FLT3 (FLT3-ITD) at a high variant allele frequency often contributes to full transformation to a highly proliferative, rapidly progressive disease with poor outcome. The FLT3-ITD mutation is targetable with approved FLT3 small molecule inhibitors, including midostaurin and gilteritinib. However, outside of patients receiving allogeneic transplant, most patients fail to respond or relapse, suggesting alternative approaches of therapy will be required. We employed genome-wide pooled CRISPR knockout screening as a method for large-scale identification of targets whose knockout produces a phenotypic effect that enhances the antitumor properties of FLT3 inhibitors. Among the candidate targets we identified the effect of XPO1 knockout to be synergistic with midostaurin treatment. Next, we validated the genetic finding with pharmacologic combination of the slowly reversible XPO1 inhibitor selinexor with midostaurin and gilteritinib in FLT3-ITD AML cell lines and primary patient samples. Lastly, we demonstrated improved survival with either combination therapy compared to its monotherapy components in an aggressive AML murine model, supporting further evaluation and rapid clinical translation of this combination strategy.Acute myeloid leukemia (AML) is a hematopoietic stem-cell-derived leukemia with often successive derived driver mutations. Late onset acquisition of internal tandem duplication in  TP53, or proliferative kinase genes, such as FLT3, c-Kit, and RAS-related family members. Acquisition of activated kinase genes often leads to rapidly proliferative disease. Despite being the most incident adult leukemia, AML clinical outcome with a common intensive chemotherapy approach not directed toward the specific driving cytogenetic or mutational feature of AML has been relatively poor due largely to inter- and intrapatient heterogeneity of the disease [Acute myeloid leukemia (AML) results from the accumulation of somatic chromosomal abnormalities, genetic mutations, and epigenetic changes that disrupt normal hematopoietic maturation and allow for expansion of undifferentiated myeloid precursor cells. Indeed, the pathogenesis of AML often involves one or more cytogenetic abnormalities that blocks hematopoietic differentiation combined with mutations in either pro-apoptotic genes, such as  disease .fms-like tyrosine kinase 3 gene (FLT3). The FLT3-ITD mutation is by far the most common of the two common mutations of FLT3, and when present at a high variant allele frequency  in the entire leukemia clonal population, it promotes a more aggressive disease phenotype and poor prognosis [FLT3-ITD with a high allelic ratio in the high risk group of patients, whereas a FLT3-TKD or a low allelic ratio (<0.5) and absence of other high risk features results in intermediate classification [FLT3-mutated AML, either in combination with standard chemotherapy as initial treatment (midostaurin) or as salvage therapy (gilteritinib) [Exportin-1 (XPO1).Approximately 30% of patients with acute myeloid leukemia (AML) have an internal tandem duplication (ITD) or tyrosine kinase domain (TKD)-activating mutation in the rognosis ,3. Indeefication . The advritinib) ,5. Howevritinib) . We hypoFLT3-ITD leukemic cells. However, despite some clinical benefit in vivo in a human clinical trial of AML, the anti-XPO1 drug selinexor was not sufficiently active as a monotherapy in relapsed disease versus other salvage agents [FLT3-ITD patients [FLT3-ITD AML patients and further extend the antileukemic benefit to the LIC compartment [XPO1 has been recognized as a potential therapeutic target in hematologic and solid tumors. Selective inhibitors of XPO1 have been shown to possess antileukemic and proapoptotic activity in AML blasts by disrupting aberrant removal of tumor suppressor proteins from the nucleus, such as TP53 . XPO1 ine agents . Given tpatients , combinapartment .FLT3-ITD, building upon prior work examining selinexor in combination with the tyrosine kinase inhibitor sorafenib, which has anti-FLT3 activity [Through genetic and pharmacologic validation, we showed that inhibition of XPO1 enhanced the activity of both midostaurin and gilteritinib ex vivo in AML primary patient blasts harboring activity . FurtherTo identify genes that, when cotargeted, produce a phenotypic effect that can enhance the effect of midostaurin treatment in AML patients, we conducted genome-wide loss-of-function screens with the human CRISPR knockout (KO) pooled library “Brunello” from the Broad Institute (Addgene#73179)  applyingXPO1  levels of XPO1 protein expression with CRISPR ribonucleoprotein in the same MOLM-13 cell line model of FLT3-ITD AML as used in screening with three different guides (p < 0.001). Because midostaurin exerts additional non-FLT3 kinase inhibitory activity, we sought to reproduce this effect with the more selective and potent FLT3 inhibitor gilteritinib, which has enhanced in vivo activity and monotherapy activity in patients with AML [p < 0.001; Based on the above results, we examined the in vitro relevance of the loss of XPO1 on midostaurin-mediated cell death in t guides a and cont guides b. Next, with AML . SimilarFLT3-ITD AML cell lines with a range of doses of selinexor together with a range of doses of either midostaurin or gilteritinib to determine the synergistic range for the combination. For MOLM-13 cells, the range of synergy was 10–200 nM selinexor with 3–50 nM midostaurin or 2–8 nM gilteritinib  having a high allelic ratio of FLT3-ITD with HS5 stroma as a support system to enhance survival for 96 h. Samples were treated with a range of doses of selinexor in combination with either midostaurin or gilteritinib and both demonstrated some level of synergy  mice. Mice were treated with 30 mg/kg gilteritinib daily, 50 mg/kg midostaurin daily, 15 mg/kg selinexor twice weekly, combinations of gilteritinib or midostaurin with selinexor, or vehicle control (p < 0.001), 25 days for midostaurin (p = 0.001), and 27 days for selinexor , 27 days for selinexor (p < 0.001), and 39 days for gilteritinib (p < 0.001). Spleen size and weight did not appear to be dramatically different for the majority of conditions (p = 0.003). Together, this data suggests that this combination could prove beneficial for AML patients harboring FLT3-ITD and merits further clinical development.The promising in vitro results led us to evaluate the in vivo efficacy in an aggressive MOLM-13 xenograft mouse model. Human MOLM-13 cells that express luciferase to allow for visualization of disease burden and dissemination were engrafted into NOD- control a. IVIS i control b. Estimanditions d,e. HoweFLT3-ITD mutations represent the most common type of AML that confers poor prognosis in AML, even when it occurs with favorable mutations such as NPM1. Strategies targeting FLT3 activation either in combination with chemotherapy using the first-generation inhibitor midostaurin or monotherapy with the more potent and selective inhibitor gilteritinib have shown favorable clinical outcome and improved survival but still a very low rate of cure. Given this observation, we hypothesized that FLT3 inhibitor therapy with either midostaurin or gilteritinib given as monotherapy or in combination with chemotherapy will be insufficient for producing a lasting remission. We therefore sought to determine if these drugs could be combined with an additional targeted drug approved for marketing by the FDA to overcome limitations such as short duration of response and relapse. To accomplish this, we utilized the novel technique of CRISPR knockout screening to provide a genome-wide, unbiased approach to identifying genes whose knockout is predicted to enhance the antileukemic effects of FLT3 inhibitors. Doing this, we were able to identify the gene nuclear exporter XPO1 as well as many genes involved in the nuclear pore, including interacting partners of XPO1. Given the prediction from our screen that XPO1 and FLT3 inhibitors would synergistically exhibit robust antileukemic activity when in combination—which was strengthened by pathway analysis—we pursued genetic target validation by knocking down expression of XPO1 protein levels. This demonstrated that while XPO1 knockdown alone, midostaurin treatment alone, or gilteritinib treatment alone were all capable of decreasing viable cells, the combination of XPO1 knockdown with either midostaurin or gilteritinib resulted in greater reduction of viable cells compared to each condition alone or vehicle control. Similarly, our data showed that inhibition of XPO1 via the slowly reversible inhibitor selinexor phenocopied the effect of CRISPR-mediated XPO1 KD. Specifically, in vitro cell line, primary cell experiments and xenograft studies combining these two classes of drugs demonstrated synergistic benefit. Collectively, this provides a therapeutic pathway for novel combination therapies using XPO1 inhibitors together with FLT3 inhibitors.FLT3-ITD patients demonstrated that XPO1 expression increased at time of relapse and inhibition of XPO1 with selinexor increased sensitivity to induction chemotherapy used for AML. In a phase I clinical trial of selinexor monotherapy for AML patients examining multiple doses, 9% of patients responded to treatment at various doses [FLT3-ITD-positive AML. Our unbiased CRISPR-Cas9 screening data and validation with two distinct chemical entities that inhibit FLT3 in AML cell lines and primary cells provides further evidence for this. Indeed, not only KD of XPO1, but also other members of this nuclear export complex were identified. Although more laborious, the application of CRISPR-Cas9 screening, which examines targets for drug synergy in a potentially unbiased and comprehensive manner, adds to the molecular pharmacologic and in vivo tools that can be utilized by drug development teams to assure the best combination strategies and drugs are utilized in clinical trials. Collectively, these data suggest a potential role for combination strategies of FLT3 and XPO1 inhibitors in clinical trials.XPO1 (Exportin-1) has been recognized as a potential therapeutic target in multiple tumor types, including AML, and the magnitude of its overexpression at an mRNA and protein level has been directly correlated with poor overall survival. Development of XPO1 inhibitors for clinical use has been somewhat challenging due to the modest therapeutic window