Mitochondrial respiratory chain (MRC) disorders are a complex group of diseases whose diagnosis requires a multidisciplinary approach in which the biochemical investigations play an important role. Initial investigations include metabolite analysis in both blood and urine and the measurement of lactate, pyruvate and amino acid levels, as well as urine organic acids. Recently, hormone-like cytokines, such as fibroblast growth factor-21 (FGF-21), have also been used as a means of assessing evidence of MRC dysfunction, although work is still required to confirm their diagnostic utility and reliability. The assessment of evidence of oxidative stress may also be an important parameter to consider in the diagnosis of MRC function in view of its association with mitochondrial dysfunction. At present, due to the lack of reliable biomarkers available for assessing evidence of MRC dysfunction, the spectrophotometric determination of MRC enzyme activities in skeletal muscle or tissue from the disease-presenting organ is considered the ‘Gold Standard’ biochemical method to provide evidence of MRC dysfunction. The purpose of this review is to outline a number of biochemical methods that may provide diagnostic evidence of MRC dysfunction in patients. The mitochondrial respiratory chain MRC;  is compo2 and H2O . Th. Th36]. chondria . In the to water ,37. Respto water . The exhto water . The stato water . The colto water ,38. MRC to water . Furtherto water . This merography . Howeverrography .The clinical determination of patient CoQ10 status is usually based upon measurements in plasma . HoweverThe most conventional methods used to determine CoQ10 status in patients are either high-pressure liquid chromatography with electrochemical detection (HPLC-ED) or ultraviolet (HPLC-UV) . It shouPatients with MRC disorders have also reported evidence of a deficit in the status of the antioxidant GSH . This deSkeletal muscle and leukocytes are the most reliable surrogates for determining patient GSH and GSSG levels . HoweverA deficiency in cerebrospinal fluid (CSF) 5MTHF status has been reported in patients with many forms of mitochondrial disease, including Kearns-Sayre syndrome (KSS). The transport of 5MTHF across the blood CSF barrier (choroid plexus) happens via an active transport mechanism . Thus, aIn order to determine the status of 5MTHF in the CSF, a lumbar punch must be performed after 4–6 h of fasting. CSF samples must be protected from light and frozen at −80 °C within 2 h of collection ,52. CSF In addition to determining CSF 5MTHF in mitochondrial disease patients, the level of ascorbic acid in the CSF should also be measured, as the antioxidant, ascorbic acid protects 5MTHF from ROS-induced degradation . Thus, aThe investigation of MRC disorders can be challenging due to struggles in diagnosis. However, there are reliable biomarkers present to assess evidence of MRC disorders that are more favourable, as they are less invasive—traditional methods include muscle biopsy and the spectrophotometric assessment of MRC enzyme activities  (ScholleFGF21 is a metabolic hormone that is produced within the liver and is expressed in the pancreas and adipocytes . The maiGDF15 is a similar biomarker to FGF21; however, GDF15 is more sensitive in detecting mitochondrial dysfunction affecting other organs ; thus, iAt present, although there are a number of biochemical assays that can provide evidence of MRC dysfunction, the ‘Gold Standard’ investigation for this assessment is still considered to be the spectrophotometric determination of MRC enzyme activities in skeletal muscle or tissue from the disease-presenting organ, if accessible . HoweverThere is a lot of interest in identifying appropriate biomarkers of MRC dysfunction in order to alleviate the need for an invasive muscle biopsy . However


Background: Living donor renal transplant involves highly technical operations in both a healthy donor and a recipient with end-stage kidney disease. Contrast-enhanced computed tomography angiography (CTA) is used to assess critical donor anatomy, but its interpretation becomes increasingly difficult as renal anatomy becomes more complex.Case Report: A related donor was denied because of prohibitive anatomy seen on the pretransplant evaluation CTA. As the donor was highly motivated to donate, CTA DICOM images were segmented to create a 3-dimensional (3D) model that could be evaluated in an immersive and stereoscopic virtual reality (VR) environment. The donor's anatomy was found to be acceptable, and he was approved.Conclusion: In live donor nephrectomy candidates, 3D reconstruction and VR visualization can be used to facilitate appreciation of complex anatomy. Aggressive utilization of LDRT is imperative to today's kidney transplant practice, requiring pushing boundaries on many criteria such as donor age, body mass index, HIV status, immunologic compatibility, and kidney donor anatomy.4Live donor renal transplant (LDRT), the transplantation of a kidney procured from a living donor, involves invasive and highly technical operations in both the healthy donor and the recipient with end-stage kidney disease. Long wait times and poor prognosis with long-term dialysis5Renal vascular anatomy and kidney size are important considerations when approving live donors and when selecting the appropriate kidney laterality for laparoscopic procurement. Obtaining detailed data regarding renal artery and vein number, length, diameter, and position is critical to planning donor nephrectomy. Relative kidney size, determined by split volume and/or split function, is important to both donor and recipient renal function postprocedure.Utilizing every available tool or technology leads to improved understanding of anatomic details. Accurate assessment of these anatomic details is paramount to planning donor and recipient operations.We present a case in which 3-dimensional (3D) visualization with virtual reality (VR) facilitated decision-making in approving a living donor and in selecting the appropriate kidney for transplantation.2, normal blood pressure, no preexisting medical problems, and a history of laparoscopic hernia repair, laparoscopic appendectomy, and jaw surgery.The donor candidate was a healthy 35-year-old male wanting to donate a kidney to his brother who had been on hemodialysis for 3 years because of focal segmental glomerulosclerosis (FSGS). He underwent standard donor evaluation at our center, including consultation with multidisciplinary transplant specialists , extensive blood and urine testing, and multiphase thin-slice computed tomography angiography (CTA), and he was assigned a donor advocate. The donor had a body mass index of 23.6 kg/mThe selection committee initially declined the donor because of the high vascular complexity of the bilateral kidneys as they appeared on 2-dimensional (2D) CTA images in the axial and coronal views. The right kidney had 2 arteries originating 1.2 cm apart on the aorta. Both arteries appeared to be bifurcating posterior to the inferior vena cava (IVC). The right renal vein was only 1.5 cm long.The left kidney had 2 arteries originating 0.5 cm apart. One artery had an early bifurcation 1.6 cm from the aorta. Additionally, the arteries crossed, and the point of crossing coincided with the branching points, making it difficult to distinguish between branching and crossing and raising the concern that 3 or even 4 renal arteries would require reconstruction. The left renal vein was 6 cm long.Additionally, gross measurements on the CTA demonstrated a significant size difference in the organs, with the left kidney being larger than the right. Discrete volume measurements were not available with the 2D CTA images. The multiple arteries and short vein on the right kidney  were deeThe patient was highly motivated to donate, so we decided to reconsider his candidacy with the aid of 3D visualization in VR to understand his renal vascular anatomy more precisely.6 an open-source software, was used to segment DICOM data and create a 3D stereolithography (STL) object. Multiple transplant surgeons viewed the STL in VR using an internally developed application and a commercially available head-mounted display (Samsung HMD Odyssey)  .VR allows for stereoscopic visualization and delineation of complex anatomy that is not possible with traditional 2D imaging modalities. The resulting volumetric data suggested only a slight difference in kidney volume measurements (53% left and 47% right) that allowed for either kidney to be potentially acceptable for donation from a size and function standpoint.As expected, the right kidney had a prohibitively short vein and 2 arteries crossing posterior to the IVC, complicating the approach.The left kidney, however, had a normal vein and 2 arteries, each bifurcating at an acceptable distance from the aorta. The points of bifurcation and crossing were clearly defined, favoring plenty of room for safe vascular stapler placement during the recovery operation. Static images were captured and shared with the multidisciplinary team at the selection conference . ConsideThe donor was appropriately consented for donation, with extra emphasis placed on the possibility that the multiple vessels might require complex reconstruction and the potential necessity of conversion to open surgery to safely recover the kidney. The donor's scans were reviewed with standard 2D modalities as well as in VR prior to his operation. He underwent left laparoscopic, robot-assisted donor nephrectomy and left the hospital on the first postoperative day. In surgery, the donor's anatomy was found to be as expected from the 3D VR visualizations. Two renal arteries fed the left kidney, with each bifurcated far enough away from the aorta to be safely stapled and divided.The recipient underwent a successful transplant, after side-to-side/pair-of-pants–type renal artery reconstruction of the 2 left arteries on the backbench. At 16 months posttransplant, the recipient maintained good graft function, despite recurrent FSGS that required treatment. He has had no ureteral or vascular complications.7 Advances in medical imaging and visualization allow for detailed understanding of discrete donor anatomy as well as planning of the donor and recipient operations. Still, on occasion, a willing donor is declined because of prohibitive anatomy demonstrated on multiphase contrast-enhanced CT.8Application of technology to living donor nephrectomy is not new, with the goal of improving the donor's safety and recovery from a highly technical yet medically unnecessary procedure. Because of the widespread proficiency in laparoscopy, minimally invasive donor nephrectomy has become the standard.11 In a comparison of CTA vascular anatomy and intraoperative findings in 2,144 living kidney donors, CTA identified the correct number of renal arteries in 97.9% of cases.10 While none of the patients had more arteries than predicted, 2.1% had fewer arteries found intraoperatively than were demonstrated on CTA.10 Thus, CTA can lead to a misinformed decision to turn down a potential living kidney donor on the basis of complex vascular anatomy as was the case with our donor.Thin-slice multiphase contrast-enhanced CT is critical in assessing donor anatomy, but its interpretation becomes increasingly difficult as renal anatomy becomes more complex. Up to 30% of potential donors have more than 1 renal artery for a kidney.12 With traditional 