To investigate whether unfolding time of Descemet membrane (DM) graft rolls changes at various fluid temperatures.ex vivo, and experimental. The study was conducted at the tertiary center for corneal disease in Adana Numune Training and Research Hospital between June 2014 and June 2015. DMs were divided into 4 categories according to baseline roll tightness and these were distributed among 4 different groups using 4 different balanced salt solution (BSS) temperatures . Sixteen donor corneas were obtained from the hospital eye bank.The study was prospective, DM roll formations may vary according to the donor cornea received. Some form tighter rolls while others can form a more open roll. No differences in roll tightness were observed in any of the DM rolls after 5 or 10 minutes in the different BBS temperatures. In all groups, neither tightening nor opening was observed in DM roll formations.Different BSS temperatures were found to have no effects on DM unfolding time in this study. Although the Descemet stripping automated endothelial keratoplasty (DSAEK) procedure involves well standardized and reproducible graft preparation and unfolding, DMEK remains challenging. The main step of the procedure, which involves unfolding the lamella to attach the graft to the posterior stroma, can be especially difficult. This step involves most of the manipulations to the graft.3Descemet membrane endothelial keratoplasty (DMEK) was first described by Melles et al.6 However, graft preparation and unfolding are less standardized. In DMEK, the graft preparation phase, injection of DM into the anterior chamber, and all the unfolding phases in the anterior chamber are performed in a fluid medium. We hypothesized that graft unfolding could be affected by the temperature of the fluid medium.DMEK provides faster and more complete visual rehabilitation compared to DSAEK.7 Although there was no animal model or other research showing that temperature change can affect DM roll tightness, we investigated the effect of temperatures up to normal body temperatures on DM rolls in a laboratory setting.We observed a spontaneous opening of tight DM rolls that were difficult to open in the DMEK surgeries of some patients, and thought that it may be due to the warming effect of the microscope light. If DMEK surgery is prolonged, the microscope light can have a thermal effect. The major protein in DM is type IV collagen. With heat, the structure of collagen is disrupted by strong oscillations that break the bonds between molecules, and this may affect DM unfolding time.This experimental study investigated whether the unfolding time of cornea DMEK graft rolls which could not be used due to positive serology changed in various fluid temperatures.ex vivo, and experimental in nature. Ethical approval was obtained from Adana Numune Training and Research Hospital, where the study was conducted. The study was carried out with 16 donor corneas which were obtained from the eye bank of the tertiary center for corneal disease in Adana Numune Training and Research Hospital between June 2014 and June 2015. The donors’ ages were noted. The donor corneas were not appropriate for implantation due to positive results in serological tests (HbsAg+ or antiHCV+).This study was prospective, Corneoscleral buttons were excised and stored in a corneal chamber which contained Eusol-C  at 4 °C for 14 days. DM tissues were found to have no endothelial pathology by biomicroscopic examination . The death-to-preservation time was not longer than 12 hours without refrigeration and the tissue was used within 7 days of harvesting.8 Corneoscleral buttons were positioned onto a Barron vacuum punch to prevent shifting during DM preparation . All DM grafts underwent superficial trephination using an 8.0 mm punch, which is one of the most preferred diameters in DMEK surgery. The DM edges were stained with 0.06% trypan blue solution and the donor rim was filled with 23 °C balanced salt solution (BSS). Stripping was performed in this medium using tying forceps, working from edge to center, and the DM was harvested. The DM roll was then restained with trypan blue for 60 s (All tissues were prepared by a single surgeon (Y.K.) in the operating room using the submerged cornea technique immediately before evaluation as described previously.for 60 s .The DM grafts were divided into 4 categories according to initial roll tightness. Accordingly, 1/4 DM roll was very tight (about 1-2 mm width), 2/4 DM roll had about 3-4 mm width, 3/4 DM roll had about 5-6 mm width, and 4/4 DM roll was nearly completely open (about 7-8 mm width).Using tying forceps, the prepared DM rolls were placed into closed glass containers containing BSS at different temperatures. Changes in DM roll formations and sizes were observed and photographed by ophthalmic surgical microscope at the beginning, at 5 minutes, and at 10 minutes.9 The ambient temperature was ~23°C in the operating room where the current study was performed. In another study, it was determined that the mean temperature in the anterior chamber was 23.6 °C.10 Therefore, 23 °C is a logical BSS temperature for stripping of DM graft. Considering the temperature range between 8 °C (4-8 °C is storage temperature of donor tissue) and 36 °C (human body temperature) suitable for this study, we evaluated temperatures of 8, 16, 23, and 36 °C to determine the optimal BSS temperature for DM grafts.The temperature of the operating room and liquids (relatively) in the room may vary between 22 and 24 °C during eye surgery.The study was conducted in 4 different groups, using 4 different BSS temperatures: 3 (20.0%) DM grafts were placed in 8 °C BSS (Group 1), 4 (26.7%) in 16 °C BSS (Group 2), 4 (26.7%) in 23 °C BSS (Group 3), and 4 (26.7%) in 36 °C BSS (Group 4). BSS temperatures in all groups were checked with a thermometer and kept stable throughout the 10-minute observation period. Changes in fold tightness of the DM grafts in BSS at different temperatures were observed for 10 minutes. In DMEK surgery, shorter DM graft unfolding time is desirable to ensure less endothelial loss and favorable postoperative prognosis. Thus, the effects of BSS temperature on the unfolding time of DM grafts in 10 minutes were observed and recorded in this study.SPSS version 16.0 software  was utilized in the analysis of the data. The analysis included nonparametric tests. Median and range or mean ± standard deviations (SD) were demonstrated through descriptive statistics. The distribution of proportions was analysed using chi-square distribution. Statistical significance was accepted as p<0.05.The median donor age (16 corneas of 8 donors) was 69 years . The male/female ratio was 6 (37.5%)/10 (62.5%). There were no significant differences between the groups in terms of age and gender .The median time from death to corneal removal was 7 hours , while the median time from preservation to surgery was 5.5 days . There were no statistical differences between the groups in terms of time from death to corneal removal or preservation to surgery .Cause of death was cerebrovascular disease in 3 donors, myocardial infarction in 2 donors, and chronic kidney disease in 3 donors. It was thought that cause of death would affect DM roll formation. However, there were no statistical differences between the groups in terms of the donors’ cause of death (p=0.238).All tissues were stripped in the operating room and prepared immediately before evaluation. The median DM peeling time was 7.0 minutes , with no statistically significant differences between the groups (p=0.946). Of the 16 corneas, the DM roll could not be obtained from only 1 (in Group 1). This lacerated DM was excluded from the study.Prepared DM roll formations can vary according to the donor cornea. In the current study, some formed a tighter roll, while others formed a looser roll. At time of collection, there was no statistical difference between the groups in DM roll tightness in the 23 °C BSS (p=0.273) . In all 11 The related literature indicates general rate of failure as 5.2%.12 In our study, we could not obtain DM graft from only one donor cornea (6.25%). There were lacerations resulting from the tightly cohesive nature of the stromal surface. This donor’s cause of death was chronic kidney disease with diabetes mellitus, which is consistent with the information in the literature.11DMEK preparation might be affected by some systemic conditions. Research shows that DMEK preparation can have higher failure rates with tissues from donors with diabetes mellitus  and hyperlipidemia or obesity. It is reported that failure rate can be reduced by eliminating the tissues from donors either with diabetes mellitus or with hyperlipidemia or obesity.13 In the current study, preoperative findings were similar and differences between groups were not statistically significant. Therefore, we were able to investigate only the effects of different BSS temperatures on DM roll formation in this study.Donor age, systemic diseases, and cause of death may have an impact on DM graft preparation, complications during harvesting, and DM roll formation.3 Another study found no correlation between corneal donor characteristics and the degree of difficulty of unfolding with graft lamella older than 49 years. The same study indicated that there was a significant association between more difficult graft unfolding and rates of graft detachments and endothelial cell loss.14 It was reported that graft orientation in DMEK surgery can be visualized and assessed with live intraoperative optical coherence tomography. In addition, faster graft positioning with less graft manipulation was reported in the presence of severe corneal edema.15 Different ways to facilitate graft unfolding are still being sought.A statistically significant correlation was reported between relatively early postoperative endothelial cell loss and unfolding time, with longer unfolding time associated with greater endothelial cell loss.When the DMEK graft is separated from the corneal stroma, it forms a roll with the DM inside and the endothelium facing outward. This formation is associated with type IV collagen in the DM and the behaviors of endothelial cells after leaving the cornea. More studies which demonstrate how graft properties affect graft unfolding time are needed. We were unable to find any animal model or other research in the literature showing that temperature change can affect DM roll tightness.18 An in vitro study showed that delivering DMEK tissue trifolded with the endothelium inward reduced surgical trauma to donor cells and facilitated spontaneous unfolding.17 However, despite the maneuvers developed, there is no standardized method of graft unfolding which affects postoperative success, and DM unfolding time can be prolonged in some cases. Moreover, the factors affecting DM unfolding time have not been determined. Existing methods used for DM unfolding result in direct or indirect mechanical trauma to the graft, which can cause endothelium injury. Therefore, less traumatic methods need to be developed.As the prognosis in the postoperative period depends on the remaining number of endothelium cells, DMEK surgery and DM unfolding times are important. The DM roll should be opened in the anterior chamber with minimum endothelial loss and attached to the recipient stroma. Recently, several maneuvers for atraumatic unrolling of DMEK grafts have been described.In some cases where we had difficulty in DM unfolding, in time we observed spontaneous unfolding. Supposing that this could be associated with heating under the microscope light, we put DM rolls obtained from this study into different BSS temperatures and observed whether any would open spontaneously. We refrained from any other maneuvers that would affect opening and investigated only the effect of the temperature. However, no changes were observed in DM rolls at 8 °C, 16 °C, 23 °C (BSS temperature we use routinely) or 36 °C BSS temperatures within the 10-minute period.19 Consequently, using BSS at a lower temperature may be advantageous and rational for DM graft since the higher BSS temperature offers no benefit in terms of DM graft unfolding time.The DM rolls demonstrated neither tightening nor opening in our study. We had anticipated that 36 °C, which is normal physiological body temperature, would promote DM roll unfolding. However, we did not observe such an effect. Although we did not demonstrate the effect of BSS temperature on the DM rolls at the molecular level, it was observed that this effect did not change unfolding time of the graft. In a study using porcine corneal endothelial cells, it was found that the eye irrigation solution stored in the refrigerator better protected the corneal endothelial cells from heat damage than BSS stored in air-conditioned room.This study has some limitations. First, we used biomicroscopic examination to detect endothelial pathology and we could not count endothelial cells. If we had a chance to perform specular microscopy, endothelial examination would be more valuable than biomicroscopic examination. Second, delivery of the tissue could not be simulated using an anterior chamber.In conclusion, different BSS temperatures were found to have no effect on DM unfolding time in this study. Other methods which are less traumatic and facilitate DM unfolding should be investigated in order to reduce endothelial cell loss.