that exists with current therapeutic agents. Selinexor was the first slowly reversible XPO1 inhibitor to enter clinical development and is currently marketed for third line treatment of multiple myeloma in combination with dexamethasone. Preclinical studies from several groups have demonstrated that selinexor (KPT-330) or its second-generation molecule eltanexor KPT-8602) have in vitro and in vivo activity in AML preclinical models and inhibit leukemia initiating stem cells. Studies have demonstrated synergy with other agents including topoisomerase 2 inhibitors, BCL-2 inhibitors, hypomethylating agents, and broad kinase inhibitors that include FLT3 (sorafinib) . Additio602 have 2 dosing used in the phase 1 AML study. Even for lower doses of selinexor, these symptoms can occur. Gilteritinib is a more selective and potent inhibitor of FLT3 that is approved as monotherapy for relapsed AML and generally lacks these gastrointestinal and constitutional symptoms. In contrast, midostaurin is a less specific and potent FLT3 inhibitor approved only in combination therapy with chemotherapy and has significant gastrointestinal symptoms associated with it. A similar earlier study observed that the less specific and potent FLT3 inhibitor sorafenib synergizes with selinexor [Clinical development of the combination of a FLT3 and XPO1 inhibitor should consider both overlapping toxicities and experience in development of each therapeutic as a monotherapy. XPO1 inhibitors such as selinexor may cause distinct gastrointestinal and constitutional symptoms that in AML patients were problematic above the 40 mg/melinexor , but likelinexor ,27. MoviNF1, FLT3, and CBL; MV4-11 cells were found to have variants in TP53 and FLT3. HEK293FT cells were purchased from Life Technologies (R70007) . Cells were propagated in filtered RPMI 1640  for MOLM-13, MV4-11, and HS5 cell lines or DMEM (Gibco) for HEK293FT cells. All media were supplemented with 10% Fetal Bovine Serum  and 1% penicillin/streptomycin/glutamine. HS5-GFP cells were kindly gifted from Dr. William Dalton (H. Lee Moffitt Cancer Center) and MOLM-13-luciferase cells from Dr. Ramiro Garzon (Ohio State University). Cell lines were validated by microsatellite genotyping , routinely tested negative for mycoplasma contamination , and were discarded after passage twenty.MOLM-13 and MV4-11 cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen  in February 2015. Our targeted sequencing panel and capillary gel electrophoresis indicated that MOLM-13 cells have variants in The human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench  . The lib6 cells/well were plated in each well of 12-well plates with total cells equal to 400 cells/sgRNA × 76,441 sgRNAs × 100/infection efficiency (%) to determine the total cells per condition. Media containing lentiviral particles was added at the predetermined ratio with polybrene and spinoculated at 450× g for 90 min. After six hours, cells were centrifuged 300× g for 10 min and resuspended in complete media. Puromycin selection was started after three days and continued for seven days of selection to remove essential and nontransduced cells. Cultures were then exposed to 30 nM midostaurin (Selleck), or dimethyl sulfoxide  as a vehicle control for three days. Cells were harvested, DNA extracted via Qiagen Blood & Cell Culture DNA Midi Kit per manufacturer’s instructions, and the sgRNA cassette retrieved via PCR as described by Broad sequencing protocol on Addgene. The P5 mix and P7 primers design from this protocol were used with the following New England Biolabs NEBNext Indexes: 17, 24, 26, 28, 29, 31, 33, 34, 35, 36, and 42. Sample sequencing on an Illumina HiSeq4000 was performed at the Institute for Genomic Medicine at Nationwide Children’s Hospital, Columbus, Ohio.For screening, 2.5 × 10Analysis of resultant FASTQ utilized Snakemake  to organFollowing manufacturer protocols, ribonucleoprotein (RNP) complexes were formed using Alt-R CRIPSR-Cas9 cRNA, tracrRNA, and Cas9 . RNP and Alt-R Cas9 electroporation enhancer were added to MOLM-13 cells and electroporated in Solution SF (Lonza V4XC-2012) on a Lonza 4D-Nucleofector System with pulse code EH100. The following Alt-R CRISPR–Cas9 sgRNAs targeting XPO1 were used as a pool: GGTTGAAACCGGTTCAGACT, GAGAGGGGACGAATCAAGGT, and TCCCAAGCTCTCCACCGAGG.Extracted proteins from whole-cell lysates were resolved by SDS–PAGE, then transferred onto nitrocellulose membrane as previously described . Blots wCell proliferation and death were determined via MTS and Annexin V-FITC flow-based assays as previously described .FLT3-ITD cryopreserved primary cells collected during apheresis of AML patients with different co-occurring mutations  was added after 96 h and incubated an additional five hours. Leukemic cells were then transferred to a new plate and formazan dye was quantified by absorbance (490 nm). The dose range of midostaurin had to be limited because doses greater than 100 nM caused stromal detachment. A next-generation sequencing targeted capture panel was used to determine mutational status of samples, which was visualized with Oncoprint [utations  were obtncoprint ,37.4 MOLM-13 luciferase-tagged cells were injected into the tail vein of NOD-em26Cd52Il2rgem26Cd22Prkdc/NjuCrl (NCG) mice from the Charles River Laboratory. Three days postengraftment, mice begin treatment of 50 mg/kg midostaurin  daily gavage in 6% drug w/w gelucire(R)44/14 , which was aliquoted and mixed with drug weekly; 30 mg/kg gilteritinib (MedChemExpress HY-12432) daily gavage in 6% drug w/w gelucire(R)44/14 , which was aliquoted and mixed with drug weekly; 15 mg/kg selinexor  gavage in 0.6% w/v pluronic F-68 (Life Technologies 24040032) and 0.6% w/v plasdone K-29/32  on two consecutive days every week; or combinations thereof. Aliquots of gelucire were stored at 4 °C and warmed each day to 44 °C in a heat block, then diluted with sterile water to a concentration of 12 mg/mL.Animal experiments were carried out under protocols approved by the Ohio State University: protocol number 2015A00000043-R1 and OLAW assurance A3261-01. Institutional Animal Care and Use Committee. A total of 10Appropriate dose was determined daily based on weight with a maximum volume of 200 µL for oral gavage. Animal technicians who were blinded to treatment groups routinely monitored mice for Early Removal Criteria . At 10% weight loss, mice were given a two-day drug holiday.For CRISPR screening, statistics were calculated using MAGeCK software . In combp-values were adjusted for multiple comparisons within each drug combination using Holm’s procedure.The mathematical synergy and all other statistical analyses aside from those described above were performed using SAS/STAT software, version 9.4 of the SAS System for Windows . For XPO1 KD experiments, dependencies among observations from the same biological replicate and the interaction between parent/KD and drug were accounted for using mixed effects models. For CFU assays, differences between conditions were assessed after fitting negative binomial models to the count data. The primary endpoint of mouse survival experiments was overall survival (OS). Kaplan–Meier methods were used to estimate median survival time in each group. The log-rank test was used to assess differences in OS between groups. For spleen weight, differences between groups were estimated using analysis of variance (ANOVA) methods, and All sequence data are available in the Gene Expression Omnibus via accession number GSE143314.Using a large, unbiased CRISPR-Cas9 knockout screen we identified many genes whose knockout is predicted to enhance the antileukemic effect of FLT3 inhibitors midostaurin and gilteritinib. Among these genes we selected XPO1 for further validation because of the potential for rapid clinical translation of combination therapies of gilteritinib with FDA-approved XPO1 inhibitor selinexor to augment antileukemic activity.FLT3-ITD AML cell lines, primary patient samples, and in an aggressive xenograft mouse model. This work shows the power of using large CRISPR screens to identify combination therapies relevant to a particular targeted therapy for translation to the clinic, demonstrates how pathway analysis can be used to strengthen predictive power of the screens, and uses multiple orthogonal approaches to validate the predicted synergy. Thus, this work serves as a template for development of rational combination strategies using CRISPR screening and—in application to the particular case of FLT3 inhibitors—supports the further clinical development of cotherapy with FLT3 inhibitors, especially gilteritinib and selinexor.Knockdown of protein levels of XPO1 with midostaurin or gilteritinib treatment reproduced the synergy demonstrated during the genome-wide screening. Replacement of genetic perturbation of XPO1 with selinexor also reproduced the expected synergy. Furthermore, the combinations of midostaurin and selinexor or gilteritinib and selinexor were effective across


