distinct isoamylase complexes on the fine structure of starch. Given that the isoamylases of different subunit compositions have been shown to have distinct functionalities in cereal endosperm, it is reasonable to hypothesise that the differences in amylopectin between species may be partly dependent on the isoamylases they contain. All three transgenic lines had starch structures that were similar, but not identical, to Arabidopsis wild-type starch . The linTaISA1) protein was able to revert the phytoglycogen-accumulating phenotype of the rice sugary-1 mutant (lacking OsISA1) back to a starch-accumulating phenotype TaISA1, the amylopectin CLD was similar – but not identical – to the wild type rice amylopectin. Recently, it was shown that the maize ISA1 (ZmISA1) protein expressed in the Arabidopsis isa1 and isa1isa2 mutants were able to restore exclusive starch synthesis in Arabidopsis, similar to our observations. Interestingly, the authors observed a greater variability of the starch content, with some transgenic lines producing even more starch than the wild type, which was not the case in our plants. However, this raises the possibility that the isoamylase may somehow determines not only the starch structure but also the amount produced. Interestingly, the ZmISA1 expressing plants produced starch with the same amylopectin CLD as the Arabidopsis wild type Our results are consistent with the findings of other studies. The wheat ISA1  plants were grown in greenhouse at 23°C, 70% relative humidity at night and 26°C, 80% relative humidity at day with a minimum 14-h photoperiod and minimum light intensity of 250 μmol photons m−2 sec−1. Leaves for the extraction of transient starch were harvested from four-week-old plants. Mature, dry seeds from these plants were used for storage starch purification. Potato (Solanum tuberosum cv. Agata) tubers were obtained from a local market and used for storage starch purification. For transient starch isolation, tubers were sprouted and leaves harvested after four weeks of growth in a chamber  at constant 22°C, 70% relative humidity, with a 12-h photoperiod and a light intensity of around 250 μmol photons m−2 sec−1. Maize  plants were grown in a chamber (Kälte 3000 AG) at constant 22°C, 70% relative humidity with a 12-h photoperiod and a light intensity of around 250 μmol photons m−2 sec−1. Leaves for transient starch were harvested from four-weeks-old plants. Purchased seeds were used directly for storage starch purification.Rice . Proteins were separated by SDS-PAGE using standard methods, transferred to PVDF membranes, probed with antibodies against AtISA1 AtISA2 2, 1 mM CaCl2 and 5 mM DTT. Reaction were stopped and activities visualized by application of Lugol solution .Soluble proteins were extracted from leaves (150 mg) of five-week-old plants by homogenization in 1 mL of medium containing 100 mM MOPS, pH 7.2, 1 mM EDTA, 5 mM DTT, 10% (v/v) glycerol, 1 x Complete Protease Inhibitor Cocktail (Roche), using all-glass homogenizers. Homogenates were subject to centrifugation  and α-amylase , and released glucose was measured spectrophotometrically The extraction procedure was the same for all plant species and tissues and was performed using perchloric acid as described previously Pseudomonas amyloderamosa isoamylase  and Klebsiella planticola pullulanase (Megazyme) were carried out for 2 h at 37°C in 10 mM Na-acetate, pH 4.8. The resulting linear glucans were analysed by HPAEC-PAD as described previously Starch and phytoglycogen in the insoluble and soluble fractions from the perchloric acid-extracted plant material, respectively, were used for glucan structural analysis. Volumes containing 100 μg of starch or phytoglycogen were used from each of four individually extracted plants. Samples were boiled for 10 min prior enzymatic digestion. Debranching reactions with OsISA1 and OsISA2 coding sequences were amplified by PCR using attB-site containing primers  was also used to calculate the difference plots in Fig. 5 B to D. A) CLDs of the lines isa1-35S::OsISA1 A (open circle), isa1-35S::OsISA1 B (open square) and isa1-35S::OsISA1 C (open triangle). B) CLDs of the lines isa1isa2-35S::OsISA1 A (open circle), isa1isa2-35S::OsISA1 B (open square) and isa1isa2-35S::OsISA1 C (open triangle). C) CLDs of the lines isa2-35S::OsISA2 A (open circle), isa2-35S::OsISA2 B (open square) and isa2-35S::OsISA2 C (open triangle).(TIF)Click here for additional data file.Table S1Primers used for Gateway cloning of OsISA1 and OsISA2.(DOCX)Click here for additional data file.