Purpose: Every year, an increasing number of international patients seek medical care in the United States (U.S.), yet little is known about their impact. Based on single institution experiences, we wanted to explore the perceived impact of international pediatric patients on large academic U.S. pediatric intensive care units (PICUs), as they are already taxed systems.Methods: To explore current perceptions, seven geographically diverse institutions who advertise care for international patients on their websites and have ≥24 PICU beds were identified after IRB approval was obtained. We consented and interviewed PICU division chiefs or medical directors from each institution regarding their demographics and international patients. Common themes were identified.Results: Participating institutions were diverse in geographic location, census, and resource allocation strategies. Five of the seven institutions reported the presence of a formal international patient program. Four of those five reported an increase in international patients receiving PICU care over the past 5 years. International patients sought complex surgeries, advanced cancer treatments and metabolic/genetic evaluations. We identified three primary domains that require further exploration and research: (1) cultural and language differences leading to barriers in providing optimal care to international patients (2) institutional financial considerations, and (3) perceived positive and negative impact on the care of local/domestic patient populations.Conclusions: The presence of international programs raises a number of important ethical questions, including whether clinicians have a greater duty to serve residents of the local community as opposed to international patients when resources are limited. Further exploration is warranted. A family from another country brings their 7-year-old son to an academic pediatric institution in the United States for experimental chimeric antigen receptor T-cell therapy for refractory leukemia. During his hospitalization, he develops multi-organ failure requiring extracorporeal life support (ECLS). Unfortunately, he dies based on neurological criteria (“brain death”), but the embassy of his home country (the payer) refuses to allow discontinuation of ECLS.Every year, an increasing number of international patients seek medical care in the United States (U.S.) –3. Interpediatric patients are now seeking care in the U.S. Of the over 50,000 total international patients who sought care in the U.S. in 2015, children accounted for 52% of all international patients who required inpatient services  admission. Given additional baseline constraints on PICU resources , 8, we eTo explore areas requiring further investigation regarding the potential impact of international pediatric patients on PICUs at a multi-institutional level, we reached out to several U.S. pediatric academic institutions. Seattle Children's Hospital IRB approved the study under exempted status given the lack of identifiable information collected. Written consent was not required but verbal consent was obtained from the division chiefs or medical directors at the start of the interview. Selective sampling was used to identify seven geographically diverse academic, free-standing pediatric institutions who advertise care for international patients on their websites and have ≥24 PICU beds. The number of beds was used as a proxy for unit size, assuming that larger PICUs were more likely to have larger international patient populations. A PICU clinician and ethicist approached either a PICU division chief or PICU medical director from each institution. All institutions that were approached agreed to participate. Although other institutions would have met the aforementioned criteria, the participation of those initially approached provided geographic diversity for this pilot data.perceptions of the international patient population, including the presence/absence of a formal international program, countries represented, treatments sought, and payment type. Importantly, all answers were provided as estimations. Notes were taken during the interviews but were not recorded. When all the interviews were completed, notes were reviewed by the primary interviewer to identify common themes and questions requiring further exploration about potential impact of international patients on PICU care. These themes and questions were then vetted by the author group.Baseline demographic data, ICU staffing and capacity, catchment area, presence of other nearby PICUs, and distribution strategies between local PICUs were collected from each participant using a written survey. Semi-structured interviews were completed by phone or in-person to gather detailed information about PICU leaders' Four PICU division chiefs and three PICU medical directors participated from seven distinct sites. Number of PICU beds ranged from 24 to 55 and average daily patient census ranged from 20 to 55. Average annual PICU admissions ranged from 1,400 to 3,800 patients. ECLS capacity varied from three to eight circuits per institution.estimated total number of international patients ranged greatly. One institution reported only one international patient requiring PICU admission in the past year whereas another institution reported ~3–8 international patients requiring PICU care per week. Patients represented many countries. Respondents identified complex surgeries, advanced cancer treatments and metabolic/genetic evaluations as common reasons for seeking international medical care  cultural and language differences leading to potential barriers in providing optimal care to international patients, (2) institutional financial considerations, and (3) impact on the care of local/domestic patient populations see .Respondents expressed concern that cultural differences potentially affect the quality of care for international patients in the PICU. All institutions have interpreter services but only one institution has a formal training in cultural diversity for staff. Importantly, respondents observed that although many domestic patients come from diverse cultures, cultural differences among international patients are magnified in the absence of local community support. Additionally, some respondents perceived that international patients/families often had different understandings and expectations about what is possible in U.S. hospitals. Since five of the international programs offer experimental treatments for life-threatening illnesses that failed conventional therapy, some international patients die in U.S. hospitals. Respondents expressed that differences in end-of-life cultural norms can lead to conflict between clinicians and families. Specifically, three institutions experienced challenges discontinuing ECLS for patients with irreversible diseases. One institution changed its consent form to explicitly permit the medical team to discontinue ECLS therapy when continuation is deemed inappropriate.Although not included in any of international program mission statements, four respondents expressed concern that institutional financial incentives play a role in recruiting international patients. Respondents perceived that most international patients are financed through government sponsorship or self-pay, and worried that this approach may be more profitable compared to private insurance or Medicaid. However, one respondent expressed concern that unanticipated PICU stays often yield additional expenses, resulting in financial deficits for the institution.Four out of seven respondents reported perceptions that international patients impact the PICU. However, only one respondent perceived a direct impact on the local population, which was improved cultural humility. Five respondents worried about how international patients might impact their future ability to care for the local population given anticipated programmatic growth. One respondent reported that despite the international program growth, no additional PICU resources were added. Several respondents perceived that international patients requiring PICU admission had long lengths of stay and required additional resources to navigate cultural and language differences. One respondent reported that hospital admission for international patients is often declined due to continuous lack of PICU capacity. Another institution accepts international patients even when resources are limited and uses double room occupancy. This respondent observed that local families seemed more likely to be placed in double rooms because of the perception that international families do not find room sharing acceptable.The seven participating institutions were diverse with respect to their geographic location, census, size of the international programs, and resource allocation strategies. While we recognize the small sample size and lack of representation of international patient program directors, the responses of the clinicians to our questions highlight a number of important ethical dimensions that require further exploration. We also acknowledge that providing incredibly valuable and potentially lifesaving therapies to children who lack access in their home countries is very important, and therefore understanding how to optimize outcomes for these programs and how they impact the care of the local/domestic population is crucial.The concerns raised by the clinicians regarding the potential impact of cultural differences on the quality of medical care provided, highlight the importance of further exploration and investigation into how international patient programs assess potential cultural differences prior to accepting new patients. Addressing these differences a priori may reduce cultural conflict during the hospital stay. For example, predictable cultural conflicts such as those surrounding end of life care, already exist within our diverse domestic population. On a daily basis in the ICU setting, we often grapple with how we can best honor the values of patients and families when their belief systems differ significantly from our own. In caring for the international population, cultural conflicts may be further intensified. Arguably, the candidates who are best served by program enrollment are those international patients who are most likely to benefit from a requested treatment that is not otherwise available to them. However, what if the risk for mortality is >80%, even if one receives the experimental treatment? Many families in this situation are so hopeful for survival that they are not able to comprehend the high risk of death, especially in a geographic location far from their family and local community. In these cases, what preparation and counseling should be offered to these families in anticipation of the possibility of a very poor outcome? Furthermore, the potential lack of exposure to Western medical practice opens the doors to additional conflict. For example, in some countries, unlike the U.S., “brain death,” or death by neurological criteria, is not accepted as equivalent to death, legally or medically . To addrall patients and their