Outcomes following total knee arthroplasty (TKA), whether clinical, radiological or survival analysis, have been well-studied. Still, there are some concerns about patient satisfaction with the outcome of the surgery and factors that might contribute to a suboptimal result. This study aims to determine if there is correlation between primary TKA malalignment and early patient-reported outcome measures (PROMs).Sixty patients, who had primary TKA and a minimum of 2 years of follow up, were recruited for a detailed clinical and radiological examination. Knee alignment was measured in the coronal, sagittal and axial planes. Normal and the outlier measurements of the patients’ knees were defined and the clinical results (PROMs) compared to see if there was a statistically significant difference.Correlation between postoperative limb malalignment in the coronal and the sagittal planes and PROMs was not significant. Conversely, there was significant negative correlation between all types of malrotation and PROMs.Although malalignment has been linked to inferior outcome and implant survival, our results showed that coronal and sagittal limb malalignment has no significant effect on early PROMs. However, all types of component rotational malalignment significantly worsen early PROMs. Although total knee arthroplasty (TKA) is a well-established orthopedic procedure with documented success; patient dissatisfaction after surgery is still substantially high . Out of The real value of patient-reported outcome measures (PROMs) is to have a full view of the patients’ functional limitations, pain levels, mental health state, and expectations from their treatment, thus improving communication between surgeons and patients with the potential for improving postoperative outcomes. Although clinical and radiological evaluations are vital in reaching the proper diagnosis, PROMs should always be assessed as we are treating patients not the x rays .The effect of malalignment on the outcome after TKA remains controversial. The majority of authors link malalignment to decreased implant survival , 5. OtheOutcomes after total knee arthroplasty (TKA), whether clinical, radiological, or survival analysis, have been well-studied. Still, there are some concerns about patient satisfaction with the outcome of the surgery and factors that might contribute to a suboptimal result. This study aims to determine if there is correlation between primary TKA malalignment and early PROMs. Our hypothesis is that malalignment after primary TKA, especially rotational malalignment, has a direct effect on the early PROMs.This is a retrospective cohort study that included 60 patients with primary TKA (60 TKAs). After searching our Arthroplasty Unit database, we contacted 145 patients who had undergone primary TKA and had a minimum follow-up period of 24 months: 60 patients (about 41% of those contacted) were available for clinical and radiological examination. Of these, 46 patients were female (77%) and 14 were male (23%). The mean age was 57 ± 9.6 years. The underlying cause of their knee problems was osteoarthritis (49 patients (82%)) and rheumatoid arthritis (11 patients (18%)). Patients who had undergone revision TKA, previous knee surgery such as high tibial osteotomy, previous knee trauma, or postoperative complications such as infection and intraoperative complications such as fractures were excluded to avoid confounding bias.The senior author had performed all surgery in these patients using a medial parapatellar approach. Initially the distal femoral cut was made according to the valgus correction angle (VCA), which was individualized according to the difference between the anatomical and mechanical axis as demonstrated on the full-length radiograph obtained in the weight-bearing position. The inter-epicondylar line was used to define the rotation of the femoral component. The tibial cut was made using the external guide. The tibial component was then rotated to the proper rotation depending on the tibial landmark, which is the medial third of the tibial tubercle. A cemented Nex Gen Legacy posterior stabilized (LPS) flex fixed bearing  was used in all knees. According to our institutional postoperative follow-up protocol, all patients were clinically and radiologically evaluated at 6 weeks and at 3, 6, 12, and 24 months.Clinical assessment: this was based on the following scores; Knee Society Score (KSS) , Knee OsRadiological assessment: this was performed as described subsequently.All 60 patients were recruited for a simultaneous detailed PROMs evaluation and radiological examination including both x-rays and computer tomography (CT) at a minimum of 24 months postoperative follow-up (range 24–132 months postoperative) by an independent researcher in the period from June 2017 to December 2017. Assessments were as follows:Coronal mechanical alignment of the lower limb was measured by calculating the hip-knee-ankle (HKA) , 15 axisThe anterior cortical line angle  was calcWe followed the protocol described by Berger et al.  to evaluFemoral component rotation was assessed using the axial CT image of the femur at the level of the femoral epicondyles. We used the surgical transepicondylar axis, which is the line connecting the medial epicondylar sulcus to the lateral epicondylar prominence. The posterior condylar line of the femoral component was schemed as a line drawn by connecting the posterior margins of the medial and lateral posterior component condylar surfaces. The angle between these two lines was then measured to obtain the posterior condylar angle Fig. . The femTibial component rotation was assessed from three axial CT images through the tibia. The geometric center of the tibial plateau was located from an axial image just below the tibial component. The geometric center is axially transposed to an axial image through the prominence of the tibial tuberosity and a line is drawn from the geometric center and the tuberosity, the tibial tuberosity axis. The tibial component axis was drawn at the level of the component polyethylene as a line perpendicular to a line drawn along the posterior surface of the component polyethylene. The tibial tuberosity axis was then axially transposed to this image and the angle between these two lines was measured Fig. .Fig. 4a The relationship between the tibial tubercle and tibial articular surface was used to assess tibial component rotation, whether internally or externally rotated. The tip of the tibial tubercle is 18° externally rotated from the tibial articular surface. The tibial component was considered neutral in both men and women when internally rotated 18° (±2.6) in relation to the tip of the tibial tuberosity [Combined component rotation was measured by adding together the femoral and tibial component rotation angles.Component mismatch was calculated by subtracting femoral from tibial component rotation.t test to compare the means of dichotomous variables, and analysis of variance (ANOVA) to compare the means of variables with more than two categories. Correlation was tested to determine the association between variables (Spearman’s rank correlation). A p value equal to or less than 0.05 was considered significant.Data were verified, coded, and analyzed using SPSS version 21 . Descriptive statistics  were calculated. Statistical significance was tested using the independent Sixty patients were eligible for our study. Limb alignment was assessed in the coronal plane: optimum coronal limb alignment (± 3°) was identified in 39 TKAs (65%), with outliers identified in 21 TKAs (35%) . In the sagittal plane, 15 TKAs (25%) were found to have optimum sagittal limb alignment (0–5°) and 45 TKAs were considered outliers. Hyperextension was identified in 31 (51.7%) TKAs and flexion deformity was identified in 14 (23.3%) TKAs.In the assessment of component alignment in the axial plane, optimum femoral component rotation (3.5° and 0.3° ± 1.2 in relation to sTEA in men and women respectively) was identified in 16 TKAs (26.7%) and 44 TKAs were considered outliers; there were 16 (26.7%) TKAs with an internally rotated and 28 (46.6%) with an externally rotated femoral component. Optimum tibial component rotation  was identified in 28 TKAs (46.6%) and 32 TKAs were considered outliers; there were 22 (36.6%) TKAs with an internally rotated and 10 (16.8%) with an externally rotated tibial component. Furthermore, combined component rotation  was identified in 15 (25%) TKAs and component rotation mismatch was identified in 17 (28.3%) TKAs. Table Correlation between postoperative limb malalignment in the coronal plane and the sagittal planes and patients’ satisfaction scores was negative and minimal, and was not statistically significant.r) was − 0.55 , − 0.58 , −0.062 , − 0.59  for KSS, KOOS-JR, OKS, and SF-12 scores, respectively  was − 0.23  and − 0.33  for KOOS-JR and SF-12 scores, respectively  was − 0.47  and − 0.49  for KSS and SF-12 scores, respectively. Furthermore, there was significant, moderate, negative correlation with combined component rotation  was − 0.48  for KOOS-JR  and all patient satisfaction scores. The correlation coefficient  after a minimum of 2 years follow up.At a minimum of 1 year follow up, Longstaff et al.  showed tBlakeney et al.  prospectAlso, Huang et al.  showed tContradicting these results, Stucinskas et al.  investigMatziolis et al. , in a caSimilar to our results, all other reports that studied sagittal component alignment failed to identify correlation between implant malalignemt in the sagittal plane and patient outcome , 8.As regards component rotation, several authors –27 have Czurda et al.  observedAlso, Lutzner et al.  studied Keeping the component rotational mismatch within ±5° during TKA is needed for controlled knee axial rotation during flexion. Mismatch above or below this range results in measurable kinematic differences and causes different patterns of axial rotation motions during passive knee flexion .Although the average amount of internal rotation of the femoral component in our study was 0.86 ± 0.2°, which is far below the average in the aforementioned studies, it was significantly associated with worse functional outcome. On the other hand, the average for internal rotation of the tibial component, combined internal rotation, and mismatch was 6.22 ± 0.7°, 7.42 ± 1.1 °, and 9.31 ± 1.9°, respectively, and these values are close to the average values for malrotation in the same studies.The results of this study confirm that malalignment in the coronal and sagittal plane has no effect on PROMs at short-term follow up, a finding supported by many authors. It seems that muscles adapt to longstanding deformity in the way that it can easily become accustomed to residual deformity after TKA. Component rotational malalignment alters patellofemoral and knee flexion-extension kinematics from the first day postoperative. This explains the high incidence of early pain and inferior outcome measures when component malrotation is evident.The main limitation to this study is the short follow-up period. A prospective study design with a longer follow-up period will be needed to track the changes in the PROMs and determine whether or not these changes persist in the midterm and long-term follow up. Another important limitation to this study is that there are many other confounding variables such as body mass index (BMI) and psychological factors, etc., which may affect the outcome after TKA.Although malalignment has been linked to inferior outcome and implant survival, our results showed that coronal and sagittal limb malalignment has no significant effect on early PROMs. However, all types of component rotational malalignment significantly worsen early PROMs.