2D imaging, length, width, and depth of each kidney may be measured and multiplied to approximate the kidney's gross volume. Nuclear medicine imaging can be performed to measure the split function of each kidney but requires additional costly or inconvenient testing. Additionally, the results of nuclear medicine split function testing have been shown to be similar to and possibly inferior to CT volumetry at predicting postdonation glomerular filtration rate.13Regarding volume measurements, 3D reconstruction and VR viewing allow for more precise volume measurements than 2D imaging can reliably provide.14 When presented with 3D models of patient anatomy, surgeons rate these as highly useful clinical tools and techniques.15 Further, a 2021 study in the live kidney donor population found interactive VR highly valuable, improving surgeons’ understanding of anatomy, decreasing preoperative anxiety, and decreasing operative time.16Surgeons generally report that they use axial, coronal, and sagittal 2D images to build a 3D model of the anatomy in their mind. Surgeons’ skill at this mental reconstruction may not be as accurate as they believe, however, as cognitive localization of renal tumors based on 2D images has been shown to be poor, and it improves when 3D modeling is provided.For this case, we chose VR because of its immersive nature and stereoscopic environment. Other modalities of 3D visualization without VR or 3D printing are available, including some used by radiology departments such as Synapse 3D (Fujifilm Corporation) and software-as-service offerings such as Ceevra. Access to these programs varies by service line and by institution. These software products were not available to the transplant team when the donor's CTA images were evaluated.Regarding the cost of the technology used in our case, while de novo startup costs could be high—approximately $100,000 for a biomedical engineer, $15,000 for US Food and Drug Administration–approved 3D segmentation software, and $5,000 for a capable computer and display hardware—many of these assets are already in place in hospital systems and universities. In our case, we developed relationships with in-house teams already using this technology, and our cost was only $250 for the time the biomedical engineer needed to create the model.17Recipient outcomes following transplantation of a donor kidney with 2 renal arteries have been demonstrated to be acceptable compared to outcomes after transplantation of a donor kidney with a single renal artery, and reconstruction techniques are typically straightforward.18 Also, the additional vascular complexity increases the risk for converting to open surgery during the donor nephrectomy, putting the donor at higher risk.11Donor kidneys with more than 2 arteries have been shown to have some inferior outcomes compared to those with 2 or 1, namely longer cold times, increased delayed graft function, and increased slow graft function, but equivalent long-term graft survival.19 With continued routine use of 3D reconstruction, the entire team  will grow familiar with the technology and how it can be applied.Liberal use of 3D reconstruction and VR viewing may elucidate additional clinical scenarios in which 3D visualization is warranted and identify precisely which conditions, pathologies, or organ systems are most amenable to 3D VR visualization. We have begun to use 3D reconstruction at our institution routinely for the kidney donor population, which has led our team to identify extrarenal structures, such as ribs, spine, and liver or spleen, as useful reference anatomy in relation to the renal vascular anatomy. In recipients, 3D visualization could be used to clarify iliac calcifications or to better appreciate the intra-abdominal space occupied by polycystic kidneys.The VR headset is cumbersome for some users, so a more convenient way of visualizing the 3D model was needed. Creation of a desktop/laptop screen viewer for the 3D models allows for increased accessibility by not requiring the headset but does sacrifice the immersive nature of VR . Nonsurg213D printing of models segmented from patient CT images is useful in select cases. 3D prints allow for improved communication regarding surgical planning and operative technique, as well as the opportunity for improved patient education, comprehension, and satisfaction.The use of 3D reconstruction with VR visualization to clarify complex renal vascular anatomy allowed a living kidney donor to donate a kidney to his brother after initially being declined by the selection committee. While these technologies may not be necessary for routine LDRT, they were instrumental in this case and should be considered in cases requiring discrete appreciation of complex anatomy or in cases with an anatomic feature that is possibly a contraindication to donation.


Tribulus Terrestris (TT) is extremely rich in substances with potential biological activity and is therefore considered to be a promising ingredient in industrial medicinal preparations based on its saponin fraction. On the other hand, studies regarding broiler chickens are limited. However, the aim of this study was to verify the safe amount of TT powder that can be used as a medicinal plant in the diet of broiler chickens by determining the growth performance and histological changes and functional tests of some target organs. In conclusion, 0.75 g/kg TT is the best dosage for the health of broiler chickens. There is a need for low dose studies to identify the mechanism of the pharmacological effect of TT on broilers. p < 0.05). Liver and kidney functional tests showed no significant effects in all feed treatments (14 days), while T1 showed lower (p < 0.05) ALT and AST levels (35 days). T1 exhibited higher weights, lengths, and weight-to-length ratios of the small intestine, and relative lengths of the duodenum (p < 0.05). Histomorphometric measurements of the ileum were higher (p < 0.05) in chickens fed the 0.75 g TT/kg diet, and except for in the goblet cell count and epithelial thickness, there were no differences between treatments (p > 0.05). In T1, hepatocytes were more normal but hepatic sinusoids were dilated, whereas in T2, lymphocytes had infiltrated around the central vein and lining endothelial cells had been lost. The kidney was improved in T1 and T2 compared with the control group. Ileal villi were shorter in T2, and some villi fused with enterocyte necrosis and inflammatory cells accumulated in the lumen. We concluded that TT powder (0.75 g/kg feed) has a safe effect and is healthy for broilers.The current experiment aimed to investigate the effects of TT powder on performance parameters and functional tests, as well as on morphological and histological changes in the liver, kidney and ileum in broiler chickens. Commercial broilers  were used, equally divided into three dietary treatments . The growth performance (1–35 days of age), absolute and relative weight, liver and kidney functional tests, intestinal morphology (14 and 35 days of age), and histomorphology of the ileum (35 days of age) were evaluated. At 35 days of age, histopathological changes in the ileum, liver, and kidney were also examined. The results showed that the growth performance and absolute and relative weights of the liver and kidney had no negative effects when dietary supplementation with TT powder was given at 0.75 g/kg diet (T1), whereas a decrease was observed at T2 ( Medicinal herbal and aromatic plants have been a gaining interest in recent years as feed additives in poultry. Medicinal herbs can alter the active compounds in final products depending on the part used  and the processing technique  ,2. SeverTribulus Terrestris (TT) belongs to the Zygophyllaceae family [e family . It is we family . TT is ae family . Rathod e family ; Daniyale family  reportede family . Additioe family  showed te family . Therefoe family ,13,14,15e family , mice [1e family , and hume family . Vincente family  reportede family  or 0.06 e family  had no se family . In anote family . Sharma e family  found the family . Other se family ,26. Although studies have shown some effects of TT, there is still much debate about possible mechanisms of action and therapeutic applications, as well as a significant lack of research data on this medicinal plant. To our knowledge, there is a limited number of studies in which the effects of adding TT powder as a whole plant to the diet on the performance and health status of broiler chickens has been examined. All previous findings are existing hypotheses from which this study was created. Therefore, the aim of the current study was to evaluate the use of TT powder on the performance, histological changes, and functional markers of the liver, kidney, and small intestine in broilers.The use of birds, sampling and analyses were performed in accordance with the recommendations of the Scientific Research Ethics Committee of King Saud University, Riyadh, Saudi Arabia .The TT was collected as a whole plant  in the valleys of the Republic of Yemen. The plants were classified in the herbarium of the College of Science-King Saud University (NO. 24519). Then, the plants were dried at a normal temperature (27 °C) until they reached a constant weight and were then ground into powder at the Research Department of Animal Production-Food and Agriculture Sciences at King Saud University.The analysis of TT powder was performed in duplicate to determine the content of nutrients  according to the methods of AOAC . The fatOne-day-old commercial Ross chickens  were used for this experiment. Birds were weighed individually and randomly divided equally into 3 treatments with 8 replicates per treatment (7 chicks per replicate) in an environmentally controlled battery with automatic electric heating. The initial temperature of 35 °C at one day of age was gradually reduced to 22 °C at 21 days of age and then maintained at this temperature and 55% humidity for the remainder of the 35-day period. Dietary treatments were supplemented with TT at three doses . The basal diet for the chickens during the starter and finisher periods was formulated according to the recommendations from the commercial practice (Saudi Arabia) see . Feed anAll chicks were weighed individually to determine their body weight at days 1 and 35 and to calculate body weight gain per treatment. Feed intake was recorded during the experimental periods to calculate the feed conversion ratio. g for 20 min and then frozen at −80 °C until analysis. These functional tests were analyzed spectrophotometrically  using reagent kits  according to the manufacturer’s instructions. At 14 and 35 days of age, blood samples were collected from 16 birds in each dietary treatment in tubes without EDTA to measure liver function tests  and aspartate aminotransferase (AST)) and kidney function tests (uric acid and creatinine). Serum was separated via centrifugation at 3000× At 14 and 35 days of age, the entire small intestine, liver, and kidney were randomly collected from 8 birds per treatment, weighed and calculated as a percentage based on live weight. The measurement of small intestine length was recorded and the relative length of intestinal segments was calculated based on the total small intestine length. The length (L) and weight (W) of the small intestine were used to calculate the W/L ratio . The mid-portions of the ileum (1.5 cm) between the Meckel’s diverticulum and the