families.These conversations may need to extend beyond the immediate family. In cases where government sponsorship is instituted, exploring what conversations should occur with the embassies prior to acceptance into the program is important. Preemptively modifying consent for care forms, and being explicit regarding cultural practices in the U.S. is critical. While respect for patient and family autonomy is the predominant model for medical decision-making in the U.S., not all cultures align with this model. Sometimes extended family members, religious leaders, or even the government may have the authority to make decisions, rather than the individual patient and family . ExploriThe financial questions raised by our participants similarly demonstrate the need for programmatic transparency. Importantly, knowledge of how international patients are expected to pay and how the generated revenue is distributed is necessary. Non-for-profit U.S. hospitals still require revenue in excess of cost. If international programs help maintain institutional financial stability, then all patients seeking care at the institution may benefit. Notably, some children within local communities lack adequate insurance coverage and would benefit from increased uncompensated care funds. Ideally, if revenue is generated from these programs, the monies could be directly used to serve those in need, both locally and internationally. Furthermore, potentially subsidizing international programs to maintain equity in access for all international patients, not just for those who can pay or are sponsored by the government, also may be important. As providing care for all international patients is too far reaching, investment in sustainable global health programs may be a way of reaching more patients in their home countries.all children or should children in the local community be prioritized? In general, if resources are limited, how should international programs be instituted so that the local/domestic community does not have reduced opportunities to access local medical care and services? Although ideally the goal of equitable access to medical care is often on the global scale  are available on request to the corresponding author.EB conceptualized the pilot study, conducted the survey and interviews, performed the initial analysis, drafted the initial manuscript, and approved the final manuscript as submitted. JC, DD, and ML-N helped to design the study questions, vetted themes and questions, reviewed and revised the manuscript, and approved the final manuscript as submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P > 0.05). Feed intake was reduced with increased energy densities (P < 0.001). Weight gain and FCR were improved with increased dietary energy densities (P < 0.0001). Starch to lipid ratios linearly increased weight gain  and feed intake  without influencing FCR (P > 0.75). Both nutrient densities and starch to lipid ratios significantly impacted on carcass weight and yield. Heavier carcass weights and higher yields were observed in broiler chickens offered diets with high nutrient density (P ≤ 0.001). Carcass weight  was positively correlated with starch to lipid ratios and this tended to be the case for carcass yield  too. However, there were interactions on lipid concentrations in carcass (P < 0.001) as broiler chickens offered diet containing the lowest nutrient density and the highest starch to lipid ratio had the highest lipid carcass concentration of 12.94%. In conclusion, protein and energy need to be considered in tandem in practical diet formulation, especially in diets containing high crystalline amino acid inclusions. The impact of lipid on feed intake and starch on carcass lipid concentrations should also be taken into consideration.Twelve experimental diets with three levels of energy densities  and fours levels of starch to lipid ratios  were offered to 288 male Ross 308 broiler chickens. All the diets were formulated to contain consistent digestible lysine to metabolisable energy ratios (0.87 g digestible lysine/MJ AMEn) and ideal amino acid ratios. Growth performance was monitored from 7 to 27 days post-hatch and parameters of nutrient utilisation  were determined from 24 to 26 days post-hatch. Apparent protein (N) and starch digestibility coefficients, carcass yield and composition were determined at 27 days post-hatch. There were no interactions between energy densities and starch to lipid ratios on growth performance and carcass weights ( The influence of protein and energy on growth performance and carcass traits in broiler chickens have been intensively studied in the literature and the investigation is ongoing because the performance of modern broiler chickens improve every couple of years as a result of successfully selective breeding program. However, starch and lipid are the two major source of energy in typical broiler diets and little information about the influence of starch and lipid on bird performance and nutrient utilisation has gained in the literature. Starch is the most abundant macronutrient in broiler diets and its primary function is to provide energy for maintenance and growth and escalating dietary starch concentrations would increase lipid concentrations in carcass. Dietary lipid for poultry, including poultry fat and vegetable source oil, has higher energy density but is more expensive than starch. Its concentration is also critical to pellet quality and feed intake. Liu et al.  reportedad libitum access of feed. Indeed, poultry nutritionists may choose to fix the ratio of protein and energy in practical least-cost feed formulation in order to conveniently change dietary nutrient densities according to the fluctuation of feed ingredient prices. This approach is suitable when broiler chickens tend to eat to a constant energy intake regardless of the energy density of the diet [Amino acids and energy are considered in tandem in the formulation of practical diets for monogastric animals  and Lewithe diet ; howeverthe diet , 7. Liu the diet  reportedThe present study is one of a series to investigate the influence of macronutrients on feed intake, weight gain and carcass composition in broiler chickens. The details of bird management, diet preparation and sample collection were similar to methodology described in Liu et al. .The feeding study comprised twelve dietary treatments with three energy density levels  and four starch to lipid ratios  as shown in TM World Minerals, Lompoc, CA, USA) was included in the diets at 20 g/kg as an inert marker to determine nutrient digestibility coefficients at the distal jejunum and distal ileum at 27 days post-hatch. Starch concentration were determined by a procedure based on dimethyl sulfoxide, α-amylase and amyloglucosidase, as described in Mahasukhonthachat et al. [Maize was hammer-milled through 6.0 mm screen prior to mixing with the other ingredients and diets were cold-pelleted and then crumbled. Acid insoluble ash . Male, one day-old chicks (Ross 308) were received from a commercial hatchery and were offered a commercial starter diet to 7 days post-hatch. They were then individually identified (wing-tags), weighed and allocated into bioassay cages, with dimensions of 750 mm in width, 750 mm in length and 510 mm in height, on the basis of body weight in an environmentally-controlled facility. There was no statistical difference on the average body weight for each cage at the beginning of the feeding study. Each of the dietary treatment was offered to four replicate cages (6 birds per cage) or a total of 288 chicks from 7 to 27 days post-hatch. Broilers had unlimited access to water and feed under a ‘23-hour-on-1-hour-off’ lighting regime for the first three days and then under a ‘16-hour-on-8-hour-off’ lighting regime for the remainder of the study. Room temperature was maintained at 32°C for the first week, then gradually decreased to 22 ± 1°C by the end of the third week and maintained at the same temperature until the end of the feeding study. Body weight and feed intake were recorded weekly from which feed conversion ratios (FCR) were calculated. The incidence of dead or culled birds was recorded daily and their body-weight was used to adjust FCR calculations.Total excreta were collected from 24–26 days post-hatch from each cage to determine parameters of nutrient utilisation which included apparent metabolisable energy (AME), metabolisable energy to gross energy (GE) ratios (AME:GE), nitrogen retention and N-corrected apparent metabolisable energy (AMEn). Excreta were air-forced oven dried for 24 h at 80°C. The GE of diets and excreta were determined by bomb calorimetry using an adiabatic calorimeter .The jejunum is reported to be the major site of glucose and amino acid absorption but the extent of nutrient digestion at the end of ileum, which is often expressed as apparent ileal digestibility coefficient, is usually reported in the literature , 12. AppTwo birds from each cage whose body weight was close to the cage mean were selected for carcass composition analyses. The carcass was weighed with feathers but without viscera to calculate carcass yield before processing. Then, the carcass was autoclaved, ground and freeze-dried to analyse for nitrogen, lipid, DM concentrations and GE as described previously.The AME values were calculated on a DM basis from the following equation:the body . ApparenAcid insoluble ash (AIA) was included in the diets at 20 g/kg as an inert marker. Apparent digestibility coefficients of starch and protein (N) were calculated by the following equation:Starch and protein (N) disappearance rates (g/bird/day) were deduced from feed intakes over the final phase of the feeding period from the following equation:Experimental data were analysed using JMP 9.0.0  and response surfaces were generated with R 3.1.3 software. The experimental units were replicate cage means and statistical procedures included analyses of variance using the general linear models and a probability level of less than 5% was considered to be statistically significant by Student’s t-test. Additionally, the response surface plots were constructed so that the effects from changing factor levels on the examined responses can be visualized and they were generated by generalized additive model with thin plate regression splines as the smoothing function, the details of analysis and interpretation of the contour plots can be reviewed in Solon-Biet et al. . Linear P > 0.30). The average weight gain and FCR for all the experimental treatments from 7 to 27 days post-hatch was 1591 g/bird and 1.407, respectively, which were clearly superior to the 2014 Ross 308 performance objectives of 1292 g/bird weight gain with and FCR of 1.463. The influence of nutrient densities and starch to lipid ratios on growth performance and carcass traits from 7–27 days post-hatch is shown in P > 0.05). Feed intake was reduced by 6.4% with increased energy densities  and weight gain and FCR were improved with increased dietary energy densities by 10.5% (1511 versus 1670 g/bird) and 15.3% (1.533 versus 1.298), respectively (P < 0.001). There