An acute shortage of senior mentors saw the Palliative Medicine Initiative (PMI) combine its novice mentoring program with electronic and peer mentoring to overcome insufficient mentoring support of medical students and junior doctors by senior clinicians. A three-phased evaluation was carried out to evaluate mentees’ experiences within the new CNEP mentoring program.Phase 1 saw use of a Delphi process to create a content-valid questionnaire from data drawn from 9 systematic reviews of key aspects of novice mentoring. In Phase 2 Cognitive Interviews were used to evaluate the tool. The tool was then piloted amongst mentees in the CNEP program. Phase 3 compared mentee’s experiences in the CNEP program with those from the PMI’s novice mentoring program.Thematic analysis of open-ended responses revealed three themes–the CNEP mentoring process, its benefits and challenges that expound on the descriptive statistical analysis of specific close-ended and Likert scale responses of the survey. The results show mentee experiences in the PMI’s novice mentoring program and the CNEP program to be similar and that the addition of near peer and e-mentoring processes enhance communications and support of mentees.CNEP mentoring is an evolved form of novice mentoring built on a consistent mentoring approach supported by an effective host organization. The host organization marshals assessment, support and oversight of the program and allows flexibility within the approach to meet the particular needs of mentees, mentors and senior mentors. Whilst near-peer mentors and e-mentoring can make up for the lack of senior mentor availability, their effectiveness hinges upon a common mentoring approach.To better support the CNEP program deeper understanding of the mentoring dynamics, policing and mentor and mentee training processes are required. The CNEP mentoring tool too needs to be validated. Defiionships . Howeverionships –40 and rionships –64.et al. [voluntary collaboration between colleagues of similar rank and experience and common academic interests on mutually beneficial structured fixed term projects. These processes often include a senior clinician who facilitates discussions, provides personalized support and feedback and oversees the mentoring process. Effective NP mentoring nurtures long-term friendships and professional collaborations between peers”. Chong et al. [personalised, internet or electronically mediated approach that is largely used to complement face-to-face mentoring to provide personalised, appropriate, specific, timely, holistic, accessible and longitudinal mentoring support to build mutually beneficial mentoring relationships between the host organization, a senior mentor and an individual mentee. Working within the confines of prevailing professional codes of conduct and standards of practice this approach is focused upon realizing the goals and needs of the mentee, the mentor, the host organization that supports and oversees the program and their relationships. Its asynchronous nature also nurtures reflective practices that helps develop deeper mentoring relationships”. Whilst Chong et al. [et al. [et al. [Combined Novice, E-mentoring and Peer mentoring or CNEP mentoring remains untested [To circumnavigate some of these concerns, a combination of near-peer or peer and e-mentoring to supplement the prevailing novice mentoring approach is proposed. Lim et al.  define ng et al.  define eg et al. ’s system [et al. ’s system [et al. ’s proposuntested , 67, 68.et al. [et al. [et al. [However in the face of a sudden shortage of mentors following the untimely demise of a senior mentor and the sabbatical by another senior mentor, the Palliative Medicine Initiative (PMI), a research-based novice mentoring program hosted by the Division of Supportive and Palliative Care (DSPC) at the National Cancer Centre Singapore (NCCS), had to put theory to the test . Buildinet al. ’s, Tan e [et al. ’s and Li [et al. ’s reviewThe PMI program was designed to increase student-led research in palliative medicine, professionalism, medical ethics, medical education, end-of-life ethics (EoLE) and health services through use of a novice mentoring approach. To do so, the PMI provided mentoring support to medical students and junior doctors through the various stages of the research and publication process. This homegrown approach was designed on the team’s own evaluation of prevailing data which later formed the basis for systematic reviews and scoping systematic reviews around the key elements of mentoring relationships , mentoriet al. [By inculcating new novice , 7, 9 meet al. ’s accounThis mix of flexibility and consistency within the mentoring approach has seen the PMI program publish over 50 mentored articles in peer reviewed journals and the presentation of more than 70 mentored posters in international medical education, medical ethics and Palliative Medicine conferences over the last 9 years .® , text messaging, Facetime®  and Skype®  meetings with the senior mentor to ensure timely, specific, appropriate, personalised, longitudinal, accessible and holistic support and feedback. A brief description of the new CNEP program is enclosed in The new CNEP mentoring approach supplements the PMI’s prevailing novice mentoring program with trained near peer-mentors who had successfully completed the PMI program. CNEP mentees and near peer mentors were also provided with access to e-mentoring options such as email, WhatsAppThe organisation structure of senior mentors, near-peer mentors and mentees may be found in Given the unproven benefits of CNEP mentoring, an evaluation of CNEP mentoring was called for. The research team consisting of a methodologist, an educationalist, a senior librarian, 2 senior clinicians, 4 freshly recruited medical students with no research experience and 4 junior doctors and 4 medical students with previous research experience in the PMI novice mentoring program discussed the issues pertaining to assessing CNEP mentoring with educationalists, clinicians, academics, mentors and administrators at Duke-NUS Medical School, Yong Loo Lin School of Medicine at the National University of Singapore, the University of Liverpool, the National Cancer Centre Singapore and Singapore General Hospital (henceforth the expert team) to delineate the research questions. The primary research question was “what were the mentee’s experiences of the CNEP mentoring program?” and the secondary question was “what was the impact of the addition of e-mentoring and near peer mentoring to the PMI novice mentoring program?”.However, there were significant limitations in addressing these research questions in the absence of effective assessment tools to evaluate mentee experiences in CNEP mentoring. To overcome this issue the expert and research teams concluded that a new tool to assess mentoring was required.A three phased approach was proposed to design a new tool and to address the primary and secondary research questions. Phase 1 involved design of a new tool, Phase 2 involved assessing the mentee’s experiences using the newly designed CNEP mentoring tool and Phase 3 involved comparing the findings of the new tool with data from the PMI novice mentoring program. This process is outlined in A Delphi study  was propsurvey questions that would be used to form the items of the Delphi survey questionnaire [The first step of the Delphi study sought to provide a set of ionnaire . To do sionnaire , 19, 20.These 5 essential activities were characterised by a total of 168 activities that were reviewed for redundancies, trimming the list to 67 items . This liA 70% consensus agreement for each item’s inclusion was set a-priori by the research team. Items with more than 70% of participants agreeing that a question should be addressed to either the mentor only, mentee only, or mentor and mentee were eligible for inclusion into the final survey instrument. After the first round of ratings, the number of items was reduced to 33. The items were then formatted into a final survey instrument and sent back to the experts for their assessment of how well each set of items reflected the domain of interest. The final survey received unanimous support from all 7 experts who participated in the second round.The second stage in the Delphi study involved cognitive interviews with 3 PMI mentees. Cognitive Interviews served to assess the ‘response process validity’, or how prospective participants interpret items within the pilot tool  and whetIn Phase 2 the new tool was piloted amongst mentees in the CNEP program. Email invites containing information about the study, the tool and an attached informed consent form was sent to 28 mentees who had completed the CNEP program and had published articles in peer reviewed journals and or had presented posters in conferences between 2016 and 2018. Invitees who agreed to participate in the survey printed out, signed, and scanned or photographed the consent form and returned it to a study team member with whom they have not worked with previously and who is not part of the team analysing the data. Upon receipt of the signed consent form, the study team member then sent a link to the survey that was hosted on a university website. mentoring).This study was approved by the Central Institutional Review Board of the National Cancer Centre Singapore Overall, 18 of 28 of those invited to participate completed the study . The profile of mentees who participated in the study can be found in Three reviewers , experienced in the use of Braun and Clarke ’s approaIn keeping with the first phase of Braun and Clarke ’s approaNext, the reviewers constructed ‘codes’ or a “feature of the data (semantic content or latent) that appears interesting to the analyst, and refer to ‘the most basic segment, or element, of the raw data or information that can be assessed in a meaningful way regarding the phenomenon’  from theIn the third phase of Braun and Clarke ’s approaIn the fourth phase of Braun and Clarke ’s approaet al. [In this fifth phase of Braun and Clarke ’s approaet al. ’s “negotet al. .The reviewers independently extracted and classified all quotations using the code books, collapsed them into themes and subthemes  and mainAn external reviewer analysed each code for consistency and accuracy . The theQuantitative analysis was limited given the small numbers of respondents.Descriptive analyses were performed on the quantitative survey data, reporting the proportion of Likert scale responses for the respective survey questions. The reviewers compared the quantitative results with the qualitative survey data to propose possible explanations for the quantitative survey responses, and highlight any contradicting data points between the qualitative and quantitative datasets .For the purposes of triangulation of coding and thematic analysis of qualitative data, the analysis was carried out by three independent reviewers. The codes and themes identified by each reviewer was discussed by the three reviewers