Uterus transplantation (UTx) can provide a route to motherhood for women with Mayer–Rokitansky–Küster–Hauser syndrome (MRKHS), a congenital disorder characterized by uterovaginal aplasia, but with functional ovaries. Based on our four successful living-donor transplantations and two resulting births, this analysis presents parameters relevant to standardizing recipient/donor selection, UTx surgery, and postoperative treatment, and their implementation in routine settings. We descriptively analyzed prospectively collected observational data from our four uterus recipients, all with MRKHS, their living donors, and the two newborns born to two recipients, including 1-year postnatal follow-ups. Analysis included only living-donor/recipient pairs with completed donor/recipient surgery. Two recipients, both requiring ovarian restimulation under immunosuppression after missed pregnancy loss in one case and no pregnancy in the other, each delivered a healthy boy by cesarean section. We conclude that parameters crucial to successful transplantation, pregnancy, and childbirth include careful selection of donor/recipient pairs, donor organ quality, meticulous surgical technique, a multidisciplinary team approach, and comprehensive follow-up. Surgery duration and blood vessel selection await further optimization, as do the choice and duration of immunosuppression, which are crucial to timing the first embryo transfer. Data need to be collected in an international registry due to the low prevalence of MRKHS. Uterus transplantation (UTx) has been demonstrated in recent years to provide a route to biological motherhood for women with absolute uterine factor infertility (AUFI), whose only other option to attain genetic  motherhood would be surrogacy . AUFI maWhile human UTx is clinically still at the experimental stage, our university hospital has gained comprehensive multidisciplinary experience with MRKHS and other genital malformations over the past 25 years, offering full-range specialist services from initial diagnosis to neovaginoplasty, treatment of uterine disorders, fertility treatment, and pregnancy and delivery care ,5,6, as Before the advent of UTx, the treatment of women with MRKHS focused exclusively on the vaginal aspect of the malformation, i.e., creating a neovagina by nonsurgical self-dilation or by one of the established surgical neovaginoplasty techniques ,11. WhilThe first successful living-donor UTx procedures worldwide resulting in healthy childbirth were performed in Gothenburg, Sweden in 2012 after more than ten years of meticulous basic research, including comprehensive animal and clinical studies . By 2019We here report on the experience gained at our university hospital, the first to perform UTx in Germany in October 2016. UTx became possible at our center through close collaboration with the team at the Sahlgrenska Academy, University of Gothenburg. This involved developing a multistep process encompassing medical and psychological interventions ,16.The objective of the present analysis was to descriptively analyze prospectively collected observational data from uterus recipients and their living donors who underwent living-donor UTx at our hospital to identify parameters relevant to the standardization of recipient/donor selection, UTx surgery, and postoperative treatment, with the aim of implementing such parameters in a routine setting. In addition, we report our experience with the choice and duration of immunosuppressive (IS) treatment, the time of first embryo transfer (ET), treatment after a miscarriage, as well as the pregnancy, obstetric, and neonatal data for the two successful post-UTx pregnancies we had at our institution. Finally, we also provide the first-ever report of ovulation induction and oocyte retrieval under immunosuppression after UTx.All uterus recipients and living uterus donors who respectively completed UTx recipient and donor surgery at Tübingen University Hospital were eligible for inclusion in the present analysis. Recipients had all undergone Vecchietti-based neovaginoplasty ,18 or crAll potential recipients and donors had been selected according to the rigorous screening procedure reported by Taran et al.  and BrucThe selection phase was followed by consultation of the Regional Council Committee for Living Donation to obtain approval of living donation in accordance with the German Transplantation Act. This involved ascertaining that the donor’s decision was completely voluntary and uninfluenced by any coercive pressure, that she received adequate information about the risks and benefits involved, and that she had a close relationship with the recipient ,19.Uterus recipient and living-donor surgeries were performed at Tübingen University Hospital by specialists in gynecological surgery, transplantation surgery, immunology, internal medicine, and anesthesiology, who were all part of our larger multidisciplinary UTx team, which additionally included specialists in reproductive medicine, maternal-fetal medicine, neonatology, psychosomatic medicine, radiology, and pathology .The present report was designed as a descriptive analysis of observational medical data prospectively collected from uterus recipients and their living donors during the analysis period from October 2016, when we performed the first UTx in Germany, through May 2020, by which time 1-year follow-up data were available for both babies born to two of the four uterus recipients. Specifically, parameters of interest included general pre-UTx baseline characteristics, medical and surgical history, preoperative diagnostic findings, smoking habits, fertility details, and other pre-UTx clinical characteristics. Further parameters of interest included details of the assisted reproductive technology (ART) procedure, UTx surgical report details such as surgical time, cold and warm ischemia time, surgical and postoperative complications and other events, postoperative follow-up observations, and data on post-UTx IS treatment, ET, post-UTx ART treatment, pregnancy, obstetric outcome, and neo- and postnatal findings. Surgical complications were graded based on the Clavien-Dindo (C-D) classification ,22,23.Our objective was to descriptively analyze relevant parameters pertaining to recipient/donor eligibility, surgery, fertility treatment, pregnancy, and later motherhood, with the ultimate goal of implementing such parameters in clinical routine to achieve standardization and ensure treatment success.In view of novel ethical challenges potentially associated with living-donor UTx, which unlike other organ transplantations is not a life-saving intervention, it was considered essential to involve the University’s Institute for the Ethics and History of Medicine from the inception of the program as described in detail by Taran et al. . The TübThe donor and recipient operations were performed using the standard laparotomy techniques involving subumbilical midline incisions, as previously described by Brännström et al. .Briefly, donor surgery involved harvesting the uterus with its blood vessels but without the ovaries. The Fallopian tubes were removed but not used in the graft to avoid extrauterine pregnancies. The ovaries were preserved in situ.The arterial vasculature comprised the deep uterine artery (DUA) on either side with a segment of the internal iliac arteries (IIAs), preferably divided just distally to the gluteal artery. The venous vasculature connected to the uterine graft to be harvested comprised 1 or 2 deep uterine veins (DUVs) on either side, attached to a segment of the internal iliac vein (IIV), and/or the proximal parts of the utero-ovarian branch, divided before the inlet of the ovarian veins, in order to preserve the ovaries in situ.Recipient surgery involved preparation by dissection of the vaginal vault and the external iliac vessels. Anastomoses were performed end to side from the segments of the graft’s DUAs to the recipient’s external iliac arteries (EIAs) as well as from the segments of the graft’s IIVs to the recipient’s external iliac veins (EIVs). In recipients with a thin DUV on one side, the proximal part of the utero-ovarian vein (UOV) was used for an additional venous outflow section on that side, either by anastomosis onto the segment of the graft’s IIV or directly onto the EIV. After ensuring proper reperfusion, the recipient’s vagina was opened and vaginal-vaginal anastomosis was performed. The uterus was affixed to the sacrouterine and round ligaments, and the bladder peritoneum of the graft was sutured onto the top of the recipient’s bladder.® ; tacrolimus target trough level 10–12 ng/mL, starting dose 0.1 mg/kg bw), mycophenolate mofetil (MMF) 2 × 1000 mg, and prednisolone, which was tapered from 100 mg/kg bw to 5 mg/kg bw over a period of 3 weeks, followed by a maintenance prednisolone dose of 5 mg. In view of planned pregnancy, MMF in Regimen B was replaced after approximately 6 months with azathioprine (AZA) at 1 mg/kg for at least 3–6 months. Adaptation of IS treatment, necessary to improve individual tolerability, involved replacement of tacrolimus with ciclosporin , target trough level 100–130 ng/mL) in combination with either MMF 2 × 1000 mg or, later, when planning pregnancy, AZA 1 mg/kg bw, and prednisolone 5 mg 1-0-0.IS regimens for UTx were adopted from kidney transplantation. Immunosuppression was achieved using the following triple-drug regimens. Regimen A, the initial triple-drug IS treatment, consisted of induction therapy with anti-thymocyte globulin (ATG) at 1.5 mg/kg body weight (bw) for 3 days and the parallel start of a triple-drug IS regimen with tacrolimus  status.Tacrolimus and ciclosporin trough levels were checked every 2–4 weeks, also during pregnancy, and prednisolone was retained at a 5-mg maintenance dose. Infectious prophylaxis consisted of cotrimoxazole for 6 months for Predetermined sets of medical history, diagnostic, clinical, surgical, and postoperative data were collected continually throughout the entire process from initial presentation to postoperative treatment and follow-up. Data were analyzed descriptively as numbers, frequencies, percentages, and percentiles. No statistical tests were performed.Recipient age ranged from 23 to 35 years. All recipients had type 1 MRKHS. All but one recipient, who created a neovagina by self-dilation, had undergone laparoscopically assisted Vecchietti-based neovaginoplasty ,18 to crDonor age ranged from 32 to 56 years. The 56-year-old mother of Recipient 4 was the only postmenopausal donor. All donors had given birth to at least two children, the maximum being four deliveries.HLA matching and mismatch analyses were performed for all recipients and their respective partners, as shown in Excluded from the present analysis was a fifth UTx procedure (prospective Recipient 2 and Donor 2) because it was aborted after procurement of the uterus from the donor but before recipient surgery was commenced on the prospective recipient. The procedure in question was aborted based on a team decision after multiple attempts to flush the uterine arteries (UAs) of the retrieved organ during back-table preparation failed to provide adequate flow, even at high pressures, hence potentially creating a high risk of transplant failure due to inadequate perfusion after transplantation and increasing the risk that blood flow in the recipient might be insufficient to sustain uterine function, particularly during pregnancy . HistopaUterus Evaluation®, Sanofi-Aventis, Frankfurt/Main, Germany). Additionally, angiography of the lower abdomen was performed to evaluate the diameters of the arterial and venous uterine vessels and potential variants of vessel course.Donor uteri were evaluated using transvaginal ultrasonography and high-resolution magnetic resonance imaging of the pelvic anatomy (T2-weighted turbo spin echo (TSE)) sequences in three planes after intravenous administration of butylscopolamine bromide  angiography of the pelvic vessels with arterial and venous phase imaging to confirm presumed vessel size and exclude any new stenosis or arteriosclerotic calcifications. The presence of atherosclerotic plaques in the IIAs, small arterial caliber, and any other significant changes of the UAs were considered markers of lesser organ quality and predictors of negative outcome. Both the DUVs and the proximal part of the UOVs were assessed. The presence on either side of at least one vein that could potentially serve as an outflow was considered adequate. For recipients, a native CT scan of the pelvis was sufficient to rule out the presence of calcifying plaques in the iliac arteries.2Ovarian Stimulation and Oocyte RetrievalPreoperatively, all four recipients who subsequently underwent successful UTx received fertility treatment for in vitro fertilization (IVF). The numbers of oocytes fertilized with sperm from the recipients’ respective partners and subsequently cryopreserved for later implantation ranged from 9 to 17 .