were significant linear relationships between starch to lipid ratios with weight gain  and feed intake . Broiler chickens offered diets with the lowest starch to lipid ratio had significantly inferior weight gain (1498 g/bird), feed intake (2098 g/bird) and carcass weight (1505 g/bird) in comparison to birds offered diets containing the other three levels of starch to lipid ratios. In contrast, FCR was not influenced by dietary starch to lipid ratios (P = 0.794). Both nutrient densities and starch to lipid ratios had significant impacts on carcass weight and yield. Carcass weight  and yield  decreased with reduced starch to lipid ratios. Broiler chickens offered diets with the highest starch to lipid ratio had significantly higher carcass yield compared to the other treatment groups (P = 0.001). There were treatment interactions on carcass GE (P = 0.002). In low density diets, broiler chickens offered starch: lipid ratio of 14 had significantly higher carcass GE than birds offered diets containing the other three starch: lipid ratios. Differently, in medium density diets, starch: lipid ratios did not influence carcass GE. In high density diets, broiler chickens offered starch: lipid ratio of 4 had significantly lower carcass GE than birds offered diets containing the other three starch: lipid ratios. There were no dietary effects on carcass protein concentrations (P > 0.50) and DM concentrations (P > 0.15). There were interactions on carcass lipid concentrations (P < 0.001). Broiler chickens offered diet containing the lowest nutrient density and highest starch to lipid ratio had the most carcass lipid (12.94%) whereas broiler chickens offered diet containing the highest nutrient density and lowest starch to lipid ratio had the least carcass lipid (8.25%).The mortality rate during the experimental period was 1.7% and it was not related to dietary treatments (P <0.001) and distal ileum (P < 0.0001). Broiler chickens offered high density diets had significantly higher apparent protein digestibility coefficients in the distal jejunum and distal ileum than broiler chickens offered low and medium density diets. Boiler chickens offered diets containing starch: lipid ratio of 4 had significantly lower apparent protein digestibility coefficient in the distal ileum (P = 0.004) in comparison to birds offered diets with all the other three ratios. Dietary nutrient density did not influence apparent starch digestibility coefficients in the distal jejunum (P = 0.497) and distal ileum (P = 0.639). Starch to lipid ratios significantly influenced apparent starch digestibility coefficient in the distal jejunum (P = 0.023) and the only significant difference was observed between birds offered diets with starch: lipid ratio of 14 and 12 (0.967 versus 0.934). There was an interaction between nutrient densities and starch to lipid ratios on apparent digestibility coefficients of starch in the distal ileum (P < 0.001). In low and medium density diets, broiler chickens offered diets containing starch: lipid ratio of 7 had significantly lower apparent starch digestibility coefficient in the distal ileum in comparison to birds offered diets with all the other three ratios. However, in high density diets, broiler chickens offered diets containing starch: lipid ratio of 4 had significantly lower apparent ileal starch digestibility coefficient compared to birds offered diets with all the other three ratios.The influence of energy densities and starch to lipid ratios on distal jejunal and distal ileal apparent digestibility coefficients and disappearance rates of starch and protein are shown in P < 0.001) and starch to lipid ratios (P < 0.02). Generally, disappearance rates of starch and protein were improved with increased nutrient densities regardless of the site of small intestine. There were positive correlations between starch to lipid ratios and protein (N) disappearance rate in the distal jejunum  and distal ileum ; starch disappearance rate in the distal jejunum  and distal ileum .Disappearance rates of starch and protein in the distal jejunum and distal ileum were influenced by nutrient densities (P < 0.005). Starch: lipid ratios did not influence AME in broiler chickens offered low density diets; whereas, in high density diets, birds offered starch: lipid ratios of 4 and 7 significantly higher AME than broiler chickens offered diets with the other two ratios. Broiler chickens offered the lowest nutrient density diet and the lowest starch to lipid ratio had the lowest AME  and AMEn . N retention reduced with starch to lipid ratios  and the lowest N retention (68.2%) was observed when the starch to lipid ratio was equal to 4 (P = 0.012). Increasing energy densities from 11.25 to 13.50 MJ/kg significantly reduced water intake by 14.6% .The influence of nutrient densities and starch to lipid ratios on nutrient utilisation in broiler chickens is shown in P < 0.0001), a 15.9% reduction in weight gain  and FCR was compromised by 13.8% FCR . The lipid-induced triggering of the ‘ileal brake’ may be the cause of feed intake reductions in diets with higher lipid concentrations. Martinez et al. [The response surface plots were constructed so that the effects from changing factor levels on the examined responses can be visualized and they were generated by generalized additive model with thin plate regression splines as the smoothing function. The influence of analysed dietary starch, protein and lipid concentrations on feed intake in broiler chickens from 7–27 days post-hatch is shown in z et al.  proposedz et al. . Consistz et al.  concludez et al. . TherefoWith the dietary protein and starch concentration comparisons in 2 = 0.005) and this suggests that broiler chickens had a constant protein intake of approximately 450 g/bird during the experimental period regardless of the dietary protein proportions. In contrast, starch intakes increased linearly  with its proportion in the diets. When the ratio of starch to non-starch dietary components increased, there was also an increase in the intake of starch. There was a quadratic relationship between lipid intake and ratios of lipid to non-lipid components. Lipid intake increased with the ratio of lipid to non-lipid components in the diets and it reached maximum of 167 g/bird when the ratio of lipid to non-lipid components equaled 0.198. This suggests that broiler chickens do not eat to constant starch and lipid intake target; however, that lipid intake reached a plateau may be due to the impact of lipid on total feed intake in broiler chickens. Increasing dietary lipid concentrations from 23.5 to 71.0 g/kg or decreasing starch to lipid ratios significantly depressed feed intake by 10.5% . This observation accords with the findings of Liu et al. [P < 0.0001) in broiler chickens offered diets containing the higher lipid concentration. Consistently, high lipid diets in the present study contained higher fibre concentrations  as shown in Simpson and Raubenheimer  advancedP < 0.001).It is straightforward that increased nutrient density improved feed conversion and energy utilisation . A furthP < 0.05) with apparent digestibility coefficients of protein in the distal jejunum and distal ileum (P < 0.05) with energy utilisation . There were no treatment effects on protein concentrations in carcass (P > 0.30). The balance between protein and energy is required for muscle protein deposition [P = 0.018).In the present study, weight gain and FCR were correlated Click here for additional data file.


Local stiffness of polymer chains is instrumental in all structure formation processes of polymers, from crystallization of synthetic polymers to protein folding and DNA compactification. We present Stochastic Approximation Monte Carlo simulations—a type of flat-histogram Monte Carlo method—determining the density of states of a model class of single semi-flexible polymer chains, and, from this, their complete thermodynamic behavior. The chains possess a rich pseudo phase diagram as a function of stiffness and temperature, displaying non-trivial ground-state morphologies. This pseudo phase diagram also depends on chain length. Differences to existing pseudo phase diagrams of semi-flexible chains in the literature emphasize the fact that the mechanism of stiffness creation matters. Local stiffness or semi-flexibility of polymer chains is probably by far the most important design concept for the creation of ordered polymeric structures. This is obviously true for (semi-)crystalline synthetic polymers which order into lamellar structures , displayproteins , as wellproteins .Theoretical and computational modeling of semi-flexible chains started very early on, e.g., by Flory’s approach to model the isotropic-nematic transition in semi-flexible polymers using a simple lattice model . The mosIn a computer simulation of semi-flexible chains, the persistent mechanism of flexibility is most easily realized by a bond angle potential that favors zero degrees in its ground state. Such a modeling approach has been used to explain the occurrence of toroidal structures in the collapsed state of stiff DNA chains ,14. The te Carlo ,20 study[N=13−55  later co[N=13−55 ,23, and A different mechanism of introducing stiffness was suggested for the modeling of proteins. In one variant, chains described as thick tubes were suggested ,25, whicL, to sphere diameter, σ, the more the bond angles are restricted to large values  ,34, a veM is the number of energy values or energy intervals for which one wants to estimate M is subtracted for all E, and only for the new energy The SAMC update of the current estimate for the logarithm of the density of states is (2)ln[g a broad plateau region with a high energy (very broad) maximum for h), which then collapse into liquid globules (configurations i and j), and, for k). For h) shows no crystalline order but begins to show toroidal segments or helical structures also visible in configuration m, which is an intermediate temperature configuration at a, b, c and g, next to hairpin structures like configuration f and tennis racket like structures like configuration d, which occur in this bond length range at high temperatures at or above the collapse transition.Combining the analyses of different micro-canonical and canonical observables, we obtain the pseudo phasediagram shown in b is of special interest because it is a true trefoil knot. In this context, configuration d is also of interest because it is the first step on the path to the trefoil knot, which is shown in a in c. Finally, the knot tightens (d) and, for very low temperatures, the two ends come into contact  is kept constant. Our very stiff chains can not fold back, and consequently the lamellar, folded structures are not found in the phase diagram. Hairpins and toroids exist at high stiffness, but they are no longer the ground state, which is a knotted structure, ideally the trefoil knot as for  like in ,23, wher like in , and thiWe have presented flat histogram Monte Carlo simulations using the SAMC approach to determine the pseudo phase diagram of single semi-flexible chains as a function of stiffness and temperature. Our model consists of overlapping hard spheres interacting with a square well attraction. The degree of overlap determines the stiffness of the chains. In this aspect, the model is close to the behavior of real polymers, where typically the van der Waals radius of the atoms is almost twice as large as the bond length. We presented pseudo phase diagrams for chains of length  methods ,44.For our model, in which stiffness is induced by steric effects, i.e., it is of entropic origin, the ground state structures for stiff chains are knotted (similar to what was found by Marenz et al.  for intemer case ,38 intro