cecal junction and the liver and kidneys (0.75 cm) of each bird were taken without stressing the tissue wall. Tissue samples were washed in phosphate-buffered saline (pH = 7.4) and fixed in 10% phosphate-buffered formalin for 72 h, then dehydrated in graded ethyl alcohol (50% to 100%) and paraffin-embedded into the action sections . Approximately 4 μm was cut using the Microtome System . Sections were deparaffinized, hydrated and stained in hematoxylin and eosin (H&E) with Alcian blue stain to count the goblet cells  using a protocol based on the modified method of Winsor and Sluys . Tissue sections were examined under a light microscope , and the required images were taken at various magnifications using a Nikon digital camera . Measurements of villus length, width, depth, goblet cells, epithelial thickness, mucosa, submucosa, and lamina propria were based on at least ten intact villi from one section (bird) with a total of 80 measurements per treatment. The following equation based on villus length and width was used to calculate villus surface area (surface area = 2π × (width/2) × length) according to Al-Baadani et al. . In addip < 0.05. All mean values for each parameter within the different treatments were reported as ± standard error of the means (SEM).The data obtained from the measurements of performance, enzymes, and morphology in the different treatments were statistically analyzed using the GLM procedures of SAS software  via one-The nutritional values and fatty acids composition of TT are presented in The results of HPLC analysis  showed tp ≤ 0.05) body weight gain and total feed intake compared to the basal diet (control), while the intake of TT at 0.75 g/kg diet (T1) had no negative effect. The feed conversion ratio was not affected by any of the treatments (p > 0.05).The results of the effect of TT on growth performance during the experimental period (1 to 35 days) are presented in p > 0.05) compared to the control, while the chickens fed 1.5 g/kg had weights lower than those in the other treatments at 14 days of age (p < 0.05). At 35 days of age, the live body displayed no effects in all treatments (p > 0.05). The absolute and relative weights of the liver and kidney displayed no effects (p > 0.05) between all treatments during the starter and finisher phase (14 and 35 days). The body weight and absolute and relative weights of the liver and kidney in broiler chickens fed TT powder are shown in p > 0.05) between the dietary treatments and the control group at 14 days of age. At 35 days of age, the chickens receiving the 0.75 g TT/kg diet (T1) had lower values (p < 0.05) on ALT and AST compared to those in the other dietary treatments. In contrast, there were no effects on uric acid and creatinine for all treatments (p > 0.05).The effects of TT supplementation on liver and kidney function tests in broiler chickens are shown in The results of histopathological changes in the livers of broiler chickens are shown in Histopathological changes in the kidneys of broiler chickens are shown in p < 0.05) than the control group. In addition, T1 and the control group had higher intestinal weight to intestinal length ratios and relative lengths of the duodenum (p < 0.05) compared to the chickens fed the 1.5 g/kg diet (T2). In contrast, the relative length of the ileum was lower (p < 0.05) in T1 and the control group. The small intestine weight and intestine weight to length ratio (W/L) were higher (p < 0.05) in chickens fed the 0.75 g TT/kg diet (T1) than in the control group, but there was no significant difference from T2 at 35 days of age.The intestinal morphology, such as the weight and length of the small intestine, the ratio between the weight and length of the intestine, and the relative length of the small intestinal fragments in broilers fed diets enriched with TT powder, are shown in p < 0.001), while the number of goblet cells and epithelial thickness showed no differences between the treatments compared to the control (p > 0.05). The length and width of ileal villi and villus surface area were higher (p < 0.05) in chickens fed the 0.75 g TT/kg diet (T1) than T2 but were not significantly different between the T1 and control groups. In addition, the results showed that the ratio of villus length to crypt depth and the thickness of mucosa and lamina propria were higher (p < 0.05) in T1 chickens than in the other diets (T2 and control). In contrast, the crypt depth was lower in T1 compared to the other feed treatments, and the number of goblet cells per 100 µm villus area was also lower (p < 0.05) but not significantly different from the chickens fed the basal diet (control). The histopathological changes in the ileum in broiler chickens are shown in TT is a pharmaceutical herb that has long been used by people as a traditional medicine to treat many diseases, including urinary tract infections, to remove kidney urolithiasis, hypercholesterolemia, and liver health, and as a diuretic medication. These traditional uses would be due to the substances it contains with potential biological activity, including steroidal glycosides (saponins), protodeacon, glycosides, alkaloids, flavonoids, and tannins, as shown by previous studies ,10,14,37The results obtained show that the addition of TT powder (0.75 g/kg) to a basal diet did not affect overall growth  compared to chickens fed a basal diet from 1 to 35 days of age, while performance decreased when they received a high amount of TT powder (1.5 g/kg). Our results are in agreement with those of Ammar et al. , who shoThere were no effects on the absolute and relative weights of liver and kidney in broilers fed diets supplemented with TT powder (T1 and T2). Our results agree with the findings of Martino-Andrade et al. , who repAccording to our results on intestinal morphology, such as the weight and length of the small intestine and the ratio of weight to length of the intestine, the absorption area was higher when the diet was supplemented with TT, especially at T1 (0.75 g/kg). Moreover, histomorphometric measurements of the ileum in broiler chickens were improved when they received TT powder, whereas ileal villus length and width, surface area, villus length to crypt height ratio, mucosa, and lamina propria thickness were lower in chickens receiving 1.5 g TT/kg feed (T2). Crypt height is an indicator of the multiplication and absorption capacity of the intestine . PreviouThe current results conclude that the use of TT powder as a whole plant contains nutritional compositions and saturated fatty acids as well as pharmacologically active components. Therefore, the addition of TT in a 0.75 g/kg diet had no negative effects on growth performance and liver and kidney morphology and improved intestinal morphology and ileum histomorphometry. Furthermore, adding TT powder (0.75 g/kg) to the diet improved the histological changes in the liver, kidneys, and ileum. The dosage of 0.75 g/kg diet used in the current study is likely to be safe and beneficial for the health of broiler chickens. However, further studies are needed to discover the proper dosages and mechanism of pharmacological effect of TT powder as a whole plant on the physiological and health characteristics of broiler chickens.


Dendrobates tinctorius) to test the hypothesis that alkaloid load and prey traits influence frog dietary preferences. We tested size preferences  within each of four prey groups  and found that frogs preferred interacting with smaller prey items of the fly and beetle groups. Frog taxonomic prey preferences were also tested as we experimentally increased their chemical defense load by feeding frogs decahydroquinoline, an alkaloid compound similar to those naturally found in their diet. Contrary to our expectations, overall preferences did not change during alkaloid consumption, as frogs across groups preferred fly larvae over other prey. Finally, we assessed the protein and lipid content of prey items and found that small ants have the highest lipid content while large fly larvae have the highest protein content. Our results suggest that consideration of toxicity and prey nutritional value are important factors in understanding the evolution of acquired chemical defenses and niche partitioning.The ability to acquire chemical defenses through the diet has evolved across several major taxa. Chemically defended organisms may need to balance chemical defense acquisition and nutritional quality of prey items. However, these dietary preferences and potential trade-offs are rarely considered in the framework of diet-derived defenses. Poison frogs (Family Dendrobatidae) acquire defensive alkaloids from their arthropod diet of ants and mites, although their dietary preferences have never been investigated. We conducted prey preference assays with the Dyeing Poison frog ( Melanophyriniscus) toads, Pitohui birds and Natricine snakes , Linepithema humile , Flies: Drosophila hydei , Drosophila melanogaster , Beetles: Stethorus punctillum , Dalotia coriaria , fly larvae: Callifora vomitoria , Musca domestica   , 28. AntAll dietary preference assays were conducted in the same room where frogs are housed, in which temperature and humidity replicate conditions of their native habitats. Before feeding trials began, all frogs were exposed to arthropod prey items six times over three weeks to reduce neophobia. Trials were conducted in a small transparent plastic bin (33 cm x 20 cm x 12 cm) stacked within an identical secondary bin with a layer of dried magnolia leaves to simulate a forest-floor environment while keeping the texture even throughout the arena. Inner sides of the arena were coated with Fluon™ liquid teflon  to prevent the arthropod prey from escaping. While piloting arena configurations, we confirmed frogs eat their regular diet of fruit flies within this setup. Of the 88 videos, 14 could not be scored due to camera malfunction. Both ingestion and an attempt to pursue or ingest prey of each type were scored using BORIS software. The scorer was not aware of the treatment group at the time of scoring.Adult frogs  were chosen at random and placed in the arena with 10 individual insects  of one of the given prey groups . Each trial was conducted for 5 min and recorded with a GoPro camera  mounted above the arena. Each frog participated in one trial with each prey group and the order in which frogs and prey were tested was randomized. Intertrial intervals were roughly 10 min and the arena was emptied and wiped down with a moist paper towel during this time. Videos of all behavior trials were scored using BORIS (Behavior Observation Research Interaction Software) . The numCallifora vomitoria), five small ants (Linepithema humile), five small beetles  and five small flies (Drosophila melanogaster). Only one prey type per category was chosen to avoid confounding taxonomic and size preferences. Each trial was conducted for 5 minutes and recorded with a GoPro camera mounted above the arena. The arena was emptied and wiped down with a moist paper towel between trials. Only female frogs  were used in this behavioral assay, as male frogs consistently exited the arena before trial completion. Why males exited the arena in this assay is unknown, although we hypothesize it may have been due to stress from many prey items.Dietary preference trials were conducted to determine frog preference for different insect taxa and how this preference may change with chemical defense . Both maTo test how preferences may change as frogs acquire chemical defenses, both male and female frogs were orally administered 15 μl of 0.01% decahydroquinoline (DHQ) in vehicle solution three times over the course of one week. The following week, these “low-DHQ” female frogs were re-tested in the taxon dietary preference trials described above. One week later, both male and female frogs were orally administered 15 μl 0.01% DHQ three times per week over two consecutive weeks, representing a “high-DHQ” treatment. The following week, female frogs were retested in a final preference trial. Previous work has established this alkaloid-feeding paradigm as appropriate in creating low and high DHQ groups , 31. EacAnt alkaloids were quantified via Gas Chromatography / Mass Spectrometry (GC/MS) to determine whether ants may carry alkaloids that could influence frog dietary preference during the feeding assays. Alkaloids were extracted by soaking a volume of 1 mL of each ant species in 1 mL of methanol placed in a glass vial . After a 1-hour incubation, 1 mL of the methanol from each ant species was syringe filtered through a 0.45 um PTFE syringe filter  into a new glass vial. These methanol extractions were incubated for 24 hours at -80°C to precipitate any lipids and proteins within the sample. A 100 uL aliquot of each sample was added to an individual GC/MS autosampler vial and the remaining samples were stored at -80°C. GC/MS analysis was performed on a Shimadzu GCMS-QP2020 instrument with a Shimadzu 30m x 0.25 mmID SH-Rxi-5Sil MS column as described previously . In brieD. tinctorius frogs that followed an identical vehicle alkaloid-feeding regime alongside the female frogs used in the behavior trials were obtained to confirm the presence of DHQ uptake. We used males that did not participate in the taxonomic diet trials to avoid stressing the experimental female frogs, which may have influenced behavior during feeding trials. We collected alkaloids from 2–3 frogs one week after each set of feeding trials. No individual male frog had its alkaloids collected more than once. Alkaloids were collected using a small pulse stimulator  at 1 volt for 30 seconds. After stimulation, the frog’s dorsal skin was wiped with a Kimwipe™  and then stored in 5 ml methanol in glass vials. From the methanol in which the Kimwipe™ was stored, 1 ml was syringe filtered through a 0.45 um PTFE syringe filter  into the new glass vial  supplemented with 25 ug (-)-nicotine . All tubes were then capped, vortexed, and stored at -80°C to precipitate lipids and proteins. After precipitating for 24 hours, the supernatant was filtered through a 0.45 um PTFE syringe filter again into a new glass vial. We first analyzed frog secretions using GC/MS as described above and did not detect DHQ. We then used liquid chromatography / mass spectrometry (LC/MS) as previously described [Skin secretions from the nine male escribed  given thCrude lipid content was quantified across the eight prey groups used in the dietary preference behavior experiments using an ether-soluble lipid extraction method  24]. Al. Al24]. Crude proteins were extracted from each of the eight prey groups. All arthropods were fresh-frozen live at -80°C and stored for 1–2 weeks. Five replicates of 10–150 mg of each arthropod type were put into 2 ml tubes, each containing 2.4 mm stainless steel beads  and 0.8 ml of cold 1x phosphate-buffered saline . Samples were homogenized at 4°C using a Beadmill 24 Homogenizer (Thermofisher Scientific) with the following settings: Speed in m/s (S): 4.5, Number of cycles (C): 10, Cycle time (T): 30s, Interval paused between cycles (D): 10s. Samples were then centrifuged at 2,000 rpm at 4°C for five minutes. The supernatant containing the protein lysate was moved into a new 1.5 mL microfuge tube and stored for 1 week at -80°C. Proteins were then quantified using a Bradford Protein assay according to manufacturer’s instructions . Samples and albumin standards were run in triplicate on a 96-well optical plate and quantified on a BioTek Synergy H1 96-well plate reader . Absorbance was measured at 595 nm and an endpoint absorbance value was calculated for each standard and sample. Crude protein concentrations of arthropod samples were calculated based on the absorbance values of samples compared to the standard curve.Statistical analyses were run in R version 4.1.1 using the glmmTMB package . To asseTo assess differences of prey interactions across control, low-DHQ, and high-DHQ groups, we used our taxonomic prey preference assay data to fit a zero-truncated generalized linear mixed models (GLMM) to compare the number of prey interactions for each prey type: ants, beetles, flies, fly larvae based on a truncated Poisson distribution of the number of “eats” or “attempts” across the 10 female frogs that were assayed. To account for individual variation in the subjects, frog ID was used as a random effect. As each frog differed in its total number of prey interactions across the four prey choices, we used “total interactions with prey” as an offset in order to account for these differences in consumption. Sex was not considered as a variable in the model, since all frogs used in the taxonomic assay were female. To check for differences in the general structure of frog diet categories across the three groups, we performed a permutational multivariate analysis of variance (PERMANOVA) on Bray-Curtis dissimilarities. We used a zero-truncated GLMM  to compare total frog interactions  across the three groups: control, low and high DHQ. The model was based on a truncated Poisson distribution as described above. To control for repeated measures in the subjects, frog ID was again used as a random effect.BioRender.com.P-values were adjusted using the Benjamini-Hochberg procedure to correct for multiple hypothesis testing across all dietary preference and nutritional analyses. Data were visualized with boxplots and barplots created using the package ‘ggplot2’ in R version 4.1.1 and infographics were created with images from Overall, frogs interacted (ate or attempted) more with smaller prey items . Frogs iThe total number of interactions across all prey types did not differ between the three alkaloid groups . When frogs without DHQ were given the choice between small beetles, small flies, small ants and large fly larvae, they interacted most with large fly larvae . Additionally, high DHQ frogs interacted less with fly larvae when compared to control frogs , although interactions with fly larvae were extremely variable across groups. Within all three groups, larvae were interacted with most frequently. There were no differences found among ant , fly , or beetle interactions  among groups.2 = 29.564, df = 7, p-value = 0.0001). Small ants (Linepithema humile), beetles  and fly larvae (Musca domestica) had a higher proportion of lipids than their larger counterparts , while large flies (Drosophila hydei) had a higher proportion of lipids than small flies (D. melanogaster) , where large fly larvae have more protein than most prey items, followed by small beetles, which have more protein than small or large ants and flies  , Table 1nd flies , Table 2D. tinctorius to prefer small prey items, reflecting their wild diet https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  3 Aug 2022Response to Editor and Reviewer CommentsWe have addressed and/or incorporated all reviewers’ comments in our revised manuscript. The major concern was interpretation of our data regarding preference for large fly larvae in our taxonomic preference trials. We have clarified this in our responses and in our revised manuscript.We have also added two additional co-authors, Aurora Alvarez-Bullya and Katherine Fiocca, who performed experiments to address reviewer concerns. These new experiments include the extraction and quantification of alkaloids from ant prey and frogs.Reviewer #1: The study appears to have been reasonably conducted and analyzed. I have only a few minor comments in that regard. I am, however, somewhat concerned with the interpretation of the results which I do not think are necessarily supported by the data. It is possible that I am misunderstanding the points the author is trying to make, in which case I believe that some additional clarity is warranted. Specifically, I am not certain that the claim that both toxicity and nutritional value are taken into account by frogs during prey selection is substantiated. Rather the results seem to suggest a single preference for high protein fly larvae. Specific comments related to these points follow:Line 99-101: This sentence is confusing.Response: Sentence has been reworded and split for clarity.Line 102-103: This prediction isn’t backgrounded so I am unsure where it comes from.Response: Thank you. This had been mentioned prior in the manuscript  (lines 91-92). However, we have fixed this sentence for clarity and added citations: “We predicted the frogs would show a greater preference towards smaller chitinous prey categories  (18-19), but larger versions of non-chitinous prey , as insect larvae tend to have higher protein and lipid content compared to chitinous adult arthropods (24).”Line 171-178: My only concern with the method is that all frogs were exposed to treatments in the same order which may be confounding. It is possible then that any differences between control, high, and low dosages are a results of experimental order. I don’t think that the control here is fully appropriate. As I understand it the control treatment was vehicle solution at the beginning of the trial to every frog. This doesn’t appear to control for time or the repeated administration of vehicle solution. I would have liked to see a control group that was given vehicle solution at the same time points as the experimental group.Response: Thank you for your input. We agree that a non-toxic control group, in addition to our within-subject control approach, would have been ideal. Unfortunately, this experiment was conducted during the COVID-19 pandemic when frogs were not available for purchase. Therefore, our approach was the optimal way to test preference at a variety of alkaloid ingestion levels with limited animals. We discuss the limitations of this approach at length in the discussion.Line 179: It would be helpful to know how many videos were discarded from the dataset.Response:Of 88 videos, 14 could not be scored. This has been added to the manuscript.Line 272-276: This section is hard to follow.Response: We have rewritten the paragraph with more context and explanation surrounding our statistical reports. To summarize, control frogs preferred interacting with fly larvae more than high DHQ frogs. Across all three groups, the total number of all prey interactions did not differ. We acknowledge that our heading was misleading, and we have adjusted it accordingly.Line 312-315, 346-351: I think you need a reference here. Also I wonder if this results is due to biomass of ants being dominant in most natural settings rather than prey preference exactly. The authors do discuss this a bit later on in the discussion but I think is perhaps worth highlighting in more detail. The findings here really support the idea that prey availability not preference might be the main factor determining diet. It also occurs to me that there is an alternative hypothesis here that frogs can determine prey alkaloid content. Because all of the prey items