Urological complications after kidney transplantation are mostly related to the ureteroneocystostomy, often requiring interventions with additional costs, morbidity and mortality. Our aim was to assess risk factors for urological complications in deceased donor kidney transplantation. Between January 2000 and December 2011, 566 kidney transplantations were performed with deceased donor kidneys. Recipients were divided in a group with, and a group without urological complications, defined as the need for a percutaneous nephrostomy catheter or surgical revision of the ureteroneocystostomy. Univariate and multivariate analyses were performed. Univariate analysis showed increased number of male donors (p = 0.041), male recipients (p = 0.002), pre-emptively transplanted recipients (p = 0.007), and arterial reconstructions (p = 0.004) in the group with urological complications. Less urological complications occurred in recipients on hemodialysis (p = 0.005). More overall surgical interventions (p<0.001), surgical site infections (p = 0.042), urinary tract infections (p<0.001) and lymphoceles (p<0.001) occurred in the group with urological complications. Multivariate analysis showed that male recipients (p = 0.010) and arterial reconstructions (p = 0.019) were independent risk factors. No difference was found between both groups in patient or graft survival. In conclusion, recipient male gender and arterial reconstruction are independent risk factors for urological complications after deceased donor kidney transplantation. Nevertheless, graft and recipient survival is not different between both groups. Urological complications after kidney transplantation are reported to occur between 2.5% and 30% of all recipients Risk factors that contribute to the prevalence of urological complications need to be determined. So far, many factors have been described in literature, including several donor and recipient characteristics Due to the increasing number of patients with end-stage kidney disease and a continuing shortage of donors, the demand for kidney grafts led to extension of donor criteria by the Dutch Transplant Foundation. Alongside the Donation after Brain Death (DBD) donors, Donation after Circulatory Death (DCD) (category III) donors have been deemed eligible for transplantation The Erasmus MC, University Medical Center internal review board issued a formal written waiver for the need of ethics approval and the need for written informed consent.Between January 2000 and December 2011, all kidney transplantations performed with grafts from DBD and DCD (category III) donors at the Erasmus University Medical Center Rotterdam, were reviewed retrospectively. A total of 566 recipients were identified. The surgical reports and electronic patient system were screened for donor and recipient characteristics, and urological complications. Recipients were divided in two groups, one group with and one group without urological complications within 3 months’ time after transplantation. A urological complication was defined as any event leading to the placement of a PCN or surgical revision of the ureteroneocystostomy during follow-up. We argued that a PCN placement is the best possible parameter to identify those patients who had an adverse urological outcome. An increasing serum creatinine level combined with hydronephrosis on ultrasonography was reason for a PCN placement. Monitoring of the PCN position and imaging of the ureter is performed by an antegrade pyelography (APG). If leakage of the ureteroneocystostomy is diagnosed with an APG, both PCN and urinary bladder catheter are placed until the leakage stops. In case the leakage is diagnosed shortly after transplantation immediate surgical reconstruction is performed. If a total obstruction of the ureter is diagnosed with an APG, surgical intervention is inevitable. If the APG shows a stenosed ureter but contrast reaches the bladder radiological dilation of the ureter is performed. Afterwards a percutaneous nephrocystostomy catheter (PCNC) is placed for 2 weeks. If the stenosis persists a surgical ureter reconstruction is indicated and will be performed by a transplant surgeon, together with an urologist.Tacrolimus toxicity (>15 µg/l), suspected acute tubulus necrosis (ATN), treatment for rejection (methylprednisolone and/or ATG), lymphoceles, surgical site infections and urinary tract infections were scored during the first 3 months after transplantation. Besides ureteral revisions, all other re-interventions were documented: re-interventions because of re-bleeding, lymphocele drainage, transplantectomy and re-exploration because of vascular complications. Graft failure was defined as primary non-function or loss of function requiring dialysis. All recipients had a follow-up of at least one year in our center.All transplantations were performed by a transplant surgeon or vascular surgeon and transplants were engrafted extraperitoneally in the iliac fossa. In presence of multiple renal arteries (in majority of cases two arteries) a reconstruction was performed on the bench. Dependent on the length of the artery an end-to-side or an side-to-side anastomosis was created. Urinary