®, Merck, Darmstadt, Germany), whereas Recipients 2 and 3 received daily injections of 200 IU r-hFSH from day 2 of their menstrual cycle. Pituitary suppression with cetrorelix  started simultaneously on cycle day 6, and ovulation was induced with 250 μg recombinant human chorionic gonadotropin  when the size of the leading follicle exceeded 18 mm . Of the 18 oocytes obtained from Recipient 1, 17 were successfully injected using the intracytoplasmic sperm injection (ICSI) technique, of which 10 were fertilized and cryopreserved as pronuclear 2 (PN-2) stage zygotes. Out of 15 oocytes retrieved from Recipient 3, 10 were injected using ICSI, of which 2 were fertilized and cryopreserved. Of the 26 oocytes retrieved from Recipient 4, 21 were injected using ICSI, 17 of which were fertilized and cryopreserved as PN zygotes.Before UTx, Recipients 1, 3, and 4 underwent ovarian stimulation for ovulation induction according to a standard antagonist protocol. Recipient 1 received daily injections of 150 IU recombinant human follicle-stimulating hormone  ischemia, was between 63 and 86 min.Anastomosis utilized the following vessels. For arterial anastomosis, a segment of the donor’s IIA with the adjacent DUA was end-to-side anastomosed onto the recipient’s external iliac artery (EIA) on both sides. Anastomosis of the veins was performed using a segment of the donor’s IIV with the adjacent DUV. In some cases, we used the UOV, allowing the ovaries to be retained. Only in Donor 3 did we use the OV on the right side, resulting in ovariectomy on this side. Details as to which vessels were used in which case are given in Blood flow in the uterine arteries as determined by intraoperative Doppler ultrasonography and transit time flowmeter  directly after anastomosis was used to assess arterial patency and measure arterial flow, which was up to 30 mL/min in all recipients. Two Cook-Swartz Doppler Probes , placed on either side of the uterine artery, provided continuous audible and visual feedback of blood flow during the first 5–6 days after recipient surgery.No complications were encountered intra- or postoperatively, except in the last recipient (No. 5), who showed no arterial flow on the right side intraoperatively, thus necessitating reanastomosis. This involved suturing the right UA end to side onto the EIA in addition to a segment of the IIA with adjacent proximal vesical artery. No donor or recipient required any blood transfusions.Hospital stays ranged from 14 to 18 days for recipients and from 11 to 14 days for donors. All four recipients and their donors had uneventful postoperative recoveries without any early or late complications during the respective follow-up periods of 4 up to 44 months. The four recipients began to menstruate between 3 and 6 weeks post UTx, and subsequently continued to menstruate regularly.All recipients started and were maintained on triple-drug Regimen A blue in  for the After ET, Recipients 1 and 3 stayed on unchanged triple-drug IS therapy consisting of prolonged-release tacrolimus, AZA, and prednisolone .Gynecological examinations, transvaginal ultrasound, and ectocervical biopsies were performed twice weekly during the first month after UTx and subsequently at gradually increasing intervals. Ectocervical biopsies were obtained to detect rejection and were assessed according to a uterine rejection grading scale .Postoperatively, Recipients 3 and 4 each experienced one mild episode of graft rejection, which in both cases was successfully treated with a single dose of cortisone. Recipient 4 was found to have elevated liver enzymes postoperatively. Hepatitis E was confirmed and successfully treated with an antiviral therapy. Later, Recipient 4 developed a CMV infection, which was successfully treated with valganciclovir about 7 months after surgery .As indicated in Similarly, all embryos obtained from oocytes harvested from Recipient 3 degenerated during the thawing process before ET. Therefore, postoperative restimulation became necessary in both patients.As in preoperative ovulation induction, the following, similar, standard antagonist protocol was used postoperatively.®, Merck, Darmstadt, Germany) from cycle day 2 onwards. Pituitary suppression with 0.25 mg ganirelix  or cetrorelix (Cetrotide®), respectively, started simultaneously on cycle day 6. Ovulation was induced with 250 μg r-hCG (Ovitrelle®) when the leading follicle exceeded 18 mm in diameter and respective estradiol levels were 1790 and 3246 pg/mL.At post-UTx months 24 and 15, Recipients 1 and 3, respectively, received 150 IU r-hFSH .Ovulation induction yielded 14 oocytes for Recipient 1 and 15 oocytes for Recipient 3. Normozoospermia enabled oocytes to be fertilized using standard IVF, leading to 9 fertilized oocytes for Recipient 1 and 6 fertilized oocytes for Recipient 3. Blastocyst culture was performed with a limited number of 3 fertilized oocytes in accordance with the German Embryo Protection Act (ESchG). Supernumerary fertilized eggs were cryopreserved for future use. The luteal phase was supported by administering 600 mg progesterone daily  was performed at the level of the EIVs, resulting in both ovaries and vessels being located in a clearly cranial position than in anatomically normal IVF patients.Following the ovarian restimulation and oocyte fertilization procedures, Recipients 1 and 3 both underwent a fresh single-blastocyst transfer, resulting in successful intrauterine pregnancies. Both recipients continued triple-drug IS treatment  throughout pregnancy.Recipients 1 and 3 both experienced successful pregnancies. Neither recipient had any major health problems such as diabetes or hypertension during pregnancy and childbirth. In both recipients, the umbilical artery pulsatility index was always normal. Recipient 1 was admitted to hospital 13 days before obstetric surgery (week 32 + 2) due to inguinal pain and cervical and retroplacental hypervascularization. Recipient 3 was admitted 4 days before cesarean section (week 35 + 6) due to reduced amniotic fluid and the child being diagnosed as small for gestational age (around the 10th percentile). She had previously been diagnosed with reduced cervical length (minimum length 25 mm) without contractions.Single biopsies from both pregnant uterus recipients at week 20 of pregnancy revealed no sign of rejection. Both women gave birth to cephalically presenting healthy boys. Recipient 1 delivered her baby at week 35 + 1 after preterm rupture of membranes in the absence of any signs of infection or labor. In Recipient 3, delivery took place as scheduled. Both babies were delivered by midline-incision cesarean section.Recipients 1 and 3 and their children were discharged from hospital 10 days and 34 days after admission and 6 days and 13 days after cesarean section, respectively. Breastfeeding began immediately in Recipient 1 and was stopped 12 months after delivery. Recipient 3 was unable to breastfeed due to previous mammillary surgery. The baby born to Recipient 1 was kept on the neonatal ward for 8 days due to premature rupture of membranes, hypoglycemia, and hypothermia. The infant subsequently developed completely normally, exhibiting bodyweights of 2.900, 6.310, 7.270, and 8.995 kg at ages 1, 4, 6, and 12 months, respectively. The child of Recipient 3 received neonatal care, including continuous positive airway pressure treatment, for three days due to preterm labor, hypothermia, and respiratory maladaptation. The infant exhibited completely normal development with bodyweights of 3.475, 5.950, 8.010, and 9.400 kg at ages 1, 4, 6, and 12 months, respectively, but required a 2-day hospital treatment for obstructive bronchiolitis at age 11 months.Menstruation resumed at 8 months and at 6 weeks after cesarean section in Recipients 1 and 3, respectively. The transplanted uteri were viable in all recipients as per the end of the analysis period. No events occurred that would have necessitated explantation of the graft.Worldwide, 54 living-donor and 19 deceased-donor UTx procedures had been performed by September 2019, resulting in the birth of 18 and 3 children, respectively, as reported at the 2nd Congress of the International Society of Uterus Transplantation (ISUTx) . Our uniEBL in our trial was 100 mL in all donors. As in the series of nine UTx procedures reported by Chmel et al. , no donoThe greatest recipient EBL was 500 mL. Blood loss in Recipient 5 was markedly higher than in all other recipients. This was due to the need for reanastomosis because the main branch of the UA was cut during an attempt to preserve the major posterior branch of the IIA on the right side in Donor 5. This was not discovered during back-table preparation as blood flowed to the uterus via collateral branches between the UA and the vesical artery. The latter proximal part had been preserved on the segment of the procured IIA. However, inadequate blood flow in the UA was discovered on the patch after revascularization. The UA was then dissected to achieve adequate length for an additional anastomosis. It was sutured end to side to the external artery in addition to a segment of the IIA with adjacent proximal vesical artery.Adequate flow and pulsation were then detectable by flowmeter and palpation. The complication arose because the UA had the first proximal part embedded in the IIA’s vascular wall and hence the outlet was not visualized during procurement. A lesson to be learned here is that when the angiogram indicates a short distance between the first major branch and the UA, it may be preferable not to try to preserve the iliac branches on that side. If this anatomical situation is present bilaterally, which was the case in this particular donor, the risk of not leaving any proximal branch of the IIA must be weighed against the risk of revascularization failure of the UA on one side, since performing an end-to-side anastomosis between the UA and the EIA is a much more difficult task.As UTx involves non-life-saving organ donation, only a minimal risk to the living donor is acceptable . In our In our UTx program, Donor 2, whose retrieved uterus proved unsuitable for transplantation, developed hydronephrosis (C-D IIIb), presumably due to thermal injury and consecutive stricture of the ureter. Other studies reported three instances of ureteral complications occurring in living donors. Fageeh at el.  and ChmeWe observed two mild rejection episodes, one each in Recipients 3 and 4. This observation is in concord with published