offered here were not defended it is possible that you wouldn’t see predicted prey choice if they “know” all the options are alkaloid free.Response: Thank you, references have been added. We appreciate your thoughtful interpretation of the data, and agree that prey availability is likely a dominating factor in what poison frogs consume, and that there is a likely abundance of ants in comparison to other arthropod prey. We have added another sentence to the discussion which highlights this, as well as your point that true preference may not have been adequately tested given that alkaloid-laden prey items were not presented to the frogs in feeding trials:“It is possible that frogs’ foraging behavior would have been different when presented with ants from their natural habitat, and we acknowledge that our experimental design does not allow us to assess true preference, which must be tested using native prey items. “Line 333-334: references please.Response: Reference has now been added.Line 360-362: It isn’t clear why this is contrary to predictions? Why would you predict that alkaloid supplemented frogs should be bias against protein rich foods?Response: Thank you. This is because in the wild, poison frog diet usually consists of >50% ants. This has been clarified in the text with citations added.Line 440-441: Has this been demonstrated? A naïve preference for defended prey, because as captive bred frogs that have never been exposed to nature prey sources they may not associate any species with being defended unless it was innate.Response: No, this has not been demonstrated, rather it reflects what we have surmised from our data since we demonstrated that prey categories differ in nutritional quality. Therefore, we have edited this paragraph to reflect the speculatory nature of the statement: “Prey choice becomes more complex when considering highly proteinous prey, like fly larvae, in the presence of lipid-rich small ants, which frogs might innately associate with chemical defenses. Although this tradeoff has not been demonstrated, we have surmised this tradeoff from our data since we found that prey species used in this experiment differ in nutritional quality. "Line 449-452: I am not convinced your data supports this conclusion. It seems like there is no evidence from your experiments that dietary preference explains diet of wild populations.Response: We did not intend to imply that our results directly explain diet in native populations. Rather, we suggest that what we learned from innate behavior in a controlled, laboratory setting should be considered in the context of poison frog prey consumption and in tandem with our previous knowledge. Although ants make up the majority of poison frog diet, our results suggest that they consume protein-rich prey opportunistically, which implies that ants are likely more abundant/available when compared to a protein-rich prey item, such as fly larvae.Line 455-459: Again if I understand your results right, I don’t think this is supported. The preference toward larvae suggests a preference for high protein not high lipid prey items doesn’t it?Response: Your interpretation of the results is correct. We intended to write this as speculation regarding how poison frogs would behave in their natural habitats in the presence of both prey items, and have reworded it to clarify that this is merely speculatory. Although it is known that frogs consistently eat small ants and fly larvae, we include this speculation because we feel it is important in the context of what we know about poison frogs in their natural habitat, and that this new insight may be informative. We know that poison frogs likely encounter (and therefore eat) many more ants than larvae in their natural habitats, as previous literature shows >50% ants making up most poison frog stomach contents. Since the small ants in our experiment are the most lipid-rich prey item, we propose the possibility that those in their native diets may also be lipid-rich. Yet, in our controlled lab setting, they choose to interact the most with large larvae, the prey item with a substantially higher protein content than all prey categories. Reviewer #2: The authors designed several experiments to better understand foraging decisions by a frog that acquires its chemical defenses from diet. Overall, I think the ideas presented in the paper are significant, and represent a new and important direction towards an understanding of organisms that acquire chemical defenses. The literature review presented in the paper, and the synthesis of the experimental results as they pertain to current knowledge in the study system are well presented in the discussion. My main concern with the paper has to do with the biological relevance of some of the results, and what they might mean in a natural system. I also have several questions surrounding some of the methodology, all of which are aimed at improving the overall study. I present my concerns below. To the authors credit, they do address in the discussion some of the limitations of their lab experiments as they pertain to natural systems, which I think is important. Although just an opinion, I think the most significant results in the manuscript are in relation to the protein and lipid content analysis of different prey items. These results are novel (at least within this system), and are more widely applicable to natural systems.General: I would specify fly larvae throughout as all the prey species exhibit a larval stage.Response: “Larvae” has now been changed to “fly larvae” throughout the manuscript.Fig 2b: I am having a hard time interpreting this figure. The caption doesn’t seem sufficient to explain what is being show. Does this not look like a preference for adult flies and not larva?Response: Thank you, that is correct. We agree that the caption was not sufficient and have expanded it for clarity.Arthropod prey used in experiment.The authors used two different ant species in their feeding experiments, Camponotus vicinus and Linepithema humile, which are assumed (to the authors knowledge) not to be chemically defended. It is possible that these species do not contain alkaloids, however the present authors recently report in Moskowitz et al. (2020) the presence of alkaloids in other species of ants in the same genera. Although there are many exceptions, the presence of alkaloids in ants are commonly found to be genus specific. Therefore, it seems possible that the ants used in the present study also contained alkaloids. To eliminate this possibility, the two ant species used in the present experiment should be examined for alkaloids, as the presence of alkaloids would significantly impact the findings of the study.Response: Thank you for this suggestion. First, we would like to note that we misidentified our large ants in our initial manuscript submission, which had been identified as Camponotus vicinus, but are in fact Liometopum occidentale (Velvety tree ants). To address these concerns, we examined the chemical profiles of both the small and large ants and have confirmed that they are free of frog alkaloids. This is now noted in the manuscript. The raw data files and spreadsheet will be available on DataDryad and included upon acceptance. Size mediated dietary preference assay.Why did the authors use both “eat” and “attempt to eat” events? If the goal of the study was to understand what the frogs would eat when given a choice, then why use “attempt to eat”? I am sure there is a good reason, but in the absence of more information on the category “attempt to eat” , it is not clear. Also, what are the raw numerical differences between these two categories? Did most frogs eat, and only a small subset attempt to eat? Or, did most frogs attempt to eat? Given the aim is dietary preference, it seems that the eat category would be the only relevant category to measure. A failed attempt to eat could be interpreted as a mistake by a frog, but it could also be a decision by the frog not to consume a particular prey item. Again, without more information, this is difficult to assess.Response: Thank you for noting this. We use attempts because we are looking at preference from a time and energetic investment from the frog’s perspective. Since our frogs are willing to spend time and energy pursuing prey items  over others, we felt it was important to include their attempts in our preference quantification, because attempts exemplify preferential interaction with the prey. In size trials, 943 of 1733 behavior events (eats or attempts) were “attempts to eat” (54%). In taxonomic preference trials, 189 of 409 behavior events (eats or attempts) were “attempts to eat” (46%). We have added an explanation regarding why we used “attempts” in the manuscript. The raw numbers of attempts and eats across all trials are available on our supplementary spreadsheet.Alkaloid feeding and taxon dietary preference assay.Can the authors offer any ideas on why all of the males in this experiment exited the arena before trial completion? This result is especially strange given that 9 males did not exit the arena in the size-mediated experiments. Also, the fact that this part of the experiment only involved females should be added to the discussion.Response: The size feeding trials include a total of 10 insects in the arena per trial, while the taxonomic feeding trials include a total of 20 insects in the arena per trial. Given that there are double the amount of insects , frogs in taxonomic trials experienced much more sensory stimuli in taxonomic trials. We hypothesize that the male frogs were quick and consistent in their reaction to leave the arena, due to these stimuli. However, we cannot confirm this and are unsure of why this happened. The following sentence was added to the methods: “Why males exited the arena in this assay is unknown, although we hypothesize it may have been due to stress from many prey items.”Lines 103-105: (1) “non-chinous” should be “non-chitinous.” (2) As written, the prediction is a bit confusing and it is not entirely clear what the authors mean.Response: (1) Fixed. (2) A clarifying statement has been added to supplement our predictions: “We predicted the frogs would show a greater preference towards smaller chitinous prey categories  (18-19), but larger versions of non-chitinous prey , as fly larvae tend to have higher protein and lipid content compared to chitinous adult arthropods (24). We next tested preference for prey taxonomic identity and nutritional value and predicted D. tinctorius would prefer ants, which make up the majority of poison frog stomach contents in the wild . “Lines 128-133: A minor point, but there is no information on where the fruit flies originated.Response: Line 139, we state that the fruit flies originate from the company, “Josh’s Frogs.”Lines 143-144: Something is missing or incorrect. How is it possible that “one week before the trials began”, frogs were “exposed to arthropod prey items six times over three weeks”?Response: Thank you for noting this mistake. The “one week” portion of text was incorrect, and has been removed.Lines 143-149: Were the frogs exposed to the novel arthropod prey items in their larger terraria, or were they first put in experimental arenas? Also, is it known if frogs will eat “normally” after being removed from a larger terrarium and placed into a smaller arena? Was a control performed, in which frogs were simply fed