continuity was established by either an intravesical Postoperatively, immunosuppressive therapy consisted of prednisolone (50 mg a day), tacrolimus  and mycophenolate mofetil (1000 mg twice a day). Basiliximab was used as induction therapy. Prednisolone was tapered and discontinued 4 months after transplantation. A prophylactic dose of 480 mg cotrimoxazole per day was given to prevent urinary tract infections. Cefazoline was given perioperatively. Standard dose of 12.000 U heparin daily was given during the first 5 post-operative day. Valganciclovir treatment was given to patients at risk for CMV infection or reactivation. Initial episodes of acute rejection were treated with methylprednisolone, 1000 mg a day for 3 days, ATG was given on indication.Statistical analysis was performed using IBM SPSS version 20.0 . Variables studied are presented in The manuscript is conducted in accordance to the principles expressed in the Declaration of Helsinki. Approval of our local ethics committee was not required for this study.Between January 2000 and December 2011, 566 kidney transplantations were performed with grafts from both DBD and DCD donors. An overview of the baseline characteristics is shown in Of the total 566 recipients, 117 received a PCN. In 15 recipients a PCN was placed because of leakage and in 102 because of hydronephrosis on ultrasonography. An endoscopic dilatation of the ureter was performed in 4 recipients, in 3 patients successfully and in 1 recipient an additional surgical revision was required afterwards. A surgical ureteral revision was required in 31 recipients who previously received a PCN and in one recipient a surgical ureteral revision was required without a prior PCN placement based on leakage of the ureter shortly after transplantation. Choice of re-implantation was in 30 cases a new ureterovesicostomy, in one patient a pyelovesicostmy and in one a ureter-ureterostomy. Mean graft survival of the group with a surgical ureter reconstruction was 5.57 years (inter quartile range of 2.14–9.27). In 83 recipients, the PCN could be removed without additional intervention.Comparisons of the overall complications according to absence or presence of urological complications are presented in All odds ratios regarding urological complications using univariate and multivariate analysis were presented in In total 118 (20.8%) recipients developed a urological complication and 5.7% of all recipients (32 out of 566) underwent a surgical ureteral revision. Of all recipients who developed a urological complication, 73 had a DBD donor graft and 45 a DCD. Mean graft survival time was 4.02 years with a standard deviation of 3.47. Minimum graft survival was 0 day due to primary non function and maximum was 12.1 years. Mean patient survival was 5.13 years. Death censored graft survival regarding urological complications was presented by a Kaplan-Meier curve . No signIn our study, 20.8% of all recipients of a kidney graft from a deceased donor had a urological complication as defined by PCN placement or surgical ureteral revision. As PCN placement is considered as a minimally invasive event in our center, the threshold to use a PCN is low, either for therapy, or for diagnosis. Eventually, only 32 recipients (5.7%) underwent a surgical ureteral revision for leakage or stenosis and graft survival was not worse in the population with urological complications.It has been suggested that urological complications are caused by an insufficient blood supply to the ureter. Excessive dissection of the site known as ‘golden triangle’  should therefore be avoided during graft recovery. Damage of this triangle might lead to necrosis of the distal ureter in 70% of the cases Potential risk factors for urological complications including age, prolonged cold ischemia and recipient Diabetes Mellitus were reported not to play an important role in the occurrence of urological complications There is growing evidence on the superiority of the extravesical ureteroneocystostomy when compared with the intravesical technique, with or without additional routine stent placement Although the mean graft survival of DCD donors is suspected to be shorter than that of DBD donors, there are no studies on the occurrence of urological complications in those groups. Therefore, the finding that grafts from a DBD donor were not superior to DCD donor grafts with respect to urological complications  is an imAnother important finding in our study was the fact that primary non-function of the graft or graft loss within 3 months was significantly lower in the group without urological complications . This prBased on our study of kidney transplantations from a deceased donor, we conclude that recipient’s gender and arterial reconstruction are independent risk factors to develop a urological complication. However, donor type (DBD and DCD), primary non-function, type of anastomosis, and the presence of multiple transplantations could not be defined as risk factor in our univariate and multivariate analyses. This means that donor type and surgical anastomosis technique are less important factors for the urological outcome, which is in contrast to what one might argue.