data ,22. The The present study is the first to report childbirth after implantation of a cryopreserved embryo obtained by fertilization of oocytes retrieved from a uterus recipient (Recipient 1) while under post-UTx triple-drug immunosuppression. The attempt was successful even though the ovaries were iatrogenically attached in a cranial position during transplantation. We performed IVF according to standard IVF procedures in Recipients 1 and 3. Antagonist protocols were chosen and stimulation was performed using low dosages of recombinant FSH to avoid ovarian hyperstimulation syndrome. Controlled ovarian hyperstimulation rendered the enlarged ovaries easily accessible via the standard transvaginal route. This avoided the use of alternative routes involving transabdominal or laparoscopic ovarian puncture, although these are feasible options when the ovaries are placed outside the female pelvis. Placement of the intravaginal ultrasound probe for needle aspiration of oocytes beyond the vaginal anastomosis was also feasible without difficulty, as was the exposure of the cervix for ET. Ovarian stimulation, oocyte collection and fertilization, and subsequent embryo development were unimpaired in these immunosuppressed patients. Single ETs were performed to minimize the risk of a multiple pregnancy as best possible.According to the German IVF registry, the ongoing pregnancy rate per ET can be expected to be 33% in women under the age of 30 years . Eight wAntenatal complications were observed in Recipients 1 and 3, who presented with preterm prelabor rupture of membranes and oligohydramnios, respectively, necessitating delivery at gestational weeks 35 + 1 and 35 + 6. Other research groups reported 3 cases of pre-eclampsia, 2 cases of cholestasis, and 1 case each of preterm prelabor rupture of membranes, pyelonephritis, and subchorionic hematoma between weeks 13 and 22 of gestation, and central placenta previa with accreta at week 21 of gestation ,34,35,36The infants born to Recipients 1 and 3 in our UTx program both required initial neonatal care, one for 3 days due to hypoglycemia and hypothermia, and the other for 8 days due to respiratory maladaptation. However, the infants’ subsequent development was unremarkable at the routine 9- and 12-month checkups, with both children achieving catch-up growth in weight, length, and head circumference. Deliveries after UTx reported to date have all occurred between gestational week 31 + 6 and week 37, and all children were delivered by cesarean section ,34,35,36This report presents relevant, hitherto unpublished original data from four patients who successfully underwent living-donor UTx for AUFI due to MRKHS, providing a detailed analysis of patient data from real-life clinical experience with UTx, including basic characteristics such as age, weight, and smoking habits, HLA matching, surgical details such as the vessels used, operative time, EBL, IS regimens, the IVF protocol, successful ovarian stimulation under IS, and data on pregnancy, childbirth, and neonatal health and development. It thus contributes substantially to the growing body of data on a pioneering, new, and continually evolving surgical technique and treatment option for women with AUFI, in particular those with MRKHS. However, the small size of our study, which is due to the low prevalence of MRKHS, may limit the overall generalizability of our results. Moreover, it should be borne in mind that living-donor UTx is clinically still at the experimental stage.This report presents additional data on parameters considered relevant to the success of UTx, ART treatment, and subsequent pregnancy and childbirth. Most notably, our work has shown that it is possible to achieve and prolong pregnancies successfully under triple-drug IS treatment. Moreover, we report the standardization of triple-IS regimens with initially 5–6 months of Regimen A  or switched to Regimen D  if necessary) followed by Regimen B  for maintenance immunosuppression. Of note, this is also the first study to report successful ovarian stimulation, oocyte retrieval, IVF, and ET in living-donor uterus recipients under triple-drug IS therapy, resulting in pregnancy and delivery of healthy newborns.As previously reported, living uterus donation is associated with an estimated 20% risk of C-D grade III or grade IV surgical complications ,22,23. IWith UTx clinically still in the experimental phase, numerous questions remain to be answered. For instance, when is the right time to remove the graft if pregnancy and childbirth are not achieved? Is a surgical curettage possible in the event of a miscarriage, and what risks would this involve? Crucially, what type of IS treatment is best given for what period of time before making the first attempt at ET? In the case of an uneventful pregnancy, when is the optimal time for delivery so as to avoid an “emergency delivery” while also preventing extremely early preterm delivery? Finally, how can surgery duration and blood vessel selection be further optimized?Despite the continuing need for further studies to overcome the limitations of small studies, large UTx trials are unlikely to be conducted in the foreseeable future due to the relatively small numbers of eligible patients worldwide. Data therefore need to be collected in an international registry as proposed and recently initiated by the International Society of Uterus Transplantation (ISUTx).


Freesia hybrida. Herein, we investigated the dynamics of the major form of carbohydrates and related enzyme activities and profiled the transcriptome of freesia corms at four developmental stages with the aim to reveal the relation between the expression of genes involved in the metabolism of carbohydrates and the accumulation of carbohydrates in corm developmental stages for further exploring the mechanism on the starch and sucrose metabolism regulating the formation and development of corms in F. hybrida. The content of starch, sucrose and soluble sugars followed an overall upward trend across the corm developmental stages. Activities of the adenosine diphosphoglucose pyrophosphorylase, starch branching enzyme and β-amylase generally followed the pattern of the starch and sucrose levels. Activities of sucrose phosphate synthase increased from corm formation till the initial swelling stage and subsequently reached a plateau. Activities of invertase and sucrose synthase peaked at the later rapid swelling stage. These suggested that the starch and sucrose dynamics paralleled corm swelling under the action of metabolic enzymes. A total of 100,999 unigenes were assembled in the transcriptomic analysis, and 44,405 unigenes of them were annotated. Analysis based on Clusters of Orthologous Groups suggested that carbohydrate transport and metabolism (9.34% of the sequences) was prominent across the corm developmental process. In total 3,427 differentially expressed genes (DEGs) were identified and the enrichment analysis detected starch and sucrose metabolism as a critical pathway in corm development, especially at the rapid swelling stage. Further, DEGs encoding key carbohydrate-metabolizing enzymes were identified and correlated to enzyme activities and carbohydrate accumulation. The results construct a valuable resource pool for further molecular-level studies, which are helpful for metabolic regulation of carbohydrates and improvement in F. hybrida.Starch and sucrose metabolism plays a crucial role in the formation and development of bulbs in bulbous plants. However, these mechanisms remain unclear and unexplored in the corms of  Freesia spp.), a species of Iridaceae family and indigenous to South Africa (F. hybrida). Corms are the propagative organ, thus the quality of corms determines the healthiness of the plant and then the ornamental value of freesia. Previous studies suggest that development of storage roots was paralleled with complex physiological activities including carbohydrate, enzyme, and hormone dynamics  and tulips, fructans co-occur with starch to fulfill different functions (Freesia viridis) did not store fructans in the corm.Starch is a major storage form of carbohydrate and an energy carrier in plant storage organs. For instance, starch content was proposed to be a key trait for judging lily bulb quality . During he bulbs . At the he bulbs . Therefocoldness , which ccoldness . As a recoldness . Neverthcoldness . In bulbunctions . By contTulipa edulis stolons was accompanied with a sugar consumption and starch production via the action of enzymes. Nerine sarniensis bulbs at different developmental stages. The significance of metabolic enzymes in carbohydrate metabolism in developmental bulbs was further revealed in oriental hybrid lily ‘Sorbonne’ (Gladiolus hybridus (Starch and sucrose metabolism is closely related to a series of key enzymes. Sucrose synthase (SuSy) catalyzes the reversible synthesis of sucrose, but hydrolytic activity is suggested to play a leading role . Sucroseorbonne’  and Gladhybridus .Still, there is a lack of information in molecular mechanisms related to carbohydrate metabolism in freesia corm development. Genes related to enzymes involved in starch and sucrose metabolism remain unexplored. In view of this, in the current study, the dynamic changes of levels of starch and sucrose and key enzymes involved were monitored and the transcriptome was profiled during freesia corm development to reveal functions of major carbohydrates regulating freesia corm development. The next-generation high-throughput sequencing method was employed in this study to sequence the transcriptome of freesia corms. This sequencing method applies not only to plants with known genomes but also to those lack of reference genomic sequences, and has already been successfully attempted in diverse plants for transcriptomic profiling .To our knowledge, this is the first thorough investigation of starch and sucrose metabolism across corm development in freesia at the physiological and transcriptomic level, which is of special importance for quality control of corms and informative for development of proper cultivation and breeding practices of freesia.F. hybrida ‘SN Huangjin’ was planted in late October, 2016 in a farm on Minhang campus, Shanghai Jiao Tong University, China. Corm tissues were first sampled at the 60th day after mother corm embedment and the following samples were collected at an interval of 10 days, resulting in 14 sampling events . Our previous work detailed the biological characteristics of the plant and divided the developmental process of the new corm into four stages, i.e., the formation (60–90 d), initial swelling (90–120 d), rapid swelling (120–140 d) and maturation (140–190 d) stages  stages . PictureContents of the carbohydrates and activities of corresponding enzymes were determined in corms collected at 14 sampling events. Sucrose was determined according to Corms sampled at 60, 90, 120 and 190 d, representing four developmental stages of new corms  respectively, were selected for transcriptomic analysis. Three biological replicates for each stage were used. RNA was extracted using RNApre Pure Plant Kit  following the manufacture’s protocol. An aliquot of 80 µL DNase I composed of 10 µL DNase and 70 µL RDD was pipetted into the spin columns, which stood still for 15 min at the room temperature, to remove the genomic DNA. An equal portion of RNA extracted from each developmental stages  was subject to library construction, sequencing and assembly. Quality of RNA was detected using a NanoDrop 2000 spectrophotometer  and gel electrophoresis (1%). Clear bands and appropriate A260/A280 (1.9–2.1) and A260/A230 (>2.0) values obtained in this study suggested that the RNA quality was good enough