their normal diet of fruit flies in the experimental arenas? I think this would strengthen the findings.Response: Thank you. The frogs were introduced to novel arthropod prey in their larger terraria, not the smaller arenas. During our pilot phase of testing various arena set-ups, frogs were tested to make sure they ate their normal diet of fruit flies within the arena. A sentence stating this has been added to the manuscript. Line 152: I think “five big” should read “five large”.Response: Thank you, this has been fixed.Lines 167-168: Why was a limited prey selection chosen for the alkaloid feeding experiment, as compared to the size-mediated experiment? Adding some clarification would strengthen this portion of the experiment.Response: Thank you. This was done to avoid confounding taxonomic and size preferences, which we believed was necessary to test separately. We narrowed down the categories as follows. From each prey category in the size-mediated preference assays, we chose one species to represent each of the four categories. The species chosen per category was whichever one was chosen most by the frogs during the size mediated assays. This has been clarified in the manuscript.Lines 171-178: Did the authors examine the frogs for alkaloid content at the end of the experiment? Although there was a small effect of alkaloid “load” on prey interaction, the differences in alkaloid amount between frogs is not reported. My concern is that there was not a difference in alkaloids between treatments, or that the differences were too small to be biologically relevant. The authors cite Alvarez-Buylla et al., which conducted a similar type of alkaloid feeding experiment over the course of 5 days. However, in the present paper, the “low-DHQ” treatment was fed to frogs 3 times, whereas the “high-DHQ” treatment was fed to frogs 6 times. The difference between treatments is only 3 feedings (that occurred over a longer time period for the high treatment), which may not have been large enough to result in a difference in alkaloid content between frog treatments. Comparing the alkaloid content in frogs between treatments would provide more clarification.Response: Thank you for this comment. We have the following points in reply:1. (Minor) First, we would like to point out that the difference between low and high DHQ groups was six feedings, not three feedings. 2. (Major) We have now analyzed the DHQ content of the frogs used in this study. Through the experiment, skin alkaloids were quantified from male frogs that followed an identical vehicle control + alkaloid-feeding regime alongside the female frogs that were used in the trials. These frogs did not participate in the final diet trials, and their alkaloid collection happened at the end of the experiment using an electric pulse stimulator at 1 volt for 30 seconds. We first analyzed these samples using GC/MS, which is the method we use for quantifying alkaloids in frogs from the wild. However, DHQ was undetectable with that method, which highlights the reviewer’s concern that DHQ levels were very low.3. (Major) Since DHQ was undetectable using GC/MS, we next used LC/MS methods for DHQ detection given this approach is more sensitive. We confirmed DHQ presence in frogs using this method. The raw data files generated from LC/MS machinery will be available on DataDryad upon submission. 4. (Major) We acknowledge that behavioral differences may have been more apparent if alkaloid load differed more substantially between groups. However, we have limited knowledge of how alkaloid load influences foraging behavior, and we have added this as a suggestion for future research in the discussion. Line 179-180: (1) “Videos” should be “videos” (2) How many videos could not be scored due to camera malfunction? The number or percentage should be reported. Also, how does this change the overall sample size of the experiment?Response: Thank you for noting this. Of 88 videos, 14 could not be scored, and this has been added to the manuscript. Sample size does not change substantially, mainly because most malfunctions happened during the size trials , in which each individual frog was tested 4 times – once per prey category. Line 254: (1) Why are the results heading different than the method headings? (2) The heading is confusing, as both small and large things are prey. But, more importantly, the result stating that there “was no preference for small versus large ants or larvae within this behavioral assay” contradicts the heading.Response: (1) Thank you, the heading format has been fixed. (2) Thank you for noting this inconsistency. 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Data for Asian kidney transplants are very limited. We investigated the relative importance of prognostic markers in Asian kidney transplants by using Korean Organ Transplantation Registry (KOTRY) cohort. Prediction models were developed by data-driven variable selection approach. The relative importance of the selected predictors was measured by dominance analysis. A total of 4854 kidney transplant donor-recipient pairs were analyzed. Overall patient survival rates were 99.8%, 98.8%, and 91.8% at 1, 3, and 5 years, respectively. Death-censored graft survival rates were 98.4%, 97.0%, and 95.8% at 1, 3, and 5 years. Biopsy-proven acute rejection free survival rates were 90.1%, 87.4%, and 87.03% at 1, 3, and 5 years. The top 3 dominant predictors for recipient mortality within 1 year were recipient cardiovascular disease history, deceased donor, and recipient age. The dominant predictors for death-censored graft loss within 1 year were acute rejection, deceased donor, and desensitization. The dominant predictors to acute rejection within 1 year were donor age, HLA mismatched numbers, and desensitization. We presented clinical characteristics of patients enrolled in KOTRY during the last 5 years and investigated dominant predictors for early post-transplant outcomes, which would be useful for clinical decision-making based on quantitative measures. After the introduction of brain death legislation and the establishment of regulatory agencies, deceased donor kidney transplant programs showed a decline in the early 2000s. However, with the effort to promote deceased organ donation and transparent allocation, deceased donor kidney transplants have rapidly increased, accounting for about 45% of total kidney transplants in recent years4. Accompanying this expansion of transplant volume, electronic claim databases became good resources for transplant research in South Korea, from which several good reports were produced overviewing Korean kidney transplants6. However, data based on claim reports or administrative databases generally lack many clinical details, and they should be supplemented by observational cohort or registry data.The first kidney transplant in South Korea was conducted in 1969, and the procedure was popularized in the 1990s. The number of kidney transplants has been increasing since the 1980s. In past eras, most kidney transplant programs were based on living donations, and the deceased donor kidney transplant program operated in some centers9. The Korean Organ Transplantation Registry (KOTRY) has operated as an observational cohort of organ transplantation since 2012. In 2014, we reported the first nationwide retrospective data summary of 4,500 kidney transplant cases that had been performed from 2009 to 201210. Based on that project, a prospective observational cohort involving five different organ transplants  started in 2014 under the same name (KOTRY)11. We provide a data summary of 5 years of enrollments of kidney transplant donor-recipient pairs in this paper. In addition, we investigated major dominant predictors of early kidney transplant outcomes including survival of patients and grafts, occurrence of acute rejection, and estimated glomerular filtration rate (eGFR) of transplanted grafts.In the transplant field, nationwide and international transplant registries have provided many valuable data resources and led to the development of a clinical science of transplantation23. In the present study, we compared the relative importance of clinical predictors based on a data-driven approach in addition to the 5 years outcome reporting of KOTRY.In terms of statistical modelling, most clinical epidemiological studies have used inferential methods based on the knowledge of experts and predefined hypotheses. On the contrary, the data-driven approach does not depend on prior hypotheses, which are usually used to build a prediction or prognostic model. Prognostic models in kidney transplants are an active area of research; however, the studies were scarce that compared the relative importance or the relative weight of clinical predictors for post-transplant outcomesp < 0.001). Females accounted for 40.6% of recipients. More male recipients received deceased donor kidneys. Mean body mass index (BMI) was 23.1 ± 3.6 kg/m2; mean systolic blood pressure before kidney transplant was 139.2 ± 20.8 mmHg. The proportion of current smokers was 8.6%. As comorbidities, diabetes were present in 29.8% and hypertension in 89.7% of recipients. The proportion of cardiovascular disease was 6.1%, which was higher in deceased donor kidney transplant recipients. History of malignancy was present in 6.6%. The most common cause of end-stage renal disease (ESRD) was chronic glomerulonephritis (33.3%), followed by diabetic nephropathy (23.5%). Hemodialysis was the most frequently used dialysis modality before transplant (70.9%). Preemptive transplantation was performed in 24.0% of living donor kidney transplants. Mean waiting time for deceased donor KT was 68.7 ± 38.0 months. Re-transplantation was done in 7.8% of cases. Mean number of HLA mismatches was 3.4 ± 1.8. Mean panel reactive antibody positivity percentage was 11.7 ± 24.3 in class I and 11.7 ± 24.8 in class II. As an induction agent, Basiliximab was used in 80.6% of kidney transplants, and ATG was used in 31.8% of deceased donor kidney transplants. Tacrolimus was the main calcineurin inhibitor (96.2%). Early steroid withdrawal was done in 2.0% of patients. Donor data was described as cases  , where LASSO might not show its strength in variable selection . In addition, we explored baseline predictors to early outcomes and reported dominant factors influencing patient and graft outcomes. Dominant factors for 1-year patient survival were found as predictors associated with recipient’s age or recipient’s comorbidities. Although infection was the most common cause of death, top dominant factor for 1-year patient survival was cardiovascular disease history. Aging might be intermediate process between infection as leading cause of death and cardiovascular history as leading predictor for patient 1 year mortality. For 1-year graft survival, dominant factors were predictors associated with immunologic risks and donor and recipient’s comorbidities. It was interesting to see that donor age was found to be the most dominant factor influencing graft function at 1 year, followed by post-transplant acute rejection and BKVAN.When the KOTRY was launched, annual transplant numbers were 1,400. At the design stage of KOTRY, an annual enrollment of 1,200 cases was aimed at to cover more than 80% of total kidney transplants in South Korea. However, the recent rapid increase in kidney transplants has resulted in KOTRY covering about 50-60% of total kidney transplants