Despite advances in management of immunosuppression, graft rejection remains a significant clinical problem in solid organ transplantation. Non-invasive biomarkers of graft rejection can facilitate earlier diagnosis and treatment of acute rejection. The purpose of this study was to investigate the potential role of heparan sulfate as a novel biomarker for acute cellular rejection. Heparan sulfate is released from the extracellular matrix during T-cell infiltration of graft tissue via the action of the enzyme heparanase. In a murine heart transplant model, serum heparan sulfate is significantly elevated during rejection of cardiac allografts. Moreover, expression of the enzyme heparanase is significantly increased in activated T-cells. In human studies, plasma heparan sulfate is significantly elevated in kidney transplant recipients with biopsy-proven acute cellular rejection compared to healthy controls, recipients with stable graft function, and recipients without acute cellular rejection on biopsy. Taken together, these findings support further investigation of heparan sulfate as a novel biomarker of acute cellular rejection in solid organ transplantation. Organ transplantation is the definitive treatment for end-stage organ disease. Despite advances in our understanding of immune mechanisms and immunosuppression therapy, graft rejection remains a primary driver of long-term graft failure, with 25–50% grafts lost within 5 years , 2. In tThe “gold standard” for diagnosing transplant rejection is the detection of immune injury (e.g. lymphocyte infiltration) on graft biopsy. In kidney transplantation, biopsies are typically performed when serum creatinine (sCr) levels become significantly elevated over baseline. However, sCr is an imperfect indicator of allograft rejection for multiple reasons: it rises late in the process of immune injury, the magnitude of change above baseline can be minimal, and it is not specific for rejection. Protocol biopsies performed at designated intervals can detect rejection prior to elevations in sCr, but are invasive and present additional risk to patients and to the allograft, making this a suboptimal screening approach.Non-invasive biomarkers of acute allograft rejection are thus necessary to facilitate its early diagnosis and treatment. Although the search for an alternative biomarker has remained elusive, improved understanding of the cellular mechanisms involved in rejection may help inform rational biomarker development. For example, a critical determinant of allograft rejection is the capacity of T cells to extravasate into allografts from the vasculature. Barriers to T cell extravasation into tissues include: 1) the endothelial cell glycocalyx that regulates exposure of adhesion molecules, 2) the subendothelial basement membrane that prevents T cell penetration into tissues, and 3) the extracellular matrix that limits T cell mobility within tissue. A major structural component of these barriers is heparan sulfate (HS), a molecule that is degraded by a specific glycosylase, heparanase (Hpase) . TherefoPreviously, we have shown that HS is a biomarker of alloimmune injury, mediated by alloreactive T cells during graft-vs-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) . A sentiC57BL/6 and BALB/c mice were obtained from the National Cancer Institute at Charles River. All experimental procedures involving the use of mice were done in accordance with protocols approved by the Animal Care and Use Committee of Duke University.An established murine heterotopic heart transplant model was used , 9. AlloGeneral anesthesia is induced with isoflurane using a vaporizer. The depth of anesthesia is confirmed by toe pinch. The donor animal is shaved and prepped using Betadine and alcohol. A midline incision is made with the help of surgical scissors. The thorax is opened sharply and the SVC, IVC and the pulmonary veins are localized, dissected from the surrounding tissues, ligated, and divided. The aorta and the main pulmonary artery are transected with appropriate length and flushed with cold saline. The heart is placed in cold saline and used for immediate transplantation.General anesthesia is induced with isoflurane. The recipient is shaved and prepped using Betadine and alcohol. A midline abdominal incision is made from the symphysis to the pubis. The abdominal aorta and IVC are dissected from the surrounding tissues. The lumbar vessels emerging posteriorly from the great vessels are ligated with 11–0 monofilament suture. An aortotomy is made in the abdominal aorta of the recipient and the donor ascending aorta is anastomosed to the recipient abdominal aorta using 11–0 monofilament suture in an end-to-side fashion. The donor pulmonary artery is anastomosed to the recipient IVC in an end-to-side fashion with running suture of 11–0 nylon. The distal microvascular clamp is removed first followed by the proximal microvascular clamp. Patency and bleeding are inspected, and hemostasis is performed as indicated. The abdominal muscle-fascial layer is closed with a running suture of 5–0 Vicryl. The skin is closed with interrupted suture of 5–0 polypropylene. Topical bupivacaine 0.25% (2 mg/Kg maximum) is placed to the wound margins prior to closure. Buprenorphine SR 0.5–1 mg/kg SC is administered immediately prior to surgery.After experiments were complete, animals were sacrificed by carbon dioxide euthanasia with secondary bilateral thoracotomy.Immunohistochemical (IHC) stains of murine cardiac graft tissue sections were performed on post-operative day (POD) 7 following transplantation. Cryostat sections (10 μM) were dried, fixed in methanol, and incubated for 1 hour in blocking buffer . Heparanase (Hpase) was detected using polyclonal rabbit anti-Hpase antibody  and peroxidase-conjugated anti-rabbit IgG . Slides were then developed with diaminobenzidine substrate  and counterstained with hematoxylin . Quantification of Hpase-expressing graft infiltrating lymphocytes (GILs) was performed by counting the number of cells per high powered field (200X), and comparisons were then made between syngeneic and allogeneic cardiac transplants.Immunofluorescent staining of cardiac graft tissue was performed using rabbit polyclonal anti-CD3  and anti-heparan sulfate , followed by staining with Alexa Fluor 488 conjugated goat anti-rabbit IgG and Alexa Fluor 594 conjugated goat anti-mouse IgM . Fluorescent images were obtained using a Zeiss Axio Observer widefield fluorescence microscope and MetaMorph 7 image analysis software .Murine serum levels of HS were determined by heparan sulfate ELISA kits .Purified T cells were activated by treatment with 2 μg/mL Concanavalin A  for 96 hours in RPMI media supplemented with 10% fetal bovine serum.Jackson ImmunoResearch, West Grove, PA). Blots were then incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).For Western blots, 15 μg of proteins were resolved by SDS-PAGE and transferred to PVDF membranes . The antibodies and dilutions used in these experiments were as follows: Rabbit anti-mouse Hpase , HRP-conjugated goat anti-rabbit secondary antibodies , donkey anti rabbit-647 , CD4-BV510 , CD8-PE .De-identified plasma samples from kidney transplant recipients stored in the Department of Surgery Substrate Services Core Biobank under a Duke IRB-approved specimen collection protocol (IRBs #30485 and #71689) were analyzed for this study. Blood samples were obtained at regular time points and also in the setting of a clinically significant event, such as a graft biopsy, infection, or hospitalization. Available clinical data on the subjects include medical history, immunosuppressant medications, hospitalization history, microbiology data, laboratory tests, radiologic findings, and pathology results.Peripheral blood was also collected from healthy adult donors who signed written informed consented in accordance with the policies of the Duke Institutional Review Board. None of the subjects in this study were from a vulnerable population; all subjects provided written informed consent that was freely given.Subjects were divided into the following 4 cohorts:Healthy controls (N = 12): Healthy individuals with no history of solid organ transplantation.Stable transplant (N = 24): Kidney transplant recipients with no history of clinically significant events post-transplant and no history of graft biopsy.Rejection (N = 7): Kidney transplant recipients who experienced an episode of acute cellular rejection, proven by graft biopsy using Banff pathologic criteriaNo rejection (N = 8): Kidney transplant recipients who underwent graft biopsy for elevated sCr and were found to have no evidence of cellular or humoral rejection on final pathologic analysis.Plasma HS levels were measured for all subjects by ELISA and were compared among all cohorts. For the “Rejection” and “No rejection” cohorts that had undergone graft biopsy, the average and maximum plasma HS and serum Cr levels were compared for a 28 day window (± 14 days) around the date of biopsy. The correlation between plasma HS and serum Cr were subsequently analyzed for the “Rejection” and “No rejection” cohorts.In a second analysis, serum levels of HS were measured for a cohort of kidney transplant recipients with BK or CMV viremia, to assess whether tissue damage from either of these viral infections impacted blood HS levels. No interventions or changes to the patient’s care plan were made on the basis of results obtained in this study.For murine studies, comparisons between group means were made using student’s T-test with GraphPad Prism 5.0 . A p-value < 0.05 was considered statistically significant. For human studies, Kruskal-Wallis test was used to test for the overall difference in plasma HS among cohorts. Subsequently, pairwise comparison using Wilcoxon rank sum test was conducted with Bonferroni adjustment for multiple testing. Correlation between plasma HS and serum Cr was calculated using Spearman correlation. Statistical analyses were performed using R Version 3.4.1 software .We first assessed the expression and activity of Hpase in murine cardiac grafts . Immunohistological examination of tissue sections of allogeneic heterotopic heart transplants demonstrates increased levels of Hpase present in rejecting murine cardiac allografts , compareWhile both syngeneic and allogeneic grafts demonstrate Hpase in the endothelium, an increased number of Hpase-expressing graft-infiltrating leukocytes (GILs) was present in rejecting allografts compared to syngeneic grafts .We