for library construction. RNA-seq was performed by BioMarker Biotechnology Corporation  based on Illumina . Raw data were filtered to generate clean reads which were de novo assembled by Trinity to obtain unigenes .E-value ≤ 1e−5), to NCBI Non-redundant Protein (Nr), Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic Clusters of Orthologous Groups (KOG), Swissprot and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggnog) databases for functional annotation. Unigenes were searched against the Pfam database by HMMER with an E-value ≤ 1e−10. Functional annotation by GO terms was carried out by Blast2Go (https://www.genome.jp/tools/kaas/).Unigenes were aligned, by BLASTx . Genes with FDR <0.001 and absolute expression fold change  ≥4 were deemed to be significantly differentially expressed. Identified DEGs were then subject to GO database for functional analysis and to KEGG for identification of metabolic pathways.The transcript abundance of each unigene was calculated and normalized to fragments per kilobase of transcript per million mapped reads (FPKM) value, which is the most commonly used method to estimate gene expression levels . DEGs be2O, 0.8 µL of each primer and 1µL of cDNA mix. Relative gene expression levels were acquired using the 2 − ΔCT method after normalizing to actin   and then used as the template for qRT-PCR. Briefly, the RNA was reversely transcribed in the 10 µL of reaction media containing 0.5 µg of total RNA applying random hexamer primers to synthesize the first-strand cDNA. Primers, designed using Primer 5.0 software, were provide by Qingxi Biology . Real time qRT-PCR was conducted in a reaction volume of 20 µL containing 10 µl of SYBR, 7.4 µL of double-distilled Hce gene) . Three bDescription of four stages of corm development was displayed in β-AMY activities increased from 273 to 408 µg/min/g FW during the corm formation stage (60–90 d). After transient decrease, the enzyme activity increased sharply and peaked at 120 d (appearance of flower bud), reaching 498.33 µg/min/g FW, which was followed by marked decrease in the next sampling event and ensuing increase with fluctuations during maturation stage. Throughout the corm lifecycle, an overall increasing trend of SBE activities was observed, especially at the rapid swelling stage. During the maturation stage (150–180 d), the increasing rate of enzyme activity was almost flat. The value of the highest enzymatic activity was ∼4 fold that of the minimum at the beginning of sampling. Overall, APGase activities increased across the corm development stages, especially during the formation (60–90 d) and maturation stages (140–190 d), while fluctuations were observed during corm swelling (110–140 d). The minimum (∼80 µg/min/g FW) and maximum (∼240 µg/min/g FW) values appeared at the first and last sampling event, respectively.Beta-AMY activities were rather dynamic. Specifically, SPS activities kept relatively constant (∼350 µg/min/g FW) during the formation stage (60–90 d), then increased sharply reaching maximum (∼640 µg/min/g FW) during corm swelling (100–140 d) and subsequently kept relatively stable except a marked drop at 150 d. Comparatively, the profile of SuSy activities exhibited a slightly different pattern. The enzymatic activity showed monotonous decline from 705 to 433 µg/min/g FW during the formation stage, then rose rapidly arriving at maximum (∼1006 µg/min/g FW) during the swelling period and finally dropped back to the beginning level when corms began mature. NI activities increased continuously from 60 d (88.47 µg/min/g FW) to 150 d (∼180 µg/min/g FW) followed by a sustaining drop arriving at 126.19 µg/min/g FW at the end. Averagely, AI activities followed an initial increasing and subsequent decreasing trend as corms grew except the presence of a drop at 100 d. The maximum and minimum activities were 64.2 and 123.27 µg/min/g FW, respectively.P < 0.01), which all were significantly correlated to starch content. Besides, NI exhibited a positive and significant relationship with starch content and corm dimeter (P < 0.01). Also, the correlation between SBE and NI and AGPase, and AGPase and β-AMY were significant (P < 0.01). By contrast, no significant association between NI, AI and β-AMY was observed. AI and SuSy showed no significant correlation with any other parameter at all.The sequencing statistics based on a mixed cDNA library of freesia corms at different developmental stages are summarized in In the current study, totally 44,405 unigenes were annotated with 41,175 annotated in Nr, 13,753 in COG, 25,142 in GO, 15,743 in KEGG, 23,015 in KOG, 24,441 in Swissport and 28,454 in Pfam . Figure There are 25,142 unigenes annotated in GO database, which were classifies into three big functional categories, i.e., cellular component, molecular function and biological process . The subThe expression levels of unigenes at different developmental stages were compared. As shown in GO and COG functional and KEGG enrichment pathway analysis was performed to gain further insights into DEGs. The major categories of GO terms in three pairwise comparisons  were simβ-AMY, SuSy and INV. The expression of these genes was displayed in Combining data from the literature and a keyword search in RNA-seq annotation generated a total of 39 DEGs encoding six carbohydrate-metabolizing enzymes investigated in this study, i.e., SPS, AGPase, SBE, β-AMY1 (c62599.graph_c1), INV2 (c87855.graph_c0), SPS1 (c91035.graph_c1), SuSy (c93394.graph_c2), SBE4 (c78179.graph_c0) and AGPGase5 (c93923.graph_c0). Primer sequences of the genes for PCR were specified in r2 = 0.4972, p < 0.001)  , suggestβ-AMY2, INV , SuSy, SBE4, SPS and APGase (2 and 5), which showed expression patterns resembling those of corresponding enzyme activities and carbohydrate accumulation were determined to be critical genes involved in starch-sucrose metabolism in freesia corm swelling.To identify key DEGs involved in carbohydrate metabolism in freesia corms, we correlated all 39 DEGs with carbohydrate-metabolizing enzyme activities and carbohydrate content; accordingly, actively expressed homologues, such as tulipa  bulbs and found 7% of COG annotated unigenes were involved in carbohydrate transport and metabolism. And KEGG analysis of DEGs revealed that “starch and sucrose metabolism” was the primary metabolism pathway and was most active at rapidly expanding stage. Similarly, Lilium davidii var. unicolor bulblets and reported that 8% of annotated COG was associated to carbohydrate transport and metabolism. KEGG analysis of DEGs indicated that starch and sucrose metabolism was the principle pathway and was most active during bulblet formation stage. Additionally, the close association of carbohydrate metabolism with storage root development was also revealed on Lycoris sprengeri (Ipomoea batatas). . , and Sagttifolia . In the β-AMY2, INV , SuSy, SBE4, SPS, APGase (2 and 5) critical genes in starch and sucrose metabolism. The relative expression level of SBE4 was low before corm maturation but then increased by dozens of folds at 190 d, consistent with trend of starch accumulation, seemingly indicating that SBE4 could be essential for starch accumulation during corm maturation. The high expression level of AGPase (2 & 5) at 120 d and 190 d and SBE4 at 190 d, respectively, suggest that their coordinated expression may regulate starch accumulation in the storage organ. INV converts sucrose to glucose and fructose, which join the starch synthesis pathway. INV  genes were dramatically upregulated from formation to rapid swelling stages followed by a rapid downregulation, congruent with profiles of associated enzymes, indicating the important role of INV in corm development. Only two β-AMY encoding genes were derived from the transcriptome data. The expression profile of β-AMY2 generally followed that of the enzyme activities. The low expression of β-AMY2 favors starch accumulation. Although not strictly consistent, overall trend in the expression profiles of SPS (1-10) and that of the enzyme activities and sucrose accumulation generally matched, indicating that the continuous sucrose accumulation lead to corm swelling and provided energy for reproductive growth. Only one SuSy was annotated here and its FPKM profile was opposite to that of the enzyme activities which was in line with the qRT-PCR result, suggesting possible sequencing errors or a possible converse regulation since SuSy is a bi-directional enzyme.Correlation analysis shows that the gene expression levels from qRT-PCR and RNA-seq were positively and significantly related, suggesting that the FPKM values were accurate for estimation of DEGs expression. We thus compared of FPKM profiles to that of carbohydrate accumulation and/or enzyme activities and assigned Lycoris sprengeri bulbs  peaked at 120 d (the rapid swelling stage), suggesting that the starch and sucrose metabolism was the most active in this stage. By comparison, it was found that the gene expression of some other homologues listed in ri bulbs  and Tuli stolons . Here weβ-AMY, SBE and APGase. Especially during the mid- and late developmental stages, gene expression of these enzymes rose remarkably, leading to rapid starch accumulation as energy reserve in mature corms. The proposed model is generally in line with some of the reported results for Gladiolus hybridus (Taken all together, we propose a model for the starch and sucrose metabolism during corm development in freesia . Corm dehybridus , tulip (hybridus  and lilyhybridus .Herein, we investigated the dynamics of the major form of carbohydrates and related enzyme activities, and also profiled the transcriptome of freesia corms at four developmental stages. Obviously, corm development was paralleled with dynamics of starch and sucrose which were the comprehensive effects of carbohydrate-metabolizing enzymes. On this basis, corms at four representative sampling events were selected for transcriptome profiling based on RNA-seq. Transcriptomic results also showed that carbohydrate metabolism was the most active biological pathway with DEGs significantly enriched during corm swelling. Subsequently, DEGs from the transcriptome data encoding carbohydrate-metabolizing enzymes were correlated to the enzyme activities and carbohydrate accumulations and some critical genes involved in starch-sucrose metabolism were determined. The transcriptomic analysis thus provides further insights into molecular mechanisms underlying the metabolic process. To our knowledge, this is the first transcriptome analysis of starch and sucrose metabolism during corm formation and development in freesia. The results will shed light on elucidation of molecular mechanisms in corm formation and development and benefit for future improvements in breeding of freesia.10.7717/peerj.11078/supp-1Figure S1Click here for additional data file.10.7717/peerj.11078/supp-2Figure S2Click here for additional data file.10.7717/peerj.11078/supp-3Figure S3Click here for additional data file.10.7717/peerj.11078/supp-4Figure S4Click here for additional data file.10.7717/peerj.11078/supp-5Figure S5Click here for additional data file.10.7717/peerj.11078/supp-6Table S1Click here for additional data file.10.7717/peerj.11078/supp-7Table S2Click here for additional data file.10.7717/peerj.11078/supp-8Table S3Click here for additional data file.10.7717/peerj.11078/supp-9Table S4Click here for additional data file.10.7717/peerj.11078/supp-10Table S4Click here for additional data file.10.7717/peerj.11078/supp-11Table S6Click here for additional data file.10.7717/peerj.11078/supp-12Supplemental Information 1Click here for additional data file.10.7717/peerj.11078/supp-13Supplemental Information 2Click here for additional data file.