in South Korea. Still, KOTRY projects compose the largest multi-center cohorts in this country. In KOTRY, clinical details that claim data cannot capture are important resources to future research. Another strength of KOTRY is its role as a biobank. Prospective sample collection will provide invaluable research resources.24, which is reflected in the high proportion of diabetes in KOTRY. A large proportion of glomerulonephritis as a cause of ESRD could represent the results of patient selection or accessibility to kidney transplantation. Another important feature of Korean kidney transplants is the high proportion of living donor kidney transplantation. We identified that among living donor kidney transplants, 24% were preemptive kidney transplants. Compared to the high proportion of preemptive transplants with living donor kidneys, a long waiting period among deceased donor kidney transplants is another feature of Korean kidney transplants. Aside from almost unanimous standard triple maintenance immunosuppressants, ATG induction was observed as a variation. The proportion of steroid withdrawal was 2% in this data. The most common cause of death was infection, followed by cardiovascular disease. Those causes of death are compatible with the predictors selected in the data-driven approach because recipient age and history of cardiovascular disease were selected as dominant predictors for 1-year mortality. The most common pre-transplant cardiovascular disease was ischemic heart disease in this study population. However, not only ischemic heart disease was significant predictors to post-transplant 1 year mortality, but other subcategories of cardiovascular disease were also significant predictors, which implies that holistic heart function itself is important for the early post-transplant mortality not limited to the presence or absence of coronary arterial occlusive disease.The most common cause of ESRD in South Korea is diabetic nephropathy25. In terms of graft survival on the recipient side, it is interesting to see that selected predictors were donor characteristics such as donor age, donor hypertension, and donor diabetes. However, extension of this finding to long-term risk predictors needs cautious interpretation because non-modifiable donor factors could be exaggerated in early transplant outcomes. In terms of donor safety, marginal kidney function would also affect donors’ long-term outcomes; therefore, this data is evidence of the importance of proper donor selection.Recent investigations of donor safety have concerned higher lifetime ESRD risk in young donors27; however, to the best of our knowledge, this is the first study to find that donor age is the most dominant factor influencing acute rejection in a quantitative comparison. Donor age has near-linear pattern of log odds increment, which might explain its dominancy in the regression-based dominance model. The best cutoff value which we can classify post-transplant acute rejection within 1 year with was 48 years old. However, it might be different from what we think as safety line of donor age, because the increment of log odds value is observed even in the late 20 s of donor age. In addition, the most profound increment of log odds of post-transplant rejection within 1 year from donor age was observed in the after 60 years old of donor age, especially among deceased donors. HLA incompatibility was the second most dominant predictor for acute rejection. This finding could be epidemiological evidence supporting the importance of passenger leukocyte and its memory, or the vulnerability of aged endothelial cells to ischemia reperfusion injury and damage-associated molecular pattern expression29. Desensitization was selected as an important predictor for acute rejection, which implies that although mitigation of immunological risk was performed by desensitization, residual risk still persisted. We anticipate that the details regarding desensitization will be investigated in future studies.It was interesting to see that donor age was the most dominant factor influencing acute rejection. Several publications pointed out the significance of donor age as a risk factor for acute rejection31. In this study, its importance to predicting post-transplant eGFR was high.Dominant predictors to 1-year post-transplant recipient’s eGFR were donor age, acute rejection within 1 year, BKVAN within 1 year, and recipient BMI. We might interpret this as a mixture of kidney function and risk of rejection because donor age and body mass index can affect eGFR directly via muscle mass and intermediate outcomes including acute rejection, or that BKVAN directly represents the damaging process to the transplanted kidney. The importance of donor kidney-recipient weight gap was a well-known factor to post-transplant eGFRThe limitations of the study are as follows. First, this project enrolled about 50% of total kidney transplant patients in South Korea. Informed consent was required; therefore, information bias might exist. For example, recipients with poor compliance could refuse study enrollment, and urgent transplants performed during weekends or late at night might not have been enrolled in this project. Second, dominance of predictors was based on variable selection in traditional stepwise regression, which is not completely independent as to the randomness of entering variables. We tried to overcome this limitation by applying regularized regression methods (LASSO), which were unsuccessful due to having a large number of cases compared to selected predictors. However, we think this quantitative comparison of the relative importance of variables is a significant contribution to the transplant field.In conclusion, we presented clinical characteristics of patients enrolled in KOTRY during the past 5 years and investigated dominant predictors for early post-transplant outcomes by comparing relative contributions to the outcome prediction. The dominant predictors to recipient mortality within 1 year were deceased donor, recipient age, and recipient history of cardiovascular disease. The dominant predictors to death-censored graft loss within 1 year were deceased donor, desensitization, and donor hypertension. The dominant predictors of post-transplant 1-year recipient’s eGFR were donor age, acute rejection within 1 year, and BKVAN within 1 year. Finally, the dominant predictors to acute rejection within 1 year were donor age, HLA mismatches, and desensitization.11. In brief, data on pretransplant evaluations, immunologic risks, induction and maintenance immunosuppressants, every kidney biopsy result, every treatment of acute rejection, graft function measured as eGFR, post-transplant cardiovascular events, post-transplant infection events, and the survival of patients and grafts were collected. As large-volume centers are participating in KOTRY, the numbers of organ transplantations performed in KOTRY-participating centers were 83% for kidney transplantation. However, because this nationwide cohort is based on patient’s informed consent and prospective follow up, KOTRY enrolls about 1,200 new kidney transplant cases per year, which reaches about 55% of annual total kidney transplantation in South Korea. Although some selection bias might exist for the patient enrollment, KOTRY data was shown to be compatible with the nationwide post-transplant hard outcome based on administrative claims32. For the details of post-transplantation outcome, KOTRY is the only source of Korean transplant recipient’s data in national scale. For this study, the dataset of patients who received kidney transplants from 2014 to 2018 was used. A total of 4,854 kidney transplant recipients were analyzed. Mean duration of follow-up was 36.4 ± 15.5 months.The Korean Organ Transplantation Registry (KOTRY) is a nationwide solid organ transplant cohort launched in 2014. The design and methods of KOTRY were described in detail in a previous report34. For the time to event outcomes and binary outcomes, least absolute shrinkage and selection operator (LASSO) or backward stepwise selection were used. LASSO is a statistical methodology for variable selection and penalization. During the coefficient shrinkage of LASSO methodology, some coefficient goes to exactly zero value, which resulted in variable selection. For the LASSO, chosen optimum lambda values were one standard error apart from the lambda value of minimal partial likelihood deviance at the iterative cross-validation35. A total of 19 covariate candidates for prediction model construction were as follows: recipient age, donor age, recipient sex, donor sex, recipient’s history of diabetes, recipient’s history of cardiovascular disease, recipient’s history of cancer, pre-transplant systolic blood pressure of recipient, pre-transplant body mass index of recipient, donor’s diabetes history, donor’s hypertension history, waiting time to kidney transplant, pre-transplant body mass index of donor, deceased donor, total number of human leukocyte antigen (HLA) mismatches, desensitization, anti-thymocyte globulin (ATG) as an induction agent, and smoking status of donor and recipient. When all covariables were entered into the prediction model for the death-censored graft loss, c-statistic was 0.671, which was comparable to previous prediction studies18.We tried to derive a best prediction model for post-transplant outcomes  from baseline (pre-transplant) covariables. For the variable selection, we applied various approaches depending on the availability of existing methods and the character of the variables. For continuous measures, the Furnival-Wilson leaps-and-bound algorithm determined by Akaike’s information criteria was used for variable selection37. Because dominance analysis could be applied to the generalized linear model, we applied dominance analysis for continuous outcomes (post-transplant eGFR) in the form of linear regression, or for binary outcomes  in the form of logistic regression. Because panel reactive antibody were tested in 62.1% of recipients, we did not include panel reactive antibody from the previous tests. We separately conducted the same process as a sensitivity analysis including panel reactive antibody as one of the predictors, which made total dataset reduced to 3,019 donor-recipients pairs. Panel reactive antibody was dropped during the backward variable selection and did not remain in the dominance analysis to rank the relative importance. Continuous data are presented as mean with standard deviation. Categorical data are presented as count with percent. Cox regression for time to event data was performed under the proportional hazard assumption. Splines were applied to formalize non-linearity in the statistical models. For the optimal cutoff value estimation, Liu’s method was used38. Statistical analyses were performed using Stata software  and R .After a model was built, we applied dominance analysis to rank the relative importance of each selected variable to target outcomeThe study protocol was approved by the Seoul National University Hospital institutional review board (IRB No: H-1902-138-1014). Data analysis was done with de-identified datasets. Patient privacy was preserved in all instances, and the study methods complied with the tenets of the Declaration of Helsinki. All participants provided their written informed consent.Supplementary Figure 1.Supplementary Figure 2.Supplementary Information.