in online and face-to-face meetings. In addition, the findings of each reviewer and their combined analysis was reviewed by an experienced external reviewer well versed in the topic at hand. To further ensure theoretical validation, the results of the analysis was compared with prevailing data. An iterative process was employed which meant that any new codes identified meant that all the survey data were reviewed to verify the classification and ensure complete data extraction.This manuscript adheres to the SQUIRE guidelines .Thematic analysis of the responses to the open-ended questions in the survey revealed 3 themes including CNEP mentoring process, the benefits and the challenges of CNEP mentoring. The themes expand upon the descriptive statistical analysis of specific close-ended and Likert scale responses of the survey, which will be discussed in tandem.There are three sub-themes delineated–“mentoring stages”, “e-mentoring’s role” and the “near-peer mentoring’s role”.There are 6 subthemes to mentoring stages. These include recruitment, aligning expectations, mentoring process, mentoring relationships, mentoring environment and near peer role.i. Recruitment. Eighty-nine percent  of mentees selected their own senior mentor whilst only 33% (n = 6) of mentees selected their near-peer mentor. Mentor selection was based upon a variety of factors. Prior interactions with the mentor and recommendations from colleagues were important considerations as were the mentor’s personal attributes, experience and common personal and professional interests (nterests .In most cases the near-peer mentor was often determined by the senior mentor (n = 12). The role of the senior mentor was determined by research, clinical and educational experiences n = 18, .In 89% (n = 16) of case mentees saw the senior mentor as the consultant/attending. The near-peer mentor was in most cases a senior resident or resident n = 12, .ii. Aligning expectations (n = 11). Once mentees and mentors agreed to work together, they met to align expectations (n = 18), availability (n = 18), goals (n = 15) and timelines (n = 12) and establish their roles and responsibility . n = 11, . Menteesiii. Mentoring process. The PMI CNEP mentoring program is built around a structured research process. This research-based approach guided the mentoring approach and ensured that it is easily understood by mentees, near-peer mentors and senior mentors as well as the host organization and applied in a consistent fashion. The regular steps involved in the research process including the design, data gathering, review of the study findings, analysis of the data, manuscript preparation, and ending with reflection of the research process ensured a systematic mentoring trajectory replete with clear stages that can be evaluated longitudinally.iv. Evolving mentoring relationship (n = 17). A key feature in the mentoring process was change. This was primarily the result of changing schedules and priorities, access to training and support and the inevitable difference in the focus of each stage of the research based mentoring process. The personal and professional needs of the mentee also changed the mentoring relationship  Review milestones (n = 2)“It (mentee role) did (change), based on changes and needs of the relationship.”(Mentee 1)b) Resolve issues (n = 3)“I am thankful that they are always there to patiently communicate ideas with me, remain open and honest in all interactions, and willing to clarify any misunderstandings so that my relationships with them continue to be built on trust.”(Mentee 15)c) Realign goals and expectations (n = 1)“Set good deadlines and negotiate where needed.”(Mentee 4)d) Provide specific training and resources (n = 1)“to be proactive and communicate well with mentors on goals, responsibilities and expectations”(Mentee 18)v. Mentoring environment (n = 17). Change within the mentoring relationship however was possible given the impact of the mentoring environment and the mentee’s own attitudes and motivations , psychological and emotional issues (n = 9) and in addressing their professional identities along the course of the mentoring process n = 9, .vi. Transitioning to a near-peer mentor role (n = 3). As mentees progressed, their mentors evaluated the development of their mentees and their suitability as near-peer mentors.“Yes, I slowly changed from the role of a mentee to near-peer mentor under their guidance. My (senior) mentor's role stayed the same, but with the change in my role as near-peer mentor, my (near-peer) mentor's role also changed to become senior near-peer mentor, whereby my (near-peer) mentor will mentor me to become a better near-peer mentor.”(Mentee 15)One of the motivations to take up the role of a near-peer mentor is to pay it forward.“I became a near-peer mentor for other juniors too- paying it forward”(Mentee 3)As the mentees reflected upon their mentoring experiences and growth, the majority of the mentees had a positive mentoring experience, with 94% (n = 17) of them feeling a sense of belonging, teamwork and collegiality.The primary role of electronic mentoring was to supplement face-to-face meetings (n = 10). Mentees felt face-to-face helped build connections and appreciated the nonverbal communication it provided. As a result, mentees believed electronic mentoring could not replace face-to-face meetings (n = 10).“Convenient but somewhat more impersonal; meant to complement other aspects of mentoring”(Mentee 9)Electronic-mentoring however did enhance access to support and feedback (n = 11).“Faster/ more efficient communication and dissemination of information”(Mentee 9)The asynchronous nature of Electronic-mentoring also facilitated reflective practice (n = 1).“We do not necessary have to reply immediately and can always take time to reflect prior to replying”(Mentee 15)Electronic-mentoring was also seen as an important platform for tracking and disseminating information (n = 1).“Have details always accessible for better follow up and work.”(Mentee 4)89% (n = 16) of the mentees felt that the near-peer mentor played a pivotal role in the development of personalized and nurturing mentoring relationship. Effective near-peer mentors were committed, empathetic, non-judgemental, supportive, caring approachable and accessible (n = 17).“A more junior mentor may be more relatable/approachable to a medical student”(Mentee 3)“Perceived as less authoritative/threatening, more collegial environment as responses are more polite, especially helpful climate for novices who may be more shy.”(Mentee 4)Near-peers facilitated the timely and personalized support (n = 12).“One can rely on the secondary mentor should the (senior) mentor be unavailable … multiple mentors may provide a more holistic experience to mentees.”(Mentee 13)Near peer mentors provided an alternate source of support (n = 9).“Spent more time understanding mentee's concerns and sharing valuable experiences and insight”(Mentee 3)The near-peer mentors also provided oversight (n = 3).“(Having a near-peer mentor) definitely helps to keep track of work more closely”(Mentee 16)Mentees listed the benefits of the program in There are a number of specific challenges to use of the PMI’s CNEP mentoring approach. These are:The presence of multiple avenues of communication may become a source of misunderstanding (n = 1).“Communication and clarification regarding more complex/confusing matters is easier when done face-to-face compared to electronic-mentoring (only)”(Mentee 13)Near peers may also be the source of misunderstandings (n = 1).“(near-peer) and (senior) mentors may have differing views/instructions/perspectives which may confuse medical students”(Mentee 13)Use of CNEP mentoring blurred professional and personal boundaries (n = 1).“Invasion into private time/after hours, but this is universal of communication technologies”(Mentee 4)This contributed to stress amongst 67% (n = 12) of mentees and a negative impact upon their work life balance amongst 28% of mentees n = 5, .An expansive program also raises concerns about ethical practice in the mentoring program .Details of issues faced such as ethical issues, unfair treatment and mentoring malpractice were not expanded upon.et al. [Based upon advice from the expert team and stakeholders as part of the sixth phase of Braun and Clarke ’s approaet al. ’s study et al. [Whilst rather superficial given that these comparisons involve qualitative data it is nonetheless reassuring that the themes and accounts detailed in both studies were similar. In addition it lends weight to the validity of the tool designed here given that Krishna et al. ’s study et al. [In Krishna et al. ’s study,To address its primary research question, this study has adopted a three phased research approach to circumnavigate the lack of effective assessments tools to evaluate CNEP mentoring. Phase 1 identified and extracted specific activities deemed essential in mentorship programs to evaluate the effects of NP and e-mentoring. Through the Delphi process, a new tool was created and piloted through use of Cognitive Interviews.Phase 2 saw application of the new tool to capture a mentee’s perspective of the PMI’s CNEP mentoring program. This data is consistent with prevailing accounts of mentoring experiences in novice mentoring programs in Palliative Medicine , 20, 21.In Phase 3 data from the new tool on mentee’s experiences in CNEP experiences were compared with mentee accounts of their experiences in the PMI’s novice mentoring program. These comparisons revealed that CNEP mentoring like the PMI novice mentoring process progressed through the mentoring stages of recruitment, alignment of the mentoring process, data collection, data analysis and preparation for publication. Here similarities between PMI’s previous and present mentoring approaches reaffirm the validity of comparing the findings of the two studies to help discern the impact of changes to the novice mentoring program.The presence of mentoring stages suggests the presence of two aspects of CNEP mentoring. First is that progress from one stage to another is competency-based and second, CNEP mentoring is dependent upon a structured mentoring approach. In turn these findings have two important implications. A competency-based approach ensures that mentees only progress upon attainment of required skills, reiterating the notion of each stage is a ‘circumscribed sequential projects’ with ‘specific goals and competency requirements’. It also ensures consistent mentoring experiences and clear end points that help alignment of expectations, goals, timelines, expectations and roles and responsibilities . ConsistHowever, it is also clear that the framework can ill afford to be rigid given the need to contend with personalization of the mentoring process. Efforts to personalise the mentoring process was evident in catering meeting and training schedules to the mentee’s abilities, availability and goals. Personalization of the mentoring process was also evident in