Dear JBIC Reader, dear Colleague:I am excited to see the galley proofs of the first 2020 issue of JBIC, my first issue as Chief Editor of this journal. I am grateful to our previous Chief Editor, Larry Que, and Debbie Schoenholz as the editorial assistant, who were a superb team to lead and develop the journal for the past 20 years. Both have been professional and extremely helpful to enable a smooth transition with the beginning of this new decade.Along with the change in the Editor’s office, you can expect more changes to come: On the Associate Editor’s team, Valeria Culotta has taken on a more demanding role in her institution with the beginning of 2020, and will no longer be handling manuscripts for JBIC. Thank you, Val, for your excellent service, and good luck with your new position. Efforts are underway to appoint a replacement for Val, and as with all of the latest news from JBIC, you will hear this first on our newly established Twitter account  – so follow the tweets!It is my aim to maintain JBIC’s standing as the premier journal for new findings and developments in Bioinorganic Chemistry. JBIC serves as the major link within our global community, and it is our window to the outside world. Together with the Team of Editors we will work hard to increase its impact, and make sure that JBIC is a journal that all members of our society are proud of, and proud to publish in.JBIC will maintain its roots in classical Bioinorganic and Medicinal Inorganic Chemistry, but we will also openly embrace new developments, for example in bioinorganic nanotechnology, the use of big data, or applications of digital chemistry to problems related to Bioinorganic chemistry. I would very much welcome suggestions for topics and authors, be it for reviews, commentaries and original articles, and of course I am always available for informal consultation before submission.But finally, it is excellent science, hard, reliable data, and good service to our authors, reviewers and the community that will determine JBIC’s future standing. I am thus asking you to submit your excellent work to JBIC, cite articles in JBIC where appropriate, and make colleagues in neighbouring disciplines aware of us. At JBIC, we will make every effort to make your publishing experience with us a pleasant and rewarding one—so that it is with the same pleasure like mine today that you flip through the pages of every new issue of our journal.Enjoy reading!Nils Metzler-Nolte.January 2020.


Many medical specialties use scientometrics to assess the impact of publications, journals, and authors. The aim of this study was to analyze and compare trends of publications from a hospital medical center to publications from a college of medicine connected to that hospital and compare collaboration rates between them to other domestic and international institutions. We used Elsevier’s SCOPUS database to compare Penn State College of Medicine (PSCOM) publications to Hershey Medical Center (HMC) publications, analyzing 31,856 total publications. We hypothesized that HMC and PSCOM have room to improve on both internal and international collaborations. Our results show that despite PSCOM’s international collaboration being nearly three times higher than HMC, overall international collaboration is less than 2%, far below the US national average. Scientometrics measures and analyzes scientific literature and is a subset of bibliometrics. Many medical specialties use bibliometrics to compile, use, and review the most-cited works . This isThe aim of this study was to identify historical trends in Penn State College of Medicine (PSCOM) publications and compare them to the trends of Hershey Medical Center (HMC) publications, especially as it relates to collaboration. We hypothesize that given their physical, financial, and leadership connections, in addition to shared research aims, that the collaboration rate between PSCOM and HMC will be higher than with all other institutions combined, that their top authors and most cited articles will be the same and that they will have similar publication rates in journals. We also hypothesize that international collaboration for PSCOM and HMC will be higher than the national average because of both institutions' focus on global health rotations and research projects.A comprehensive search within Elsevier's SCOPUS was performed on May 2, 2020. Institution search was performed using the Boolean query "Penn State College of Medicine" and variations yielding Affiliation ID 60027671. Another search was performed using the Boolean query "Milton S. Hershey Medical Center" and variations yielding Affiliation ID 60013671. Data were collected, sorted, and analyzed by topic, affiliation, journal name, highest citations, authors' publications, and H-index. The Penn State Cancer Institute was considered a separate institution. All collaborating affiliations were obtained for both HMC and PSU. Calculated international collaboration rates in percentage were obtained by dividing all publications from international institutions by the total number of publications.PSCOM has 18,327 total publications by 4,535 authors, and HMC has 16,259 total publications by 4,135 authors.Research topicsFor both PSCOM and HMC, the top three publication topics are Medicine , Biochemistry, Genetics and Molecular Biology , and Neuroscience . The bottom three topics for PSCOM were Chemical Engineering (1%), Multidisciplinary (1%) and Health Professions (1%), whereas the bottom three topics for HMC were Materials Science (1%), Chemical Engineering (1%), and Multidisciplinary (1%) , Journal of Virology (257), and Cancer Research (156). The top three journals in which HMC chose to publish were the Journal of Biological Chemistry (193), Antimicrobial Agents and Chemotherapy (163), and Cancer Research 140) . In the 2020 Indicators Report published by the National Science Foundation, 39% of US peer-reviewed publications have international collaborators, with China being its closest collaborator, followed by the United Kingdom -6. DakikIn the process of improving international collaboration, some factors might explain the potential obstacle to reach a higher international collaboration rate. The Penn State system is a public institution with most of the research activity being federally funded, and it is subjected to federal government regulations for international research collaboration. The complicated approval process of the international component might hinder HMC and PSCOM's motivation for international collaboration .PSCOM and HMC share an organic connection and geographical proximity. Yet, of their combined 31,856 publications, only 5,659 (17.7%) were collaborative works between these two institutions . This is surprising given the graduate student workforce available to PSCOM, which consists of many MD and MD/PhD students who also have clinical duties at HMC, and the number of HMC physicians who hold professorship positions at PSCOM.The most prolific authors from each institution are likewise dissimilar, as are the most cited works. Likewise, although the top journals that PSCOM and HMC publish in are similar, they are not the same and have different rates of publication . Overall, all of our null hypotheses are not rejected, as our analysis provides data contrary to our hypotheses that given their physical, financial, and leadership connections, in addition to shared research aims, that the collaboration rate between PSCOM and HMC will be higher than all other institutions combined, that their top authors and most cited articles will be the same, and that they will have similar publication rates in journals, as well as data contrary to international collaboration being high.Our limitations include our search being limited to articles contained within Elsevier's database, which, although fairly comprehensive with 70,000 institutional profiles and 1.4 billion cited references, does not capture the entirety of everything ever published. In addition, though we report 5,659 collaborative papers, this number may be lower because one author may have affiliations at both PSCOM and HMC. This stresses the need for more collaboration.This study provides a snapshot in time of PSU and HMC publication statistics. Both institutions are producing high-quality research published in top national scientific journals. However, both institutions should make it their goal to foster greater international collaboration as well as collateral collaboration, as an average of 1.30% international collaboration is significantly lower than the US publication average and is not reflective of the Penn State system’s research potential. A 17.7% collaboration rate between sister institutions that share similar goals and climates is likewise something that can be improved upon. The authors suggest that this study be repeated for other sister institutions throughout the US to compare collaboration rates. This study should also be repeated every few years to note trends and measure efforts to improve collaboration.


Increasingly grandparents provide primary or supplementary care for their grandchildren globally. In view of the challenges faced by grandparent caregivers, a number of intervention programs were designed to act as a buffer to the potential negative impacts on their well-beings. A recent meta-analysis  shows that interventions for grandparent caregivers have small to moderate effect sizes for increases in social support and parenting skills. However, little is known how the multi-component interventions work. The aim of this review is to categorize the key components of intervention programs and determine the direct effect of components on grandparent caregivers’ outcomes . A component-level approach is adopted to disentangle the ‘active ingredients’ of the interventions. Peer-reviewed articles were searched via five electronic database (published before January 2020). A keyword search was performed based on combinations of two groups of terms: (1) grandparent caregivers and (2) intervention, training, and support group. Studies adopting an experimental or pre/post design and reporting quantitative changes in grandparents’ outcomes were included. Studies focusing on the special needs of grandchildren were excluded. Fourteen interventions were identified from over 20 included studies. Preliminary analyses show that different physical and mental outcomes of grandparent caregivers were predicted from key components of education, skills training, relaxation and psychosocial support. With the goal of developing evidence-based interventions for grandparents raising grandchildren, this synthesis offers insight into the mechanisms of an intervention, which informs its generalizability or applicability to a new context.