also examined whether HS was degraded in cardiac allograft tissue during T cell infiltration. Detection of CD3+ T cells and HS in rejecting cardiac allograft tissue by immunofluorescence shows dissolution of the abundant intercellular HS by advancing T cell infiltrates , Fig 1D,In light of the findings above that suggest degradation of HS is mediated by activated T cells, we examined mouse T cells in vitro to investigate changes in Hpase expression during the activation process. Purified mouse CD4+ and CD8+ T cells were activated using Concanavalin A (ConA), a pan-T cell activator. Both naïve CD4+ and CD8+ T cells demonstrated a robust increase in Hpase expression upon activation , Fig 5.In order to investigate the potential role of HS as a biomarker of kidney allograft rejection, we measured plasma HS levels in prospectively collected samples from kidney transplant recipients. Plasma HS levels were compared among four cohorts: healthy controls (N = 12), stable post-transplant recipients (N = 24), recipients with biopsy-proven acute cellular rejection (N = 7), and recipients who had undergone biopsy for elevated sCr but without evidence of rejection (N = 8).We observed significantly elevated plasma HS levels in patients with biopsy-proven acute cellular rejection in comparison to other cohorts , Table 2When comparing the sCr levels in the “Rejection” and “No rejection” cohorts, we did note a significant elevation in sCr in the “Rejection” cohort, as expected . There wIn clinical practice, elevations in sCr due to viral infection cannot be distinguished from acute rejection without obtaining a biopsy. In order to establish that HS was specifically associated with acute rejection and not viral infection, we examined HS serum levels in patients with CMV and BK viremia. Both of these viruses are carefully monitored in kidney transplant recipients as they have potential to lead to graft nephropathy. Interestingly, BK viremia did not result in any detectable HS elevation and CMV viremia produced only a mild increase, even at very high titers .Despite advances in immunosuppressive therapy, 25–50% of solid organ transplants fail within 5 years, primarily due to immune-mediated graft rejection , 2. WhilIn kidney transplantation, measurement of serum creatinine (sCr) is currently the primary approach in monitoring renal allograft function. However, sCr performs poorly as a biomarker of rejection for several reasons: 1) elevation of sCr occurs late in the process of immunologic injury, 2) the magnitude of change above baseline can be subtle and often less than 2-fold, and 3) elevation of sCr is non-specific for immune injury and can be elevated for other reasons such as dehydration. A biomarker more specific to early immune mediated graft injury would be a significant improvement over current methods for assessing graft rejection. Ideally, such an assay would be measured from a readily available patient sample (e.g. blood), would be reproducible in clinical laboratories, and would be easily interpreted to inform clinical decision making.In this study, we investigate the potential use of HS as a biomarker of acute cellular rejection. HS is a highly sulfated polysaccharide that is a common component of the extracellular matrix, basement membrane and vascular glycocalyx. Due to the high degree of sulfation, HS carries a substantial negative charge under physiologic conditions and is repulsive to similarly charged cell membranes. Consequently, HS acts as both a physical and an electrostatic barrier to cell migration. Because HS also binds a multitude of proteins, including cytokines, chemokines and growth factors, its degradation affects numerous biological functions, including cell signaling and motility , 11–13. In mammals, HS is degraded exclusively by the enzyme heparanase (Hpase), an endo-β-D-glucuronidase , 14, 17.In this study, we demonstrate that Hpase-mediated release of HS has significant potential as a sensitive and specific biomarker of acute rejection. Our murine cardiac transplant studies demonstrate increased expression of Hpase in rejecting cardiac allografts along with disappearance of tissue HS in areas of T cell infiltration. Accordingly, both naïve CD4+ and CD8+ T cells express Hpase at baseline and demonstrate increased Hpase expression following activation. Furthermore, serum HS becomes highly elevated during allogeneic, but not syngeneic cardiac transplantation, indicating that Hpase activity is specific to the alloresponse and not related to surgical injury or non-specific inflammation.In human kidney transplant recipients, we similarly found consistent elevation of plasma HS preceding the diagnosis of renal allograft rejection by biopsy. Amongst patients with rejection, plasma HS levels were increased early (>1 week before biopsy) and with a high magnitude relative to baseline (>10-fold higher). In contrast, patients that underwent graft biopsy, but had no histologic evidence of rejection, universally had baseline serum HS levels. Furthermore, BK and CMV viremia did not produce an elevation of HS levels, indicative of the specificity for HS for alloimmune-mediated injury. Most importantly, since all biopsies in this analysis were performed for cause (elevated sCr), these results indicate that had HS been used as the criteria for obtaining a graft biopsy, patients would have been diagnosed and treated earlier for graft rejection.In conclusion, HS appears to be a sensitive and specific biomarker of allograft rejection. We propose that Hpase-mediated degradation of HS represents a new avenue of investigation in transplantation and holds promise for the development of biomarkers and novel strategies for reducing graft rejection. Specific inhibitors of Hpase may have the potential to lessen T-cell mediated pathology by limiting their access to the extravascular domain.


Pythiogeton isolated from wild rice exhibiting rot and dieback of roots and stalks in California is described. Pythiogeton manoomin sp. nov. is characterized by coenocytic hyphae, club-like appressorium, and terminal or intercalary sporangia, which are often a short distance from the end of supporting hyphae. The protoplasm is discharged through a discharge tube into an elongate transient vesicle, which soon disappears, leaving the naked protoplasm to differentiate into reniform zoospores. Pythiogeton manoomin also produces thick-walled pigmented chlamydospores, not found in other Pythiogeton species. In greenhouse trials, Pg. manoomin did not infect economically important crops such as rice, bean, chard, corn, carrot, lettuce, oat, radish, sweet pepper, tomato, or wheat. Phylogenetic analysis based on ITS data supports the conclusion that this organism is a new species that is most closely related to Pg. ramosum. In this paper, we describe morphological characteristics, temperature–growth relationships, pathogenicity, and phylogenetic relationships that support the description of this taxon as a new species, Pythiogeton manoomin sp. nov.A new species of urn:lsid:zoobank.org:pub:4C63AAA4-4D4A-4679-A344-79B75121A5C6 Pythiogeton (Pg.), together with Pythium and Phytophthora, belongs to the family Pythiaceae of the Oomycota. Minden first described members of the genus in 1916, and since then, Pythiogeton species have been reported in the USA , Pg. microzoosporum , Pg. oblongilobum , Pg. paucisporum , Pg. proliferatum , and Pg. puliensis . Most described Pythiogeton species are saprophytic, with only three species, Pg. zeae, Pg. zizaniae, and Pg. ramosum known to be pathogenic to corn, water bamboo, and ginger, respectively  or manoomin, as it is called in the Ojibwe language, is a semi-aquatic grass species endemic to North America and China. Three species of wild rice, Zizania palustris, Z. aquatica, and Z. texana, are native to North America, and one species, Z. latifolia, is native to China. Wild rice is one of America’s oldest indigenous cultivated grains, with over 90 percent of cultivated wild rice grown in the United States produced in California and Minnesota  cv. Franklin was observed in a 16-ha field in Big Valley, Lassen County, California. Infected plants exhibiting rot and dieback of roots and stalks were in various stages of decline, including death. A pythiaceous fungus was consistently isolated from the diseased plant and was identified as Pythiogeton sp.  cv. Franklin exhibiting rot and dieback of roots and stalks were collected from Big Valley, Lassen County, California (GPS coordinates 41°08′41.93″ N 121°10′07.49″ W). Symptomatic stem and root tissues from affected plants were placed on 9 cm Petri dishes (four pieces per plate) of PARP agar , which were then incubated at 25°C in the dark for 1 week. Agar blocks 8 mm in diameter containing hyphal tips were transferred onto 9 cm Petri dishes (4 pieces per plate) of potato dextrose agar (PDA) and modified Rye B agar (RBA) containing 60 g rye-grain extract, 20 g of Difco Bacto Agar, and 20 g of sucrose as described by Caten and Jinks  from wild rice. A 3 day-old, 8 mm-diameter Rye B agar plug of each isolate was transferred onto Rye B agar plates for each of the nine temperatures evaluated. The plates were incubated at 4, 12, 15, 18, 21, 25, 28, 31, or 34°C in the dark, and the growth rate was determined daily by measuring the linear colony diameter in three places up to 5 d for each temperature. Three plates were used for each treatment, and the experiment was repeated three times with at least 24 h between each experiment. The average growth rate for each isolate from the three replications was calculated for each temperature. The average growth rate of three isolates was tested using analysis of variance (ANOVA) in R version 3.3.2 (The R Foundation).The effect of temperature on mycelial growth was evaluated on Rye B agar for three The morphology and development of sporangium were observed on Rye B agar using the method described by Chang  and HuanPg. manoomin to Avena sativa L. cv. Cat Grass , Zea mays L. cv. Golden Bantam Improved , Triticum aestivum cv. Liquid Sunshine , Lactuca sativa L. cv. Great Lakes Iceberg , Beta vulgaris subsp. cicla L. cv. Fordhook Giant , Capsicum annum L. cv. California Wonder (Burpee Garden Product Co.), Daucus carota subsp. sativus L. cv. Short ‘n Sweet (Burpee Garden Product Co.), Solanum lycopersicum L. cv. Moneymaker , Phaseolus vulgaris L. cv. Ejote silvestre Contender (Burpee Garden Product Co.), Raphanus sativus L. cv. Cherry Belle (Burpee Garden Product Co.), Oryza sativa L. cv. Calrose (USDA # C.I. 8988), and Zizania palustris L. cv. Franklin was determined in the greenhouse. Ten seeds of each were planted in sterilize sand in plastic pots measuring 10 cm in diameter (750 ml). After 5 days, six 8 mm-diameter agar discs from the margin of a 7day-old culture growing on modified Rye B agar were placed in each pot. Pots inoculated with six 8 mm-diameter agar discs from a modified Rye B agar plate were used as controls. After 20 d post inoculation, root and crown tissues were examined for symptoms and disease was recorded. The experimental design was a randomized block with ten replications (blocks) in the greenhouse