determining the timelines, codes of conduct, roles and responsibilities of each stakeholder. Ensuring effective balance between a consistent approach and flexibility are personalised, appropriate, specific, timely, longitudinal, accessible and holistic assessments of the mentoring process that inform the administrators and mentors of the adaptations needed.However, the CNEP mentoring program also throws up new considerations. Unlike the PMI’s novice mentoring program that employed mentee-initiated matching that saw mentee’s select their own mentors, CNEP mentoring employed a combination of mentee-initiated mentor matching and matching of near-peer mentors and mentees. Selection of the near-peer mentor was determined by the senior mentor and was often determined by the research topic of research. There is little evidence of any ill effects to the overall success of mentoring relationships. This may be a reflection of the undiminished primacy of the senior mentor’s role with near-peer mentors providing only practical and psychosocial support, that all near-peer mentors are experienced clinically and from a research perspective, trained and employ the same mentoring approach meaning that there is consistency within the overall mentoring process, the belief that all mentoring interactions are carried out under the oversight of the mentor by virtue of the e-mentoring platform or the fact that e-mentoring allows immediate and easy means of contact with the senior mentor. Many of these possible explanations also raise questions as to whether having NP mentoring and e-mentoring addresses the shortage of mentoring support or merely adds to the senior mentor’s workload. Yet with no failed relationships reported, it would seem that CNEP mentoring appears to be as effective as the PMI’s novice mentoring program.Once more communications play a critical role in the mentoring process, helping to align expectations, provide feedback and resolve issues. Here it would seem having both the presence of near-peer mentors and e-mentoring support enhanced communication and cultivated a more nurturing mentoring environment and relationship that enhanced the mentoring experience. The combination of NP and e-mentoring and novice mentoring is also seen as a complementary process particularly with data suggesting that neither NP mentoring or e-mentoring was seen as ‘stand-alone’ mentoring approaches. E-mentoring was ‘found to be ‘impersonal’ (Mentee 9) and did not necessarily result in a timely response (Mentee 15) whilst NP mentoring sometimes led to misunderstandings (Mentee 13) and sometimes invasions of private time (Mentee 4). It would seem novice mentoring and oversight and support by the senior mentor was crucial to the overall mentoring process.However not all the shortfalls facing NP and e-mentoring can be addressed by supplementing them with novice mentoring. The data highlights a number of ethical issues. Perhaps none are so concerning as the worries that 11% of mentees believed that there were ethical issues concerning CNEP mentoring including mentees believing that their near-peer and or senior mentor was motivated by self-interests and/or treated them unfairly. This raises questions as to why 10 of the invited mentees did not participate in the survey with 67% (n = 12) of mentees reported being stressed, 28% (n = 5) experiencing negative effects upon their work life balance and 17% (n = 3) reporting negative impacts upon their clinical work and studies. This is a concern given that CNEP mentoring is lauded for increasing personalised, appropriate, specific, timely, holistic, accessible and longitudinal support and feedback. These findings underline the need for close monitoring of the CNEP program.The need for effective assessment and oversight of the mentoring process underlines the key role of the host organization. The host organization plays a number of critical roles including recruiting and training mentees and mentors, overseeing and supporting the matching process and structuring the trajectory of the mentoring process around the research process. The other critical role of the host organization lies in its establishment of clear codes of conduct, setting out the roles and responsibilities of mentees, near peer mentors and senior mentors and ensuring effective oversight of the mentoring process. The host organization also play a critical role in supporting mentees particular when some have reported a loss of self-esteem, being treated unfairly and disillusionment. The role, ability and support for the host organization must also be evaluated and must be part of the audit and policing of mentoring processes.The absence of such holistic assessments re-emphasise the need to move away from reliance upon post-mentoring surveys and snap-shot interviews. It is clear that views and concerns of senior and near-peer mentors and representatives of the host organization are required given that data on their interactions will change the complexion of the discussion and better inform practice and policing of the mentoring program. To enable such a process a mentoring portfolio replete with both mentoring diaries that would capture all interactions between mentee and the mentors and regular input from the mentors on the mentee’s progress and general direction of the progress may be required. For the host organization, feedback loops must be closed and independent assessments of the mentee, senior and near-peer mentors are required. This can capture concerns on the part of any stakeholder throughout the course of the mentoring process and ensure personalised, appropriate, specific, timely, holistic, accessible and longitudinal support and prompt and appropriate training be provided to each party. Evaluations of the host organization itself and the mentoring program as a whole should be carried out regularly to help understand some of the influences upon individual relationships and provide a more complete picture of the mentoring relationship.Overall, CNEP mentoring does fill many of the gaps in novice mentoring but this approach can only do so when clear lines of communication, effective codes of practice and appropriate oversight is provided. As a result, like novice mentoring, CNEP mentoring needs careful and oversight by the host organization.Whilst a mixed-method questionnaire approach to appraising mentoring relationships and programs is less resource intensive and offers deeper insights into the CNEP mentoring experiences, it has not yet been validated. Similarly, whilst the themes are similar to those identified in semi-structured interviews of the PMI program, a formal validation program for the new is being planned.The use of retrospective data at a single time point a variable distance from the end of the mentoring process invites recall bias . The preThe small sample size and a uniquely structured approach around a research process may limit the applicability of these findings in other settings.It is apparent that CNEP mentoring offers a new dimension to mentoring but to realise its full potential CNEP mentoring requires clear mentoring guidelines, mentor, near peer mentor and mentee training, effective holistic and longitudinal assessment of mentoring processes and support , 98–111.


Lysyl oxidase (LOX) has been described as necessary for premetastatic niche formation in epithelium-derived malignancies and its expression level therefore correlates with risk of metastatic disease and overall survival. However, its role in acute myeloid leukemia (AML) has not been sufficiently analyzed.p-value method dichotomizing patients into a LOX-high group  and a LOX-low group .We investigated LOX plasma expression in 683 AML patients (age 17–60 years) treated within the prospective AML2003 trial (NCT00180102). The optimal cut-off LOX value was determined using a minimal-FLT3-ITD and NPM1 mutations at diagnosis. Higher LOX expression was found significantly more frequently in patients with secondary AML and therapy-related AML, in patients with French-American-British M5 subtypes, and in patients with adverse-risk cytogenetics. Comparing patients in the LOX-high group and the LOX-low group revealed a 3-year overall survival (OS) of 47 and 53% (p = 0.022) and 3-year event-free survival (EFS) of 27 and 35% (p = 0.005), respectively. In the LOX-high group significantly more patients had extramedullary AML compared to the LOX-low group (p = 0.037). Combining extramedullary AML and LOX as interacting factors in a multivariate analysis resulted in an independent impact on survival for the LOX-high-extramedullary interaction for OS  and EFS . Furthermore, in patients with extramedullary disease (n = 59) the LOX level predicted survival. Patients within the LOX-low group had an OS of 43% and EFS of 36% as compared to the LOX-high group with an OS of 13% and EFS of 6% .Higher LOX expression was associated with lower peripheral white blood cells, lower serum LDH, and a lower frequency of We hypothesize LOX expression to be a new potential biomarker to predict outcome in AML, specifically in AML subgroups such as the prognostic heterogeneous group of AML patients with extramedullary disease.NCT00180102). Patients were enrolled between December 2003 and November 2009.This retrospective study was performed with patient samples registered within the prospective AML2003 trial ( Within the last years various studies were able to demonstrate that in epithelium-derived cancers the occurrence of metastases is an orchestrated process involving hypoxia-induced mechanisms, neo-angiogenesis, recruitment of bone marrow-derived cells, and enzymatic deposition of extracellular matrices which result from certain genetic and epigenetic features of the underlying tumor cells themselves –4. The cInterestingly, these mechanisms have only been described for epithelial tumors so far. However, there is an increasing body of evidence that extramedullary manifestation of acute myeloid leukemia (AML) is often underreported at initial diagnosis and during relapse . Studiesnucleophosmin 1 (NPM1) mutated AML patients as well as mutated NPM1 in biopsied extramedullary sites in a high fraction of patients with extramedullary AML [NPM1 mutations Garzon et al. were able to demonstrate that a significant proportion of patients have an up-regulation of microRNA 10a (miR-10a) in their leukemic cells [While it is speculated that the true prevalence has yet to be defined, some reports have associated extramedullary AML with certain clinical and morphological features such as high white blood cell (WBC) count and French-American-British (FAB) M4/M5 subtypes –16 as welary AML , 21. In ic cells . While wic cells .LOX is a copper-dependent, extracellularly secreted amine-oxidase mapped on human chromosome 5q23 that has been extensively described to be involved in the initiation of a premetastatic niche in epithelium-derived malignancies and in bone marrow fibrosis as well as in the development of pediatric acute megakaryoblastic leukemia , 