Bi-axial ankle stiffness measurements showed significant intra-class correlations (ICCs) between the two visits for all ankle movements at slower  and faster  speeds. Stiffness measurements along the talocrural joint were thus shown to have significant negative correlation with active ankle range of motion . The ankle movement trainer, based on anatomical characteristics, was thus used to demonstrate valid and reliable bi-axial ankle stiffness measurements for movements along the talocrural and subtalar joint axes. Reliable measurements of ankle stiffness may help clinicians and researchers when designing and fabricating ankle-foot orthosis for people with upper-motor neuron disorders, such as stroke.This study involves measurements of bi-axial ankle stiffness in older adults, where the ankle joint is passively moved along the talocrural and subtalar joints using a custom ankle movement trainer. A total of 15 elderly individuals participated in test–retest reliability measurements of bi-axial ankle stiffness at exactly one-week intervals for validation of the angular displacement in the device. The ankle’s range of motion was also compared, along with its stiffness. The kinematic measurements significantly corresponded to results from a marker-based motion capture system (dorsi-/plantar flexion:  Modern lower-limb rehabilitation devices have been refined to evaluate joint stiffness. Lokomat , for example, has been successful in clinics for lower-limb rehabilitation in patients with neurological disorders . StudiesThe ankle joint structure is intrinsically complex with relation to functional improvements to movement in elderly populations ,10,11. TReliable ankle stiffness evaluations are therefore important to assessing the outcomes of ankle rehabilitation programs for elderly patients with neuro-musculoskeletal disorders. A custom automatic bi-axial ankle movement trainer (AMT) was developed to provide passive ankle stretching that was shown to be effective for hemiparetic gait . The AMTThus, our first objective was to evaluate the reliability of ankle stiffness measurements during ankle movement along both the subtalar and talocrural axes in elderly people, using the AMT. Then, bi-axial ankle stiffness was investigated by measuring the vertical reaction forces of the anterior, posterior, medial, and lateral areas on the moving AMT foot plate and the target foot during passive bi-axial ankle movements along the talocrural and subtalar joints, to demonstrate the test–retest reliabilities for the designated ankle movements during repeated stiffness measurements. Using these natural bi-axial ankle movements, our second aim was to investigate whether the subject-initiated active ankle ROM was correlated with ankle stiffness in the elderly. The kinematics measured with the AMT were validated with those from a conventional 3D motion-capture system. Reliable ankle static and stiffness measurements could help clinicians and orthotists provide necessary assistive devices, such as ankle-foot orthosis, to people with neurological disorders or hemiparesis. The anatomical characteristics of the ankle joints are reflected in the current mechanical design of the AMT . In the Two motors  are installed, with one executing ankle movements along the talocrural axis, and the other executing movements along the subtalar axis. The first motor controls ankle movements along the subtalar axis, which is located at the rear of the hindfoot, 42° upward in a seesaw-type cradle a,b; it iThe AMT controller unit consists of a field-programmable gate array (FPGA) with a real-time data acquisition board, integrated with a 2M gate-reconfigurable I/O FPGA board , and two modular digital positioning controllers . The two controllers operate two DC motors via a controller area network protocol. An enclosure for these controllers and other electronic components is located under the footplate. LabView  software is used to send and receive the command data and output via ethernet protocol. To operate the AMT, two motor communication and ethernet connections were securely connected for the LabView program before beginning trials.The AMT simultaneously measured underfoot reaction forces using four bar-type load cells  located under the four corners diagonally between the two rigid aluminum plates. The stiffnesses along the talocrural and subtalar axes were calculated from outputs sampled at 1000 Hz. Calibration outputs confirmed the linear characteristics of the four load cells at the four corners , located between upper and lower plates, and underfoot reaction force sensors were used to measure ankle stiffness during movement along the talocrural and subtalar joints. anklek) is defined as the slope of torques at given ankle angles with respect to the neutral ankle posture: l0 is a moment arm (distance from center of force plate to load cell), Stiffness along the talocrural joint for dorsi-/plantar flexion movements was measured with two forefoot load cells, namely forefoot reaction force (FF), as well as the two hindfoot load cells, namely hindfoot reaction force (FH). Stiffness along the subtalar axis was measured with two medial-side load cells and two lateral-side load cells. The formula below defines ankle stiffness and explains its calculation along the talocrural and subtalar axes, using torques measured by vertical reaction forces from the foot plate at given ankle rotation angles. Ankle stiffness  were recruited for this study. All participants were briefed on the procedures and provided an informed-consent form approved by the Institutional Review Board of the National Rehabilitation Center . The study protocol was registered at a clinical trial registry . Eligibility criteria were elderly people with chronic post-stroke hemiparesis (onset duration > 6 months), who could walk independently on a level surface  without abnormal muscle tone as determined by a score on the Modified Ashworth Scale (MAS) < 3. Subjects who had complications of orthopedic disorders, cognitive dysfunctions, or mental illnesses were excluded. Participants performed two identical tests to assess test–retest reliability at exactly one-week intervals. A licensed physical therapist measured the active ankle ROM for the talocrural (dorsi-/plantar flexion) and subtalar joints (inversion/eversion) during the first visit. To evaluate the ankle angle measurement with the developed AMT, kinematic measurements from the AMT were synchronized with a precise 3D motion capture system using ten infrared cameras  and compared to determine their correlation. To measure ankle movements, reflective markers were placed along lower-limb and trunk landmarks according to the plug-in gait marker setup . Foot maMotion analysis was performed using Visual3D software . The ankle angle was calculated using the joint angle between the dominant-side shank and dominant-side foot. Bi-axial ankle stiffness was then measured on two separate occasions in the span of one week, and test–retest reliabilities were evaluated using the SPSSWIN 18.0 software package. An intra-rater correlation, which indicates the within-subject reliability between the two visits, was assessed using a one-way intraclass correlation coefficient (ICC) random model . Pearsonr = 0.996; inversion/eversion: mean r = 0.985).Average passive ankle movement velocities were 2.14°/s (SD 0.43°/s) at the lower speed and 9.77°/s (SD 0.65°/s) at the higher speed. Passive ankle movement angle measurements using the AMT system showed considerable agreement with measurements obtained from the Vicon system .When the ankle attained the required position  in each direction, bi-axial ankle stiffness was calculated using the four load cells. Ankle stiffness between visits 1 and 2 did not show significant differences for the different speeds . Although active dorsiflexion ROM showed no significant correlation with stiffness along the talocrural joint for faster dorsiflexion movements, stiffness along the talocrural joint showed a significant positive correlation with active dorsiflexion ROM. When the ankle was in the designated eversion position during slower/faster movements, stiffness along the subtalar joint showed a significant negative correlation with active ankle ROM .When the ankle achieved the designated plantar flexion during slower movements, the stiffness along the talocrural joint showed a significantly negative correlation with the active ankle ROM .Patent: “Ankle Muscle Training Apparatus”, Patent No. KS10-1796916, Nov 2017, Inventors: Hogene Kim, Hwiyoung Lee, Yoon-Ho Na.


Objective: To report a new technique for Descemet’s membrane endothelial keratoplasty (DMEK) in aphakic and vitrectomized eyes. Case description: A 56-year-old man presented with corneal decompensation in the left eye after combined pars plana vitrectomy and lensectomy for the management of a traumatic eye injury. DMEK graft and recipient bed were prepared as regular. The posterior half of the donor stroma was dissected with a crescent knife to form a temporary stromal barrier tissue. The prepared donor stromal tissue was implanted into the anterior chamber (AC) to form a barrier over the iris and pupilla. Then, the endothelial graft was safely injected into the anterior chamber and deployed by gently tapping on the corneal surface. Air tamponade was applied into the AC for 10 minutes to allow the graft to attach. Afterwards, the stromal barrier tissue was removed through the main incision and the AC was refilled with air tamponade. There were no intraoperative or postoperative complications during 1-month follow-up. Conclusion: Insertion of a temporary posterior stromal tissue as a barrier over the iris and pupilla successfully provided AC stability and prevented posterior dislocation of the graft or air tamponade. This new technique was a safe and effective approach for DMEK in aphakic and vitrectomized eyes. Descemet’s membrane endothelial keratoplasty (DMEK) is a relatively new posterior lamellar keratoplasty technique that allows the surgeon to replace only the removed dysfunctional endothelial tissue layer with a new one without changing the corneal stroma. As the DMEK graft includes only Descemet’s membrane and the endothelium , it is tA 56-year-old male patient presented with corneal decompensation in the left eye after combined pars plana vitrectomy and lensectomy for the management of a traumatic eye injury. The best corrected visual acuity in the left eye was counting fingers at 1 meter. Slit lamp examination revealed marked corneal edema throughout the entire corneal tissue  into the anterior chamber. The center of the recipient cornea was marked with a 8 mm marker, which was stained by a marker pen to define the size of Descemetorhexis area. A cohesive ophthalmic viscosurgical device  was injected into the anterior chamber to form space and the Descemet’s membrane of the recipient was peeled and removed from the central 8 mm of the recipient cornea  , 2]. DM. DM2]. DAfshari et al.  reportedGroat et al.  performerd month examination [There is only one report on DMEK in an aphakic and vitrectomized eye . Ozmen amination .In the current case, we performed a new surgical technique for DMEK surgery in an aphakic and vitrectomized eye. Here, we used the posterior stroma of the donor cornea as a scaffold and barrier to stabilize the anterior chamber during the surgery. This approach allowed a stable anterior chamber during the surgery and kept the air bubble in the anterior chamber to allow the graft tissue to adhere better on the recipient stroma. Even in the absence of a lens and vitreous support, the injected donor stromal tissue could provide a good physical barrier to prevent complications and contribute to the success of surgery. The placement of a safety suture on this temporary barrier tissue during the surgery provided an extra level of security by preventing the risk of posterior displacement. Therefore, we believe that this technique is a safer approach during DMEK surgery in aphakic and vitrectomized eyes compared to the previously defined pars plana infusion method.In conclusion, the temporary placement of a half-thickness posterior donor stromal tissue over the iris and pupilla allowed a safer DMEK surgery and very good anterior chamber stability in an aphakic and vitrectomized eye.The authors declare that they have no competing interests.