Information regarding a possible competing interest of the sixth author is incorrectly omitted from the Competing interests statement. The Competing interests statement should read: "James A. Colbert was an editorial fellow at the New England Journal of Medicine. Navindra Persaud is an Associate Editor for the Canadian Medical Association Journal. Peter Gill is a member of the Editorial Advisory Board of the CMAJ. All other authors have declared that no competing interest exists. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials."


Neobalantidium coli are asymptomatic in most hosts. In humans and captive African great apes clinical infections occasionally occur, manifested mainly by dysentery; however, factors responsible for development of clinical balantidiasis have not been fully clarified. We studied the effect of dietary starch on the intensities of infection by N. coli in two groups of captive chimpanzees. Adult chimpanzees infected by N. coli from the Hodonín Zoo and from the Brno Zoo, Czech Republic, were fed with a high starch diet (HSD) (average 14.7% of starch) for 14 days, followed by a five-day transition period and subsequently with a period of low starch diet (LoSD) (average 0.1% of starch) for another 14 days. We collected fecal samples during the last seven days of HSD and LoSD and fixed them in 10% formalin. We quantified trophozoites of N. coli using the FLOTAC method. The numbers of N. coli trophozoites were higher during the HSD (mean ± SD: 49.0±134.7) than during the LoSD (3.5±6.8). A generalized linear mixed-effects model revealed significantly lower numbers of the N. coli trophozoites in the feces during the LoSD period in comparison to the HSD period (treatment contrast LoSD vs. HSD: 2.7±0.06 (SE), z = 47.7; p<<0.001). We conclude that our data provide a first indication that starch-rich diet might be responsible for high intensities of infection of N. coli in captive individuals and might predispose them for clinically manifested balantidiasis. We discuss the potential nutritional modifications to host diets that can be implemented in part to control N. coli infections.Infections caused by the intestinal ciliate  Neobalantidium coliBalantidium coli), inhabiting the hindgut of a variety of mammalian species worldwide N. coli infection is commonly reported in captive African great apes  the presence of reservoir(s) in captive facilities or/and (ii) differences in the diet of wild and captive apes Potamochoerus porcus) being in close contact with free raging apes, Pomajbíková, unpublished data]. On the other hand, it is well-known that the diet of captive great apes significantly differs from that in the wild. Captive ape diets are low in fiber, rich in starch and readily available sugars in vitro studies on N. coli demonstrated that this ciliate is able to ingest the starch particles N. coli infection in pigs, rats and humans Troglodytella abrassarti in captive chimpanzees Although pigs and rats have been used to study N. coli in captive primates N. coli in two groups of captive chimpanzees.Our long-term research on This study was authorized by managements of Hodonín and Brno Zoos, the ethics committee of the Institute of Vertebrate Biology; AS CR reviewed and approved the protocol of the study. Our research and the protocol complied with the legal requirements of the Czech Republic . Our study was non-invasive. Collection of fecal samples did not include any disturbance of the animals (see the part Sample collection and ciliate quantification). We designed the diets in close collaboration with both Zoos. For welfare and enrichment purposes we paid careful attention to feed chimpanzees variable diets of different food components each day. However, in order to design diets which differ in starch, but stay as much as possible similar in all other nutrients, these diets did not fully meet nutritional requirements of chimpanzees in captivity N. coli with no clinical symptoms (e.g. diarrhea) The study was conducted in the Hodonín Zoo, Czech Republic  from August 12, 2010 to September 19, 2010 and in Brno Zoo, Czech Republic  from November 8, 2010 to December 16, 2010. Chimpanzees in Brno Zoo could utilize three recently repaired indoor enclosures and two outdoor enclosures, while chimpanzees in Hodonín Zoo could use one indoor and one outdoor enclosure. Wooden wool was provided to the chimpanzees for bedding and the indoor enclosures were cleaned once per day by water and disinfectants. Both zoos carry out enrichment programs with focus on feeding enrichment i.e. different ways of hiding, presenting