25. DawTo date, little is known about the clinical implication of LOX expression in AML. In this study we investigated the plasma LOX expression from AML patients included in the AML2003 trial at diagnosis and correlated the results with clinical features and outcome of these patients.All available peripheral blood lithium-heparin plasma samples of 683 patients with AML (age 17–60 years) were analyzed for LOX expression. Lithium-heparin plasma samples were collected at initial diagnosis whenever possible. All patients were treated within the prospective, randomized, and multicentric AML2003 trial (NCT00180102). The study had been approved by the institutional review board (IRB) of the University of Dresden and all IRBs of participating centers of the Study Alliance Leukemia (SAL). The protocol was in agreement with the Helsinki Declaration. Written informed consent was obtained from each patient before inclusion. The risk-adapted design and inclusion criteria of the AML2003 trial have been described previously . BrieflyFLT3-ITD and NPM1 mutations were performed as previously published in detail [Bone marrow samples were processed in reference laboratories of the SAL study group. Cytogenetic analyses were performed using standard G-banding techniques and karyotyping according to the International System for Human Cytogenetic Nomenclature . Fluoresn detail , 34. Extn detail . Data waNPM1 mutated AML cell line OCI/AML3 served as internal control and for analysis of a day-to-day variability. OCI/AML3 cells were purchased from the Leibnitz-Institute – German Collection of Microorganisms and Cell Cultures .Peripheral blood lithium-heparin plasma samples were analyzed for LOX concentration using an enzymatic assay  according to the manufacturer’s instructions . All flup-value method [n = 272, 40%) and a LOX-low group . Differences of continuous variables between both groups were analysed by means of the Mann-Whitney-U-Test. Differences of the distribution of categorical variables were analysed using the uncorrected Chi-Squared-Test. The method of Kaplan-Meier was used to estimate OS and EFS. Survival distributions were compared using the log-rank test. Prognostic parameters were tested in a Cox regression model for OS and EFS. Cytogenetic karyotype, FLT3-ITD mutation status, NPM1 mutation status, extramedullary AML, and LOX expression at diagnosis were used as dichotomized variables whereas age, and logarithmic (to base 10) WBC at diagnosis, and the bone marrow blast count at day 15 after induction chemotherapy were used as continuous variables. All statistical analyses were performed using SPSS version 19.0  and R 2.15.1.Overall survival (OS) and event-free survival (EFS) were measured from the date of entry into the study to the date of event or the last follow up. Death, induction failure, and relapse were considered events for EFS. Complete remission (CR) was defined according to standard criteria . The rane method . The resFor internal validation we first identified the OCI/AML3 cell line as representing a cell line with a high LOX expression in the supernatant. We performed independent supernatant day-to-day variability measurements in triplicate on each day on three consecutive days resulting in a mean LOX concentration of 792.5 ng/mL and a standard deviation of 101.9 ng/mL with a day-to-day variability of 13%.p < 0.001), lower WBC counts , and lower serum LDH  compared to those patients with low LOX expression, respectively. Furthermore, a significantly lower proportion of FLT3-ITD mutations  and NPM1 mutations  were detected. The LOX-high group consisted of significantly more patients with therapy-related AML (t-AML) (7% vs. 4%) and patients with a preceding MDS (mdsAML) (11% vs. 4%), p = 0.001, a higher proportion of monocytic morphology, more cytogenetically defined adverse-risk karyotypes (34% vs. 16%), and less normal karyotypes (58% vs. 70%) and favourable karyotypes (9% vs. 14%), p < 0.001. In detail, single cytogenetic abnormalities that were identified more frequently among the LOX-high group were complex karyotype  (p = 0.005), del(5q) (p = 0.007), t  (p = 0.002), and other abnormalities of chromosome 11q23 (abnl (11q23)) (p < 0.001). Abnormalities belonging to a favorable or undefined risk category that were frequently higher expressed in the LOX-high group were monosomies of chromosomes other than chromosome 5 or 7 (p = 0.004), and trisomy of chromosome 8 (p = 0.001). AML with normal karyotype or inversion of chromosome 16 were less frequently observed in the LOX-high group, p < 0.001 and p = 0.022, respectively. As hypothesized initially, we could confirm a significantly higher proportion of patients with histologically confirmed extramedullary AML as defined per WHO criteria in the LOX-high group, p = 0.037.As mentioned, a logarithmic logLOX value was identified which dichotomizes the analyzed 683 patients in a LOX-low group comprised of 411 patients (60%) and a LOX-high group with 272 patients (40%). Detailed patient characteristics are depicted in Table p = 0.022), and a 3-year EFS of 27% (95% CI 21–32%) and 35% , respectively , bone marrow blast count at day 15 after induction chemotherapy, extramedullary disease, and LOX expression. In the presence of these variables LOX expression lost its influence on OS and EFS. However, since extramedullary AML and LOX seem to be related on a biological level, we introduced an interaction term in the Cox model. We identified a robust impact of LOX expression and extramedullary manifestation on OS , p = 0.025) and EFS (HR = 2.48 (95% CI 1.26–4.86), p = 0.008)  and 53%  and LOX low patients (n = 28) were about equally distributed. The clinically and laboratory baseline characteristics of the 59 patients are depicted in Table n = 31) and LOX-low (n = 28) patients, only a higher proportion of t-AML and MDS-related AML (p = 0.049), higher numbers of monocytic AML according to FAB-subtype (p = 0.003), and more patients with cytogenetically defined high-risk abnormalities (p = 0.001) remained as statistically significant different in these groups in univariate analysis. Regarding single cytogenetic abnormalities we again observed a two-fold higher rate of complex karyotypes and trisomy 8 in the LOX-high cohort . For abnl (11q23) however, a more pronounced significant difference between LOX-high and LOX-low patients was observed despite of low patient numbers in this cohort , respectively. Furthermore, also in patients with extramedullary AML the LOX expression predicted survival. Patients within the LOX-low group had an OS of 43%  and EFS of 36%  as compared to the LOX-high group with an OS of 13%  and EFS of 6% , p = 0.002 and p = 0.008, respectively, in univariate analysis . Consequently, we suggest a considerable number of unknown EM AML patients currently still classified as “normal” AML without clinical apparent extramedullary manifestations at diagnosis for which LOX could also be a potential marker for prognosis.NPM1 mutations.The multivariate analyses revealed that this association is mainly caused by a higher incidence of adverse cytogenetic aberrations and myelodysplastic features and a lower incidence of favorable cytogenetics and WT1 expression by quantitative PCR which was reported to predict extramedullary AML. Patients with extramedullary relapse had high expression levels in the peripheral blood whereas the WT1 level in the bone marrow remained negative [AML1-MTG8 expression levels were shown to possibly predict EM relapse, specifically in t  AML [Furthermore, the statistically significant interaction between higher LOX expression and extramedullary AML suggests a potentially pathophysiological relevant mechanism involved in extramedullary homing and growth of AML and may offer further insights into AML biology. The prognostic heterogeneous group of AML patients with extramedullary disease can be separated in those with superior and those with inferior survival by applying the peripheral blood lithium-heparin plasma LOX expression level at diagnosis. This might be of importance for future applications in clinical as well as in scientific settings since so far, the survival of AML patients with extramedullary disease at diagnosis has been discussed controversially. Importantly, our data need to be confirmed in an independent validation patient cohort which might prove difficult since most large AML trials do not collect and bank plasma samples. Further experimental studies are needed to address the functional modalities of how LOX is regulated and how it contributes to migratory and adhesion properties in AML. Possible caveats for future studies addressing these questions might be that few AML trials include the systematic screening, documentation and analysis of extramedullary AML - a phenomenon that already led to an underreporting of extramedullary AML after allogeneic HSCT . Even monegative . Further;21) AML .Further indicators linking LOX to extramedullary manifestation of AML in our report is certain characteristics known to be more common in extramedullary AML were more frequently observed in the LOX-high group, i.e. higher numbers of patients with monocytic morphology and certain karyotypic abnormalities that have been associated with extramedullary AML such as e.g. complex aberrant karyotypes, trisomy of chromosome 8, or 11q23 abnormalities amongst others , 45, 46.However, without the existence of a homogenous tumor population within one individual it was speculated by Fidler et al. that in epithelial cancers rare subpopulations of malignant clones exist which are endowed with some or several “metastasis-promoting functions” . FurtherWhile the three-dimensional structure of the human LOX protein has not been resolved so far, it furthermore needs to be explored which isoforms of LOX are relevant in the context of AML and whether its regulation through HIFs in hypoxia, its ability to recruit CD11b myeloid cells which adhere to crosslinked collagen and produce matrix-metalloproteinase-2 enhancing premetastatic niche formation is of relevance in AML. Also the ability of LOX to increase vascular endothelial growth factor (VEGF) expression and secretion which promotes angiogenesis needs to be reflected in this context . FinallyThe correlation between high LOX expression and extramedullary AML suggests a potentially relevant mechanism involved in extramedullary adhesion, migration, and growth of AML and may offer further insights into AML homing biology and therapeutic targeting options. However, one must speculate that due to the lack of routine screening for extramedullary AML at diagnosis, there might be a considerable amount of unknown extramedullary AML at diagnosis, especially in cases without clinical apparent extramedullary manifestations, for which LOX could also be a potential marker for prognosis. A subsequent approach in order to modify or even selectively inhibit LOX might be reasonable for an attempt to improve the clinically inferior and still dissatisfying outcome of these patients.