In the continuum limit, master equations lead to Fokker–Planck partial differential equations that represent the dynamics of physical systems in continuous spaces. Over the last few decades, nonlinear Fokker–Planck equations have become very popular in condensed matter physics and in statistical physics. Numerical solutions of these equations require the use of discretization schemes. However, the discrete evolution equation obtained by the discretization of a Fokker–Planck partial differential equation depends on the specific discretization scheme. In general, the discretized form is different from the master equation that has generated the respective Fokker–Planck equation in the continuum limit. Therefore, the knowledge of the master equation associated with a given Fokker–Planck equation is extremely important for the correct numerical integration of the latter, since it provides a unique, physically motivated discretization scheme. This paper shows that the Kinetic Interaction Principle (KIP) that governs the particle kinetics of many body systems, introduced in G. Kaniadakis, Physica A  The constant Boltzmann constant and T the temperature of the system.Nonlinear kinetics in the coordinate or velocity space or in the phase space has been used systematically over the last few decades. A popular approach involves studying the temporal evolution of a particle system while it interacts with a medium in thermodynamic equilibrium. In Ref. , the mosgeneralized Kramers equation that describes a nonlinear kinetics in the phase space. The nonlinearity is due to the presence of the two arbitrary functions f. In the case where The above equation can be viewed as a Equation  has been-entropy ,3,4, pre-entropy ,10,11,12-entropy ,15,16,17-entropy ,19,20, q-entropy ,22, plas-entropy ,25,26,27-entropy , astroph-entropy ,33,34,35-entropy , genomic-entropy , complex-entropy ,39, econ-entropy ,41,42,43-entropy ,45,46,47anomalous diffusion process. Such processes have long been observed and studied in various fields of condensed and soft matter physics [It is important to note that the right hand side of Equation , which i physics .master equation that is associated with a given Fokker–Planck partial differential equation is crucial for two reasons. First, as stated above the master equation provides a uniquely defined discretization scheme for numerical integration. Second, the master equation is physically meaningful, since it defines the process dynamics on lattices. Hence, the study of the lattice dynamics allows an improved understanding of the dynamics in continuous space.Knowledge of the at the lattice scale to univocally define the discrete kinetics and directly derive the master equation. The master equation thus obtained is clearly useful, not only for deriving the nonlinear Fokker–Planck Equation  which governs the particle kinetics [The present paper shows that the kinetics  univocalThe paper is organized as follows. In x can indicate either the velocity or the coordinate space variable. Equation =fn . EquatioEquation  describeechanics .Kramers–Moyal expansion to a balance equation or alternatively starting from a master equation [f, i.e., In the case of bosonic and fermionic statistics, Equation  was obtaequation . The samequation ,54 to deThe most general form of Equation  involvesolutions ,55,56,57Kramers–Moyal expansion (that takes into account only transitions between near neighbors) to a balance equation based on the (KIP) [The nonlinear Fokker–Planck equation in the most general possible from  has beenhe (KIP) .f and is related to the f and Subsequently, Equation  was obtaN identical particles. Under the hypothesis that only transitions involving the nearest-neighbour lattice sites are allowed during the evolution of the particle system, the probability density function i to site j.Let us consider the particle kinetics in a one-dimensional lattice gas comprising i to site j depends on the nature of the interaction between the lattice and the particle, and this dependence is taken into account through the transition ratesi and arrival j sites.The transition probability related to the hopping of a particle from site Kinetic Interaction Principle.In Ref. , it was transition probability from site i to site j. The Let us now consider the nonlinear kinetics the j and can simulate collective interactions in many body physics. For instance, the expression Pauli exclusion principle that governs a fermionic particle system [bosonic systems. Other more complicated expressions of the Haldane generalized exclusion principle originating from the fractal structure of the single particle Hilbert space.In linear kinetics, ly posed . In nonle system ,60,61. Au controls the equilibrium state: a normal diffusive regime is obtained for u below a threshold, while an abnormal diffusion regime that exhibits the Matthew effect of accumulated advantage is obtained at higher u.Another case of nonlinear kinetics corresponding to gated in , motivatgated in . In thisnearest-neighbor transition rates are master Equation  in the formoutgoing, incoming, Let us express the i and the outgoing current at node forward differencebackward differenceTaking into account the continuity relation between the incoming current at the node Based on the definition of the incoming and outgoing currents given above, the master Equation  assumes The two forms of the master equation, as given by Equation , are asydiscrete Fokker–Planck current, To write the latter equation in an even more symmetric form we introduce the When this definition is employed, the incoming Equation , we obtasymmetric finite difference operators are introducedFinally, the following first- and second-order Equation , the masEquation  assumes The latter expression of the master equation is particulary suitable for obtaining the continuous limit as Subsequently, after taking into accounts the limits quations  and 21)Δ+/Δx→∂/∂continuity equation is the nonlinear Fokker–Planck equation obtained in the continuous limit, starting from the master Equation  an anji, dequations , in termTo express the current Furthermore, we assume that the functions iFollowing the same procedure, one obtains the approximate expressions for the functions discrete Fokker–Planck currentAfter substitution of the above expressions for Equation  of the dThe above expression for the discrete current can be simplified in the continuum limit, Hence, the discrete Fokker–Planck current diffusion coefficient, defined by means ofdrift coefficient, defined by means ofThe Equation  assumes kinetic energy functionFurthermore, the introduction of the Fokker–Planck current in the continuum limit is given by the following equationThe discrete current nonlinear Fokker–Planck equationIf we insert the continuum limit of the Fokker–Planck Equation , in the Equation  we obtaiThe above equation recovers the original Fokker–Planck equation given by Equations  and 3)..3). ThusIn the following, we investigate the impact of the discretization of the time derivative in Fokker–Planck equation, as well as the impact of the space derivative discretization in the linear and nonlinear kinetic regimes. We also present an extension of the one-dimensional formalism to two spatial dimensions.We will express the Fokker–Planck Equation  as(41a)There are various numerical schemes for the discretization of partial differential equations such as the above, including finite-difference schemes, methods based on finite elements, finite-volume methods, spectral approaches, multigrid methods, etc. . Each api refers to the lattice site and the index n to the time instant.It is most straightforward to compare the master Equation  with disThree of the most common finite-difference methods lead to the following discretization schemes of (42a)finOn the other hand, the discretization of the master Equation  leads toIf we compare the master Equations  with  to the continuum model, discretization of the latter generates the master Equation (50), which is radically different from the master Equation , althougLet us now write the Fokker–Plank Equation  in the fnonlinear diffusion coefficient. If linear kinetics for boson or fermion kinetics, for The term This important result, pertaining to the ordinary diffusion that underlies the kinetics of classical bosons and fermions, naturally leads to the question whether there exist other nonlinear kinetics which are governed by standard Fickian diffusion.In the following, we focus on the Fokker–Planck Equation  in the cEquation  is subjestationary and stable solution, In terms of the generalized logarithm, the Equation  assumes It is easy to express the Equation  in the fThen, based on Equation  and takiFinally, the solutions of the two equations above for The nonlinear Fokker–Planck kinetics described by Equation , when a(quations  and 60)a(f) and The Equation  describeAs a working example, let us briefly consider the kinetics , where tThe inverse of the logarithm function, i.e., the In this case, the It is remarkable that the latter equation is different from the one proposed in . Both eqordinary Fickian diffusion, second term on the right hand-side of , we, wenonliIt is straightforward to extend the KIP-based approach for the derivation of physically inspired master equations to lattices in two and three dimensions. For example, in the case of a two dimensional square lattice whose sites are labeled by the integer indices Equation  with nea


The aim of this in vitro study was to assess the influence of surface pretreatment on shear bond strength (SBS) of an adhesive resin cement  to three different yttria-stabilized tetragonal zirconia polycrystalline (Y-TZP) ceramics: (1) Copran Zirconia Monolith HT, COP; (2) Katana ML Zirconia, KAT; and (3) Metoxit Z-CAD HTL Zirconia, MET. In total, 45 cylinders  for each type of zirconia ceramic were prepared used a computer-aided design and computer-aided manufacturing (CAD/CAM) machine (software CEREC 4.2). Each type of zirconia was subdivided into three groups and each group received a different surface pretreatment; 15 samples were not conditioned as control , 15 samples were air-borne particle abraded with aluminum dioxide particles of 50-μm size at 0.3 MPa for 20 s , and 15 samples were hot-etched with a solution of hydrochloric acid and ferric chloride . After specimen fabrication, the adhesive cement–ceramic interface was analyzed using an SBS test. Subsequently, the adhesive remnant index (ARI) was measured. Data were submitted to statistical analysis. Air-borne particle abraded specimens showed the highest SBS values for COP and KAT groups. For MET, no significant difference was reported between air-borne particle abraded specimens and untreated controls. The lowest values were detected for acid-etched groups. A higher frequency of ARI = “1” and ARI = “2” was reported in control and air-borne particle abraded groups, whereas ARI = “3” was detected in hot-etched groups. No correlation was found between ARI score and shear bond strength. Air-borne particle abrasion is considered the best treatment for Zirconia Copran and Zirconia Katana ML, if it is followed by using dual-curing resin cement.  