or enriching food provided to animals. Moreover, enclosures are enriched with species specific equipment promoting their natural way of life, including locomotion, resting etc. Chimpanzee had no close contact with humans or other animals kept in the zoos. Our previous study showed that the chimpanzees harbor During the study, we fed the chimpanzee groups two different diets: a high-starch diet  and a low-starch diet . The LoSD was composed only from low starch food items, while the HSD included also potatoes and rice . First, We froze the samples of each food item and analyzed them for crude protein, lipid, starch, ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL), by standard methods approved by the Association of Official Analytical Chemists International In both zoos we collected fecal samples 2–3 times per day from individuals in indoor and outdoor enclosures. We did not pool the samples. The collection of fecal samples was non-invasive and did not cause any disturbance to the animals. We could not assign fecal samples to particular individuals as the ethical considerations prohibited us from separating the animals for the duration of the study.N. coli under light microscope (Olympus CX 41) with magnification 400× and by FLOTAC 400 apparatus according to the FLOTAC technique protocol Neobalantidium trophozoite quantification using the FS3 Zinc Sulphate flotation solution , We immediately placed 5 g of feces in 20 ml of 10% formalin and then transferred the samples to the laboratory of the Department of Pathology and Parasitology of the University of Veterinary and Pharmaceutical Sciences Brno for examination. We quantified the trophozoites of N. coli trophozoites present in feces by a generalized mixed-effects model (GLMM) with Poisson error distribution and log link function. Zoo  was considered as the experimental unit. The numbers of trophozoites (count data) entered the model as the response variable, while the type of diet was a categorical predictor with two levels (HSD and LoSD) and zoo (Hodonín and Brno) a random effect. The GLMM was fitted in the package lme4 We tested the effect of diet on the number of N. coli; no other parasites were present. No clinical symptoms were observed throughout the whole study period. The numbers of N. coli trophozoites were higher during the HSD (mean ± SD: 49.0±134.7) than during the LoSD , z = 47.7; p<<0.001).We examined 58 samples during HSD and 67 samples during LoSD from group I, and 39 samples during HSD and 28 samples during LoSD from group II. We found only trophozoites of 3.5±6.8; . The genN. coli trophozoites, leading to increased infection intensities. In our model situation, which is much closer to humans, we also recorded lower numbers of Neobalantidium trophozoites in feces when chimpanzees were fed with the low starch diet. Although the number of experimental units in our study was low, our results provide an indication that starch-rich diet, which is not typical of wild African great apes N. coli in captive apes. We point out that a possibility that starch-rich diet might predispose the animals for clinically manifested balantidiasis needs to be further studied.Early studies, carried out nearly a century ago The assessment of the exact amount of starch passing through the small intestine to the colon of studied chimpanzees is complicated, because resistant starch is determined complexly by several factors and its analytical measurement is difficult N. coli in infected apes N. coli. Negative impact of starch-rich (and concurrently low-fiber) diet on various components of intestinal microflora of chimpanzees has been described also by other authors .Clinical balantidiasis in humans and great apes is usually treated by administration of antibiotics, though even repeated treatment does not lead to permanent elimination of Neobalantidium infection in rats and domestic pigs N. coli infections and future studies should survey the possibilities of nutritional adjustments as a part of parasite control strategies not only in livestock farming, but also in zoo husbandry and potentially in human medicine.Development of effective antiparasitic strategies requires substantial knowledge of host-parasite interactions, including direct effect of host nutrition on the parasites. To date, most of the research has focused on utilization of nutritional alternations in the control of helminthiases parasitizing livestock


The clinical trial registry information was accidentally omitted from this paper. It is registered at clinicaltrials.gov under the registration number NCT00792922.