on a 13-h photoperiod provided by high pressure sodium bulbs with daytime temperatures ranging from 80–85°C and nighttime temperatures ranging from 65–70°C. The pathogenicity test was repeated three times. Re-isolation of the pathogen from crown and root tissues was attempted for all inoculated plants, as described above. Damping-off assay was performed as above with the exception that 10 seeds were planted into sterilized sand that was pre-inoculated with 12 8 mm-diameter agar discs from the margin of a 7day-old culture grown on modified Rye B agar. After 10 d post inoculation, germinated seeds were counted, and re-isolation of the pathogen from seeds was attempted, as described above.Pathogenicity of Pythiogeton manoomin isolates  was extracted from mycelia using Qiagen® DNeasy Plant Mini Kit ™  according to the manufacturer’s protocol. The internal transcribed spacer (ITS) region was amplified by PCR and sequenced using universal ITS5 and ITS4 primers , 0.1 μL each of a 50 μM concentration of primers, and 27.6 μL sterile deionized water. The thermal cycling parameters were initial denaturation at 94°C for 5 min followed by 34 cycles consisting of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2.5 min. A final extension at 72°C for 10 min was done at the end of the amplification followed by 4°C. The PCR products were purified with GeneJet PCR Purification Kit . Sequences of the forward and reverse strand were conducted by the College of Biological Sciences UCDNA Sequencing Facility .Total genomic DNA from three http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The ITS sequence for all three Pythiogeton manoomin isolates was identical. Therefore, the sequences of Pythiogeton manoomin isolate BV1 was submitted to GenBank (KF719169). The ITS sequence (JQ610201) of Pythium aphanidermatum was selected as an outgroup. Sequences were aligned using ClustalW, and a phylogenetic tree was constructed with MEGA 5.03  cv. Franklin from a commercial agricultural field on 20 June 2012 and deposited in the University and Jepson Herbaria (UC 2060102), Berkeley, California. The living culture of the isolate was deposited and maintained at the Centraalbureau voor Schimmelcultures (CBS 145436), Fungal Biodiversity Centre, Utrecht, The Netherlands. Description of the species was submitted to MycoBank (MB 830264) and the ITS sequence was deposited in GenBank (GenBank Accession No. KF719169).United States, California: Lassen County. Isolate BV1, designated as the holotype, was recovered from root tissue of wild rice . Discharge tube 5–100 μM long and 5 μM wide, protruding from different positions around sporangia. Encysted zoospores 15–25 μM in diameter. Sexual structures absent. Cardinal temperatures  for grow– Pathogenicity of Pg. manoomin to wild rice was confirmed by inoculation followed by re-isolation. After 14 days, 84% of inoculated wild rice plants in all tests developed root and basal stalk rot, consistent with the symptoms observed in diseased wild rice in the field. No symptoms were observed on other inoculated plants. In addition, Pg. manoomin caused damping-off of 71% of wild rice seedlings tested. Pythiogeton manoomin was consistently re-isolated on PARP from symptomatic wild rice plants and seeds, but not from other inoculated plants or seeds and control plants, thus fulfilling Koch’s postulate. Pythiogeton manoomin did not infect the seedlings of Asian rice, bean, chard, corn, carrot, lettuce, oat, radish, sweet pepper, tomato, or wheat. All control plants grown in noninoculated soil remained healthy at the end of the experiment.– The internal transcribed spacers (ITS) 1 and 2 flanking the 5.8S rRNA regions were amplified by PCR and sequenced using universal ITS5 and ITS4 primers. A BLAST search of the 855 bp sequences revealed 98% similarity with a sequence of Pg. ramosum isolate Pg-164 (GenBank Accession No. JQ610190.1). The 21 nucleotide differences suggest that the isolate from wild rice may be an unreported species. Based on previous published phylogeny, all Pythiogeton species belong to the same clade, separated from Pythium, Phytophthora, and downy mildew clades. Within the Pythiogeton clade, 10 subgroups (A1-A10) have been reported . Pythiogeton manoomin can be readily isolated from plant materials  submerged in stagnant water during crop cultivation. Host range tests indicate that the pathogenicity of Pg. manoomin may be limited to wild rice and does not infect seedlings of economically important crops such rice, bean, carrot, chard, corn, lettuce, oat, radish, sweet pepper, tomato, or wheat. However, Pg. manoomin can possibly infect some wild grasses or weeds not tested here.The genus Pythiogeton manoomin is characterized by ovoid to ellipsoid sporangia in terminal or intercalary positions. Like all members of the genus Pythiogeton, Pg. manoomin sporangia release undifferentiating protoplasm through a discharge tube into an elongate transient vesicle, which soon disappears, leaving the naked protoplasm to differentiate into reniform zoospores. Appressoria form sparsely in contact with hard surfaces, (i.e. bottom surface of Petri dish). Pythiogeton manoomin is morphologically and genetically related to Pg. ramosum. Initially, descriptions of most Pythiogeton species were based exclusively on uncultured material . However, the 21 nucleotide differences suggest that the isolate from wild rice is an unreported species.Morphologically, l Minden . IsolatiBipolaris oryzae , isolated from wild rice plants exhibiting rot and dieback of roots and stalks in California, is a distinct new Pythiogeton species.Wild rice, the only cultivated cereal native to North America, is a staple food of indigenous peoples (Hayes et al.


Heterotopic cardiac xenotransplantation in the intra-abdominal position has been studied extensively in a pig-to-baboon model to define the optimal donor genetics and immunosuppressive regimen to prevent xenograft rejection. Extensive investigation using this model is a necessary stepping stone toward the development of a life-supporting animal model, with the ultimate goal of demonstrating suitability for clinical cardiac xenotransplantation trials. Aspects of surgical technique, pre- and post-operative care, graft monitoring, and minimization of infectious risk have all required refinement and optimization of heterotopic cardiac xenotransplantation over time. This review details non-immunologic obstacles relevant to this model described by our group and in the literature, as well as strategies that have been developed to address these specific challenges. Heterotopic cardiac xenotransplantation model is widely used to study the immunology of xenograft rejection and to test various immunosuppressive regimens. This model has several advantages as it facilitates immunologic monitoring through the period of graft rejection at reduced cost and complexity compared to orthotopic models. Since the native heart remains in place, rejection of heterotopic xenograft does not result in primary hemodynamic compromise. This model has additionally allowed for the successful demonstration of multiple immunosuppression regimens that have resulted in long-term xenograft survival –4. Howevde novo cross-species incompatibilities as well as the use of therapeutic immunosuppressive agents, such as anti-CD154 ligand antibody  is our standard for dysrhythmia prophylaxis. Heparinization is achieved with 500 IU/kg 3 min prior to cross-clamp. Diastolic arrest is achieved with 20–30 cc/kg of cold crystalloid cardioplegia, followed by cold static ischemia. We generally use Custodial HTK, but equivalent commercial preservation solutions, such as Belzer UW are acceptable.During implantation, care must be taken to protect bowel and bladder during exposure of the abdominal aorta at the level of the iliac bifurcation. Additionally, geometric considerations during implantation of the graft are key, as kinking of either donor aorta or pulmonary artery can lead to inflow or outflow obstruction that may result in stasis, thrombus formation, and graft failure. The pulmonary artery to inferior vena cava anastomosis is particularly susceptible to thrombosis from angulation at the time of abdominal closure. As in an orthotopic position, de-airing of the heart and establishment of normal sinus rhythm are integral to long-term graft survival, and electrocardioversion is occasionally required to establish such a rhythm as described in the arrhythmias in donor heart section below. Adams et al. described early experience with avulsion of the aortic suture line immediately upon post-operative movement of the baboon into an upright position. The authors report that particular care with replacement of bowel loops into anatomic position prior to closure prevented any further disruption of the graft secondary to shifting of intra-abdominal contents .A number of supportive measures are required to maintain optimal function of a heterotopic xenograft that would not be utilized in an orthotopic model due to its non-load bearing, relatively low-flow state. Continuous intravenous heparin is required to keep the activated clotting time to twice its baseline level to minimize clot formation. This is particularly imperative with longer surviving xenografts as formation of left ventricular thrombi are more frequently observed. In addition, continuous infusion of heparin has been demonstrated to have a mitigating effect on the development of consumptive coagulopathy, and has been utilized in other models, including the kidney xenograft model .Cefazolin is routinely utilized for bacterial prophylaxis for 1 week following placement of a central venous catheter . This line is maintained until the xenograft is explanted to allow for delivery of medications over extended periods of time , access for routine blood draws, or administration of therapies during emergent circumstances. The central venous catheter traverses through a jacket and tether system  that protects it from inadvertent damage by the baboon and allows for access by the research team from outside the baboon cage. The long-term tethering system permits relative mobility of the animal while still providing durable and reliable intravenous access for the research team . Over tiImplanted telemetry  as described elsewhere  is utiliThe disparity between the donor and the recipient size plays an important role in subsequent development of complications. A heart from a smaller pig (3–4 weeks old ~5–6 kg in weight) transplanted in a larger baboon (>10 kg) allows enough room for the heart to function without compression and fibrillation at abdominal closure. On the other hand, a larger heart in a smaller baboon appears to compress the graft, restricting its systolic function, abdominal compartment syndrome with hypotension of the recipient and induces arrhythmias leading to loss of the xenograft if not recognized and treated promptly. The relatively small size of available recipient baboons and our preference to use smaller baboons to save on costly immunosuppressive drugs limits the size of potential donor pig hearts. In our group's experience, grafts from