In recent years, the use of aesthetic materials increased more and more in the field of dentistry. This might be the consequence of the development of new fabrication techniques and of the need to satisfy patients’ desires. Considering the current restoration systems, computer-aided design and computer-aided manufacturing (CAD/CAM) ensures the production of tooth-colored restorations of both resin and ceramic materials in a short amount of time .Composite resin block materials are available for CAD/CAM technology since 2000 . During The physical and chemical pretreatments applied on the inner surfaces of the fixed prosthetic frameworks aim to optimize the cement adhesion to the restoration material. Densely sintered ceramics are based on pure alumina or zirconia. The cementation of these polycrystalline materials (considering the first to fifth generation of zirconia) was reported to be difficult .Hydrofluoric acid positively enhances the surface morphology of the glass ceramics, but it cannot be used with polycrystalline materials . This trThe cohesion of the tooth–restoration complex with a bonding technique (through the use of resin cements) involves the establishment of a durable link even within the substructures. Several experimental pretreatments of zirconia were proposed, including air-borne particle abrasion (ABPA) with alumina in some cases followed by silanization , depositThe resin cements that include in their chemical composition a functional phosphate monomer ) showed reliable short and long bonding links to air abraded zirconia. This procedure is currently the most reliable method suggested for the cementation of high-density ceramic and zirconia ,20.Accordingly, the aim of this in vitro study was to verify the main effect of ABPA pretreatment on shear bond strength for three recently developed CAD/CAM zirconia ceramics. Furthermore, considering the resin cement used on the tested zirconia ceramics, this study aimed to assess the adhesive remnant index (ARI) score in the three different conditions tested  and to evaluate whether a direct correlation occurs between this index and shear bond strength.The null hypothesis of the present investigation was that there are no significant differences in bond strength values among the three tested zirconia ceramics considering different pretreatments. The second null hypothesis was that there are no significant differences in adhesive remnant index (ARI) scores among the various groups.Three different tetragonal CAD/CAM zirconia ceramics were tested in this study. The specifications of the materials used are listed in In total, 45 cylinders of equal size  for each tested product were designed with CEREC software 4.2 platform. Samples of each type of zirconia were subdivided into three groups, and each one underwent a different surface treatment  as shown in the flow chart in -Groups COP 1, KAT 1, and MET 1: no treatment (control groups). The cementation surfaces remained unaltered and unchanged after the manufacturing process ;-Groups COP 2, KAT 2, and MET 2: ABPA  with aluminum dioxide particles with 50-μm particle size at 0.3 MPa for 20 s at a distance of 10 mm perpendicular to the sample’s surface; subsequently, samples were rinsed with an air/water vaporizer  for 10 s and finally air-dried ;-3 (800 mL of hydrochloric acid HCl and 5 mL of ferric chloride FeCl3) for 10 min at a temperature of 100 °C, subsequently washed with 90% CH3OH methanol and finally air-dried [Groups COP 3, KAT 3, and MET 3: immersion in an experimental corrosive solution of HCl–FeClir-dried .After the surface treatments, a liquid primer  containing methacryloyloxydecyl dihydrogen phosphate (MDP) was applied to all the samples with a microbrush to guarantee the subsequent adhesion of the resin cement to the zirconia ceramics. With an air syringe, light air flow was applied for 5 s, which allowed the solvent (ethanol) to evaporate. TM, GC Corporation, Tokyo, Japan), directly from the syringe inside a metallic annulus, with predetermined dimensions , which acted as a mold  at a light intensity of 1200 mW/cm2, with a wavelength of 430–480 nm, standing with the ferrule at a distance of 1 mm from the cement [After the elimination of the superficial excess cement with an Explorer 6, the samples were irradiated with light curing for 20 s using a light-emitting diode (LED) lamp .During the test, the zirconia specimens were subjected to a force applied with a steel tip, tangentially with respect to the cementation surface, until the failure of the cement , as showAfter detachment, the external surface of each zirconia’s sample, where adhesive was applied, was examined using an optical microscope to determine the proportion of residual adhesive using the adhesive remnant index (ARI) that evaluated the zirconia–cement interface. The ARI scale has a range between 0 and 3: (0) all the cement remained on the zirconia surface; (1) more than half of the cement remained on the zirconia surface; (2) less than half of the cement remained on the zirconia surface; (3) no adhesive remained on the zirconia surface .p < 0.05. Statistical analysis was performed with R software . Descriptive statistics, including the mean and the standard deviation, were calculated for each group. The normality of the data was calculated with the Kolmogorov–Smirnov test. Analysis of variance (ANOVA) was applied to establish whether significant differences in debond strength values existed among the groups. Tukey’s test was used post hoc. Significance for all statistical tests was set at p < 0.05.Finally, a frequency analysis of the ARI scores was carried out to assess the presence of differences between the residual adhesive indices of the different groups tested by means of the chi-square test. The level of significance for all tests was set at Descriptive statistics are presented in p < 0.0001). According to post hoc Tukey testing, group COP 2 (Zirconia Copran Monolith HT air-particle abraded) and group KAT 2 (Zirconia Katana ML air-particle abraded) showed significantly higher bond strengths (p < 0.05), as represented in p > 0.05). Groups KAT 1, MET 1, and MET 2 showed detachment forces not different from each other (p > 0.05) and significantly lower than groups COP 2 and KAT 2 (p < 0.05) but significantly higher than the other groups (p < 0.05). Groups COP 1, KAT 3, and MET 3 showed detachment forces that were not different from each other (p > 0.05) and significantly higher than group COP 3 (p < 0.05) but significantly lower than the other groups (p < 0.05). The lowest detachment forces were detected by group COP 3 (p < 0.05). Analyzing the effect of pretreatment, significantly higher forces were found in the groups  subjected to ABPA (p < 0.05), whereas significantly lower values were detected in the groups  subjected to pretreatment with hydrochloric acid and ferric chloride (p < 0.05). Intermediate values were detected in the control groups without pretreatment .ANOVA showed the presence of significant differences among the various groups (p < 0.05).ARI scores are presented in 2R < 0.0687) was recorded between adhesion strength (MPa) and adhesive remnant index (ARI Score) in the three different conditions tested .As shown in Zirconia is an innovative material in the panorama of modern dentistry. It combines excellent aesthetic characteristics with many exceptional mechanical properties that make it suitable for the production of restorations in the posterior areas of the oral cavity .In recent years, zirconia-based ceramics became the primary choice for the production of conventional fixed prostheses thanks to their aesthetic performance, their excellent mechanical properties, and their biocompatibility with the oral cavity . The maiConditio sine qua non to achieve complete clinical success and longevity of the restoration is the realization of a strong bond between the resin-based cement and the zirconium oxide restoration . This is3+) or the copper ion (Cu2+) leads to the breaking of the protective film of the zirconium oxide, allowing local corrosion and the creation of micro porosity. Hot etching, however, presents some limits; the vapors of the boiling solution must be aspirated by means of large hoods, and the manipulation of these caustic substances by the technician in the laboratory could constitute another important obstacle to the practical realization of this pre-treatment [Over the years, the pretreatment on zirconium oxide-based surfaces was extensively studied in order to make the bond with cement more effective and lasting. Due to the nature of the type of material, this research is very complex and still unfinished. Some authors showed that ABPA reduces the mechanical properties of zirconia , creatinreatment . The acireatment . Currentreatment . The effreatment ,38,39.According to results reported in literature, one of the purposes of this in vitro study was to confirm the main efficacy of ABPA, compared to hot etching and no pretreatment, on the shear bond strength of an adhesive resin cement to three recent zirconia ceramics. The first null hypothesis of the present report was partially rejected. As demonstrated, the adhesion values of the air-borne particle abraded zirconia were superior  or equal (MET 2—Zirconia Metoxit Z-CAD HTL) to the untreated zirconia samples  and the etched samples .It is of interest to point out that, only for Metoxit Z-CAD HTL, ABPA pretreatment caused an increase in strength bond values not statistically significant if compared to controls. Considering that values for untreated Metoxit Z-CAD HTL were even higher than those of Copran Monolith HT and Katana ML in the same condition, we can assert that the cementation system used in this report was effective as well. Therefore, the outcome regarding Metoxit Z-CAD HTL might be explained considering the chemical composition of this material or, more likely, its surface structure, which might be less responsive to ABPA. With regard to the ABPA procedure, many studies in the literature described the elimination of abraded zirconia particles before proceeding with cementation. This might be performed by rinsing the samples with water or air/water spray ,41 or thThe second purpose of this study was to assess the adhesive remnant index (ARI) score in the three different conditions tested  for each material. The adhesive remnant index (ARI) is a method of defining the adhesion failure site . It is wAdhesion strength tests were extensively documented, and they were found to be useful to test new materials before clinical use . The limThe aim of this in vitro study was to assess the influence of surface pretreatment on shear bond strength of an adhesive resin cement  to three different zirconia ceramics. Adhesion values of the air-borne particle abraded Zirconia Copran and Zirconia Katana ML were superior to the untreated zirconia, whereas they were equal considering air-borne particle abraded Zirconia Metoxit Z-CAD HTL. On the contrary, the hot-etched samples showed the lowest values. Moreover, both the untreated and the air-borne particle abraded zirconia ceramics showed a higher frequency of ARI = 1 and ARI = 2, whereas hot-etched zirconia ceramics showed a higher frequency of ARI = 3. However, no direct correlation was found between ARI score and shear bond strength.