Post-transplant cyclophosphamide (PTCy) has emerged as a promising graft-versus-host disease (GvHD) prophylaxis in allogeneic hematopoietic stem cell transplantation . However, no studies have reported the efficacy of a GvHD prophylaxis based on PTCy with sirolimus (Sir-PTCy) in patients with acute myeloid leukemia (AML). In this retrospective study, we analyze the use of sirolimus in combination with PTCy, with or without mycophenolate mofetil (MMF), on 242 consecutive adult patients with AML undergoing a myeloablative first allo-HSCT from different donor types, in three European centers between January 2017 and December 2020. Seventy-seven (32%) patients received allo-HSCT from HLA-matched sibling donor, 101 (42%) from HLA-matched and mismatched unrelated donor, and 64 (26%) from haploidentical donor. Except for neutrophil and platelet engraftment, which was slower in the haploidentical cohort, no significant differences were observed in major transplant outcomes according to donor type in univariate and multivariate analysis. GvHD prophylaxis with Sir-PTCy, with or without MMF, is safe and effective in patients with AML undergoing myeloablative allo-HSCT, resulting in low rates of transplant-related mortality, relapse/progression, and acute and chronic GvHD in all donor settings. Allogeneic hematopoietic stem cell transplantation  is an increasingly offered and potentially curative option for patients with acute myeloid leukemia (AML). Regarding GvHD prophylaxis, the success of post-transplant cyclophosphamide (PTCy) in the haploidentical setting has led to the expansion of its use also in recipients of allo-HSCT from HLA-matched sibling donor (MSD) and unrelated donor (MUD) , 2. PTCyThe aim of this study was to analyze the impact of donor type on transplant outcomes in a large series of patients with AML undergoing myeloablative first allo-HSCT using a uniform platform of GvHD prophylaxis based on Sir-PTCy.This is an observational retrospective study. All consecutive patients with AML who received a first allo-HSCT between January 2017 and December 2020 at Hospital Universitari i Politècnic La Fe , Hospital Clínico Universitario , and Ospedale San Raffaele  were included in the study.Data of patients, transplant procedures, toxicity and complications were prospectively collected in all patients and then settled in a computerized database. Clinical charts were additionally reviewed for inconsistent or missing data.For donor selection, MSD was the primary option. If not available, a MUD registry search was started. If a matched donor ≥9/10 was not readily available, the best related haploidentical donor (Haplo) was selected according to the following: absence of recipient HLA antibodies against donor antigens, male sex, younger age, matched cytomegalovirus (CMV) serostatus, matched ABO group, and, for female donors, lowest number of prior pregnancies.Conditioning regimens and GvHD prophylaxis schemes used at the three centers have already been described elsewhere , 17. SirAll conditioning regimens used in the present study are considered myeloablative according to the latest definitions , 19.+ cells (≥3 ×108/Kg) [Eight patients transplanted at San Raffaele received a single dose of anti-thymocyte globulin 5 mg/Kg on day +5 according to center guidelines due to high graft content of CD3×108/Kg) .9/L for three consecutive days, while this strategy was more recently adopted in San Raffaele (starting from May 2020).Supportive care measures have already been described elsewhere , 17. CMV9/L after transplantation. Platelet engraftment was defined as the first of 7 consecutive days with platelet counts ≥20 ×109/L without platelet transfusions. Clinical diagnosis and grading of acute GvHD (aGvHD) and chronic GvHD (cGvHD) were made according to the Glucksberg criteria [Patients were classified at the time of transplantation according to DRI , 23 and criteria  and the criteria , respectcriteria , Epsteincriteria , invasivcriteria , and BK criteria  were diacriteria .p-value <0.10 were first included in the model; a stepwise backward procedure was used with a cut-off significance level of 0.10 for deleting factors from the model. The type I error rate was fixed at 0.05 for determination of factors associated with time to event. Statistical analyses were performed using R statistical software version 4.0.4  and SPSS version 25.0 .Endpoints of the study were leukemia-free survival (LFS), overall survival (OS), graft-versus-host-free/relapse-free survival (GRFS), transplant-related mortality (TRM), cumulative incidence of relapse/progression (CIR), cumulative incidence of neutrophils and platelet engraftment, and cumulative incidence of aGvHD and cGvHD. Last follow-up was considered the 1st of July 2021. Unadjusted time-to-event analyses were performed using the Kaplan-Meier estimate , and, fop = 0.02) and a slightly greater proportion of adverse genetic category in MUD recipients (p = 0.045). Regarding transplant characteristics, donor age was also higher in the MSD cohort (p < 0.0001).Patient, disease and transplant characteristics of the overall population and according to donor type are summarized in Table + selected boost for secondary poor graft function. Cumulative incidence of neutrophil recovery at 60 days was lower in haploidentical recipients (p = 0.003)  (p = 0.008).Eight patients died without evidence of myeloid engraftment, 5 in the MUD cohort , 2 in the Haplo cohort , and one from MSD (infection). Primary graft failure was only observed in 2 haploidentical recipients with high titer of anti-HLA donor specific antibodies; both patients were successfully retransplanted from the same donor after a desensitization therapy. The remaining 232 patients achieve neutrophil engraftment at a median time of 19 days . Median time to neutrophil recovery for MSD, MUD, and Haplo was 17 days , 19 days , and 21 days , respectively. No secondary graft failure was observed. All engrafted patients achieved full donor chimerism. Ten patients  underwent a CD34p = 0.01)  for the overall population and 20 days  for MSD, 26 days  for MUD, and 33 days  for Haplo. Cumulative incidence of platelet recovery was lower in the Haplo cohort , with a median time to diagnosis of 129 days ; 3 patients developed late onset aGvHD after infusion of a CD34-selected boost administered due to poor graft function.There were 9 aGvHD that were diagnosed after day 100 without any difference according to donor source . Three of these patients developed the condition after infusion of a CD34-selected stem cell boost for poor graft function, and two patients after respectively 3 and 4 months of therapy with a FLT3-inhibitor.Fifteen patients developed an overlap cGvHD in our series at a median time of 155 days . No statistical difference was observed according to donor groups  patients at a median time of 37 days : 44 (45%) grade I, 36 (35%) grade II, 15 (15%) grade III, and 5 (5%) grade IV. No variables were associated with an increased risk aGvHD in multivariate analysis.Overall, cGvHD occurred in 100 41%) patients at a median time of 173 days . According to the NIH classification, 35 (35%) were mild, 46 (46%) moderate, and 19 (19%) severe. Results of multivariate analysis are displayed in Table 1% patienThe main post-transplant complications are detailed in Table p = 0.046), but this was not confirmed in multivariate analysis.Thirty-four patients died without prior relapse or progression at a median time of 82 days . All causes of death according to donor type are shown in Table n = 7) or in combination with marrow relapse (n = 8), with central nervous system being the most common site involved (n = 5). Patients with extramedullary relapse did not have different characteristics than those with hematological relapse. One additional patient was diagnosed with HLA loss relapse 6 months after Haplo. We did not observe significant differences in the pattern of relapses and the CIR according to the type of donor . Extramedullary relapse occurred in 15 patients, either isolated  and active disease status at allo-HSCT were the only independent risk factors found for TRM, with the latter along with MRD positive status being also associated with a higher risk of relapse/progression. The lack of statistically significant differences in TRM and relapse/progression according to the type of donor translated into an absence of significant differences also in OS, LFS, and GRFS.Although non-randomized, this study included a real-life unselected population of AML patients who underwent first allo-HSCT with a similar CNI-free GvHD prophylaxis in three European centers during the same study period. Recently reported studies have demonstrated that this approach is not only feasible and effective after Haplo, but also in allo-HSCT from MSD and MUD for patients with a variety of hematologic malignancies , 16, 17.+ content in the graft  being the main variables associated with an increased risk of cGvHD in multivariate analysis.The relatively low rates of grade II-IV and grade III-IV aGvHD, as well as moderate-to-severe cGvHD, are similar to those previously reported separately in hematologic malignancies with this GvHD prophylaxis by the Italian  and SpanIn addition to the acceptable incidence of aGvHD and cGvHD, encouraging results were obtained in terms of CIR, TRM, OS, LFS, and GRFS when compared with other series that used PTCy in patients with AML , 38, 43,The fact that the median time to neutrophil and platelet recovery appears somewhat longer in our study than with CNI-based GvHD prophylaxis is likely due to PTCy. In fact, a recently reported randomized phase III trial that compared two CNI-free approaches (CD34-selected grafts and PTCy) to standard CNI-based GvHD prevention, with tacrolimus and methotrexate, showed that grafting after PTCy was slower compared to the others .Our results are partially in contrast with a recent registry-based study in which patients with AML undergoing Haplo allo-HSCT displayed a higher rate of aGvHD and TRM, coupled with a lower incidence of relapse, compared to recipients of MSD and MUD . In factThe relatively low incidence of relapse (16% at 2 years) reported in the present study, despite the considerable number of patients transplanted with active disease 15%) or ≥CR2 (19%), should be highlighted. Although conflicting results have been reported by other authors regarding the impact of conditioning intensity on the risk of relapse –58, our % or ≥CR2In addition to the well-known prognostic factors such as DRI, 2017 ELN risk classification, and disease status at allo-HSCT, we also confirmed in our study the independent prognostic impact of pretransplant MRD status on the risk of relapse , 62. AltFurthermore, we were unable to detect any impact on engraftment, GvHD, and infections when MMF was omitted from GvHD prophylaxis, but it should be noted that this group consisted of only 28 patients, which represents 11% of the total and a third of those transplanted from MSD, a very small sample size to analyze its impact.In conclusion, myeloablative allo-HSCT for AML with Sir-PTCy, with or without MMF, as GvHD prophylaxis resulted in a low incidence of relapse/progression and TRM, acceptable rates of aGvHD and cGvHD, and good survival outcomes for all donor types. Despite the absence of major differences in transplant outcomes by donor type using this GvHD prophylaxis, a possible change in donor selection algorithm for AML patients in need of an allo-HSCT should still await confirmation of our results in prospective controlled studies. Future studies should clarify whether other donor characteristics should take priority over donor type under certain circumstances in the setting of Sir-PTCy GvHD prophylaxis.Supplementary material