larger donor pigs become compressed by the recipient's abdominal wall during closure, resulting in elevated abdominal compartment pressures. Size matching in the heterotopic model has always been a challenge, even prompting early groups to utilize cervical implantation as an alternative , 17. WhiCoagulation Dysfunction, below. The need for continuous heparin infusion further increases the risk of bleeding in the xenotransplant setting.Hemorrhagic complications in our experience are attributed to several causes. Consumptive coagulopathy is a known sequela of xenograft placement and generally occurs within 4 weeks of transplantation. In our early experience, this occurred in ~10% of our cases and is discussed in more detail in In some initial experiments, the site of the LV telemetry probe bled; this was prevented in later transplants by securing the probe tightly with pledgetted Prolene sutures . In two Use of costimulation blockade of the CD40/CD154 pathway for induction and maintenance immunosuppressive therapy has led to the recent profound prolongation of graft survival. Early experience utilizing anti-CD154 antibody was successful in immunomodulation but led to an unacceptable incidence of complications from thromboembolic events . Phase IIn addition to the bleeding associated with the single case of adhesed mesenteric vessel rupture, intestinal adhesions are a risk specific to this model. While adhesions are a risk associated with any abdominal operation, the inflammation associated with xenograft placement confers a particularly high risk of significant adhesion formation. As shown in Atrial and ventricular fibrillation, ventricular tachycardia and bundle branch block were witnessed in conditions when larger hearts were used in relatively small baboons. Pig hearts, and genetically-modified (GM) pig hearts in particular, are more prone to fibrillation than human or non-human primate hearts. Subtle differences in ion channel expression in the GM pig heart are hypothesized to account for this increased myocardial irritability . We utilTwo types of disorders were observed related to dysregulated coagulation. The consumptive coagulopathy typically seen in this model has been described by others –25; howe3. Initially small amounts of packed red cell transfusion (60–70 ml) transiently improved the condition but was not able to sustain the affect. Later we were able to transfuse large quantities of blood (20 ml/kg packed RBC's) that greatly helped in restoring the HCT to normal. Over a period of time, platelet numbers also improved in these baboons. In three cases where multiple  blood transfusions were deemed clinically necessary the recipient succumbed in association with uncontrolled consumptive coagulopathy.We observed a drop in hematocrit (HCT) to 15% in four recipients who also developed bruising over their abdomen. The platelet numbers in these baboons also dropped to 50–70,000/mmInfectious complications are of paramount significance in the immunosuppressed recipient. Routine use of prophylactics (Cefazolin for the first week following transplant and ganciclovir throughout the duration of immunosuppression) is mandatory in our model. Additionally, long-term telemetry device placement allows for continuous body temperature recording such that any febrile episodes followed by blood culture were promptly evaluated and treated. In fact, the benefit of the implanted telemetry device, primarily designed for direct pressure monitoring of the graft, was primarily through the early detection of central temperature spikes. This allowed us to overcome the majority of potential infections complications efficiently with the use of broad-spectrum antibiotics in any recipient exhibiting a fever >39°C. One case of fungal infection (Zygomycosis) did occur that was ultimately diagnosed by identification of fungus on histological analysis . No clinOne early series, prior to the use of implantable telemetry and continuous temperature monitoring, demonstrated 23 episodes of infection in a series of 16 baboons; 14 of these episodes resolved with treatment, although unfortunately 8 resulted in death of the recipient . In thisWe have found with the use of early intervention based on results from continuous non-invasive monitoring that clinically significant infections are rare, and controlled effectively with bacterial, viral, and fungal prophylaxis, as well as prompt treatment of fever with broad-spectrum coverage , 27.Two baboons suffered sudden death without any prior sign and symptoms. Necropsy excluded acute hemorrhage, pulmonary embolus, pneumonia, and intra-abdominal catastrophe, but did not yield any conclusive evidence regarding the cause of death. Ventricular fibrillation is a potential etiology that could not be excluded on post-mortem exam.The heterotopic transplant model has served as a useful tool to study the immunological barriers to xenotransplantation. This model has also allowed testing and refinement of several immunosuppressive and immunomodulatory agents and approaches, and demonstrated the clear benefits associated with various genetic modifications to donor pigs. While a number of complications remain in this model, the immunologic success has formed the foundation for experiments in the orthotopic model. The successful survival of the intra-abdominal heterotopic cardiac xenograft requires vigilance by the scientific team; so, too, will success in the orthotopic model.We recently reported the longest survival of 945 days in a heterotopic xenotransplantation model utilizing either GTKO pigs that also express human CD46 and thrombomodulin (GTKO.hCD46.TBM) , 28. ThaSome of the early hurdles were related to the tether, animal jacketing and jugular intravenous catheters. These were overcome following several modifications. We faced the problem of consumptive coagulopathy and thromboembolism in a few baboons but mostly these complications occurred within the first post-transplantation month. Some of the late complications included sudden death, uncommon infections and abdominal bleeding due to rupture of the transplanted heart or mesenteric vessels adherent to the xenograft. While the animals did undergo multiple procedures with multiple cardiac stressors, no evidence of native cardiomyopathy or depressed myocardial function in the native heart was observed in our experiments.Typical consumptive coagulopathy reported by many was not common in our experiment. We did notice signs similar to consumptive coagulopathy in two baboons. These symptoms were overcome in one case with a series of packed red cell infusions to restore normal coagulation profile. However, three other cases resulted in demise of these baboons. Based on this, we recommend that persistent or recurrent anemia in this model should be treated by graft removal or euthanasia. The coagulopathy observed was limited to a sharp decrease in platelet numbers and elevation of activated clotting time of blood. This early drop in platelet numbers is likely due to deposition in the graft. When the graft survives this insult, the numbers normalize on their own. This is inconsistent with an immune-mediated process, such as heparin-induced antibody mediated thrombocytopenia, which we have not seen in our experiments.In one case thrombosis of the intravenous catheter and subsequent occlusion of a major vessel also hampered the success of this experiment. Some baboons required higher doses of heparin infusion  to achieve target ACT levels of 2 × baseline. Thrombotic microangiopathy related to anti CD154 has been described  but in oIntestinal adhesions to the heterotopic heart caused recipient mortality in two recipients, due to bowel obstruction with aspiration, intestinal rupture, and mesenteric vessel erosion. These complications led to recipient baboon's mortality before the transplanted heart was rejected. In additional cases, bowel obstruction due to intestinal adhesions was relieved surgically when identified early by abdominal X-ray, and we recommend use of X-ray imaging early for any baboon with difficulty tolerating a diet, lethargy, or signs of discomfort or systemic illness without decreased graft function.The use of telemetry implant helped accurate measurement of graft function and facilitated early detection of graft dysfunction . The heart rate and ventricular contractility based on left ventricular peak systolic and end diastolic pressure accurately and non-invasively measured graft function. The temperature probe was very useful in detecting the early onset of infection and these infections were treated at an early stage. Therefore, this complication was not a significant factor in our experiments, in contrast to experiences reported by others . ContinuIn conclusion, long-term xenograft survival has been achieved in a large animal heterotopic model where the loss of recipients to model-associated complications often used to occurs before the loss of graft function. While most of these complications are avoided in our recent experiments, we conclude that, based on encouraging results with current GM pig lines and immunosuppressive regimens, the orthotopic model may offer new challenges but a more productive format as the basis for developing a clinically applicable regimen. With continued vigilance and experience with the intra-abdominal heterotopic model, with new GM pigs and novel target specific immunosuppressive agents, even further advancement toward a successful orthotopic xenotransplant model is within reach.All datasets generated for this study are included in the manuscript and/or the supplementary files.Results from our group are from both the National Institutes of Health and University of Maryland Medical Center and the experiments detailed in this review were approved by the respective Institutional Animal Care and Use Committees.MM, AS, BL, and LD are part of the Cardiac Xenotransplantation Program at the University of Maryland.LD participated in the performance of the research and wrote the paper. AS participated in the design and performance of the research and participated in writing and editing the paper. JC participated in the performance of the research, reviewed and edited the paper. NS and TZ participated in the performance of the research and writing the paper. BL participated in the design and performance of the research. DA, PC, and KH participated in the design of the research and in the final approval of the paper. MM designed and performed the research, participated in writing the paper, and provided final approval of the paper.DA is the CEO and President of Revivicor, Inc. The authors declare that funding was received from United Therapeutics Inc for this project. The manuscript was also approved for submission by United Therapeutics. The funder had no role in study design, data collection, data analysis, and preparation of manuscript. However, the main material (donor pigs) were provided by them and based on our agreement with them, they had to approve the manuscript for publication. All authors had part of their salary driven from this funding but had no other potential conflict of interest.