To evaluate the diagnostic efficacy of MDA-MB-231 triple-negative breast cancer with 125I]I-pHLIP (Var7) and MDA-MB-231 cells was measured at pH 7.4 and pH 6.0, and tumor-bearing mice were subjected to small-animal SPECT/CT imaging studies.  The binding fraction of [125I]I-pHLIP (Var7) and MDA-MB-231 cells at 10 min, 40 min, 1 h, and 2 h were 1.9 ± 0.1%, 3.5 ± 0.1%, 6.3 ± 0.8%, and 6.6 ± 0.3%, respectively. At pH = 7.4, there was no measured binding between [125I]I-pHLIP (Var7) and MDA-MB-231 cells. Small-animal SPECT/CT imaging showed clearly visible tumors at 1 and 2 h after injection.  At pH = 6.0, the binding fractions of [125I]I-pHLIP (Var7) could bind to MDA-MB-231 cells in an acidic environment, and small-animal SPECT/CT imaging showed clear tumors at 1 and 2 h after probe injection. [ In solid malignancies, tumor cells grow in the tumor microenvironment (TME), the pH of which is acidic and can be as low as 6.0 –5. Becau125I to achieve single-photon emission computed tomography/computed tomography (SPECT/CT) imaging in an MDA-MB-231 triple-negative breast cancer mouse model. The labeling of pHLIP with 125I was used to investigate the following: (1) its usefulness for the SPECT imaging of tumor-bearing mice, tumor diagnosis, and therapeutic efficacy monitoring and as a preliminary preparation for positron emission tomography (PET) imaging performed with 124I-labeled probes. (2) The potential of using 131I-labeled probes for tumor therapy was evaluated by analyzing the distribution of radioactivity in tumor-bearing mice and the imaging results.One study  successfThe main instruments used in this study included a gamma counter , a radiopharmaceutical dose calibrator , and a small-animal SPECT/CT imaging system .125I] NaI , chloramine-T , sodium metabisulfite , and a C18 column .The main reagents used in this study included  NaI (38.48 MBq) solution were thoroughly mixed, 20 μL of 5 mg/mL chloramine-T solution was added, and the mixture was allowed to react at room temperature for 50 s. Next, 150 μL of sodium metabisulfite solution (5 mg/mL) was added to extend the reaction for 5 min. The solution was passed through the activated C18 extraction column and rinsed first with distilled water and then with ethanol. The solution was blown with nitrogen to obtain a near-dry solution and then diluted with PBS buffer to obtain [125I]I-pHLIP (Var7). The radiochemical yield was then calculated.pHLIP (Var7) was labeled with T method . One hun125I]I-pHLIP (Var7) was determined by paper chromatography. Normal saline was used as the developing agent, and Xinhua Grade 1 chromatography paper was used as the stationary phase. An appropriate amount of [125I]I-pHLIP (Var7) eluent was aspirated by a capillary pipette and spotted at the origin of the chromatographic filter paper. After drying, the paper was placed vertically into a test tube containing the developing agent; the unfolded chromatographic filter paper was removed, air dried, and cut at intervals of 1 cm. The radioactivity count was measured segment by segment to calculate the radiochemical purity.The purity of [125I]I-pHLIP (Var7) was placed at 4°C, at room temperature (25°C) in PBS buffer, and at 37°C in mouse serum. The radiochemical purity at different time points was measured, and the stability was determined. 125I]I-pHLIP (Var7) solution was placed at 4°C and at room temperature in PBS buffer. Radiochemical purity was determined immediately after purification and at 1, 2, 4, 8, and 24 h after purificationTwo hundred microliters of purified [125I]I-pHLIP (Var7) solution was mixed with 100 μL of mouse serum and placed in a 37°C water bath. Radiochemical purity was determined immediately after mixing and at 1, 2, 4, 8, and 24 h after mixingFifty microliters of purified I-pHLIP (Var7) (20 μL volume) was added to each well. The pH inside the wells was adjusted to 7.4 and 6.0, yielding two groups of cells according to the pH value.2, the supernatant was aspirated into a tube and washed with PBS of the corresponding pH (pH 7.4 and 6.0) twice. The wash solution was also added to the supernatant tube. The cells were completely digested with trypsin, after which the cell suspension was aspirated into a tube and washed twice with PBS; the wash solution was also added to the tube. The radioactive counts of the cell tube (B) and supernatant tube (F) were measured by a γ counter. The cell binding fraction of the marker was calculated as B/(B + F) × 100%After 10 min, 40 min, 1 h, and 2 h of culture without CO6 cells/mL, which were temporarily stored in an ice box. Female 4-week-old BALB/c nude mice (16-20 g) were selected. MDA-MB-231 cells were inoculated subcutaneously using a disposable insulin syringe inserted into the right lateral thighs of the nude mice. The number of inoculated cells in each nude mouse was approximately 0.1 mL. The nude mice were raised under specific pathogen-free (SPF) conditions.A mouse model bearing a human breast cancer cell line, MDA-MB-231, was established. MDA-MB-231 cells were cultured using the conventional method. Cells were collected at the logarithmic growth phase and washed once with PBS. The cells were then diluted to prepare suspensions at a concentration of approximately 1 × 10125I]I-pHLIP (Var7) was diluted with PBS, and a total of 15 tumor-bearing nude mice with no significant differences in tumor size were randomly divided into five cohorts. Nude mice in each cohort were injected with [125I]I-pHLIP (Var7) at approximately 1480 kBq/200 μL through the tail vein. At 0.5, 1, 2, 4, and 8 h after injection, blood was collected from the orbital sinus. The mice were sacrificed, and tissues and organs, including the heart, liver, spleen, lung, kidney, pancreas, stomach, small intestine, bladder, thyroid, brain, bone, muscle, and tumors, were collected and weighed. The radioactive count was measured, and the results were converted to %ID/g.Biodistribution experiments were conducted when the tumor diameter was 0.8-1.0 cm. Purified [The machine was calibrated dailyAnesthesia Induction. The tumor-bearing mice were anesthetized with isoflurane.μL of [125I]I-pHLIP (Var7) was injected into the tail veinWhen the righting reflex of the tumor-bearing mice disappeared, 3.7 MBq/200 125I]I-pHLIP (Var7) injection, scanning was performed at 1, 2, 4, 8, and 24 h using static 10 min SPECT and medium-resolution whole-body CT. Imaging settings for the SPECT/CT and reconstruction are as follows: the time per frame was 10 min, voxel size was 0.8 mm, number of subsets was 32, and number of iterations was 15After 3H+)  and at least two smaller products at retention times of 14.5 and 15.0 min . The mas 3H]3H+) . The exp125I]I-pHLIP (Var7) was 345 ± 25 MBq/μmol.The radiochemical yield was 30.3 ± 2.2%, and the radiochemical purity was 98.6 ± 1.3%. The molar activity of [125I]I-pHLIP (Var7) had a relatively high stability at 4°C and at room temperature in PBS buffer and was still higher than 98% at 24 h. The probe stabilities at 37°C in serum for 1, 2, 4, 8, and 24 h were 97.8 ± 1.9%, 96.8 ± 1.0%, 94.5 ± 1.1%, 91.3 ± 2.6%, and 85.6 ± 2.9%, respectively ([ectively .125I]I-pHLIP (Var7) and MDA-MB-231 cells at 10 min, 40 min, 1 h, and 2 h were 1.9 ± 0.1%, 3.5 ± 0.1%, 6.3 ± 0.8%, and 6.6 ± 0.3%, respectively. At pH = 7.4, the binding fractions of [125I]I-pHLIP (Var7) and MDA-MB-231 cells at 10 min, 40 min, 1 h, and 2 h were 0.9 ± 0.1%, 0.8 ± 0.1%, 0.8 ± 0.1%, and 0.6 ± 0.1%, respectively. At pH = 6.0, the cell binding fraction at each time point was significantly higher than that at pH = 7.4 (P < 0.001)  .125I]I-pHLIP (Var7) in the tumors was highest (6.9 ± 0.4%ID/g) at 1 h after injection (P < 0.01) and then decreased with time, which were 3.6 ± 0.7, 2.0 ± 0.6, and 1.0 ± 0.4%ID/g at 2, 4, and 8 h, respectively. The distribution in the blood was 12.0 ± 1.5%ID/g at 0.5 h and decreased rapidly with time, which were 6.5 ± 1.5, 2.8 ± 0.6, 1.2 ± 0.5, and 0.5 ± 0.1%ID/g at 1, 2, 4, and 8 h, respectively. In addition, the distribution in the liver was relatively high, which were 13.9 ± 2.7, 8.0 ± 1.9, 4.0 ± 0.6, 1.8 ± 0.2, and 0.7 ± 0.2%ID/g at 0.5, 1, 2, 4, and 8 h, respectively (The distribution of [ectively . The radectively .125I]I-pHLIP (Var7); the imaging result at 1 h was better than that at 2 h, and liver imaging was pronounced  pHLIP can connect the imaging agent to the cell surface and be used for tumor diagnostic imaging. Some studies , 14–16 p125I-labeled by the chloramine-T method, and the labeling efficiency and radiochemical purity meet experimental requirements. Iodine binds to the tyrosine residue, which resides in the middle of the pHLIP (Var7) transmembrane domain and has the potential to affect insertion and adoption of the alpha-helical structure. However, ex vivo and in vivo results showed that this was not an issue. [125I]I-pHLIP (Var7) had a relatively high stability at 4°C and 25°C in PBS buffer and was still higher than 98% at 24 h, and the stability at 37°C in serum for 24 h was greater than 85%, which basically satisfied the imaging requirement. The relatively low stability at 37°C in serum may be related to deiodination activity or decomposition of the markers.Four sequences in the pHLIP peptide family have enhanced tumor-targeting properties: wild type (WT), variant 3 (Var3), variant 7 (Var7), and ATRAM . Var7 is125I]I-pHLIP (Var7) at pH = 6.0 had a significantly higher binding fraction with MDA-MB-231 cells than that at pH = 7.4. Under the condition of pH = 6.0, the binding fraction of [125I]I-pHLIP (Var7) and MDA-MB-231 cells increased with time, rapidly increasing to approximately 6.3% at 1 h and stabilizing at approximately 6.6% at 2 h. At pH = 7.4, [125I]I-pHLIP (Var7) and MDA-MB-231 essentially did not bind. This study suggests that [125I]I-pHLIP (Var7) can target MDA-MB-231 cells in an acidic environment.[125I]I-pHLIP (Var7) showed the highest uptake value (6.9 ± 0.4%ID/g) in tumors at 1 h after injection into the body and then began to decrease. In addition, [125I]I-pHLIP (Var7) had a relatively high uptake in the thyroid, stomach, liver, and blood. [125I]I-pHLIP (Var7) showed some retention in the thyroid gland, suggesting that deiodination may occur in the body. Therefore, the thyroid gland must be blocked before imaging MDA-MB-231 tumor-bearing mice. Josefsson et al. I-pHLIP (Var7) in the stomach of experimental animals may reflect iodine or iodine-labeled amino acid fragments detached from the markers. The radioactive distribution of the probe in the liver was significantly higher than that in the kidney, suggesting that the probe was mainly metabolized by the liver. The probe was mainly composed of nonpolar amino acids (13 nonpolar amino acids and 11 polar amino acids) with relatively poor water solubility, which is not conducive to excretion by the urinary system. The time-radioactivity relationship of [125I]I-pHLIP (Var7) in the mouse models revealed rapid blood clearance, with 12.0 ± 1.5%ID/g at 0.5 h after injection of 1480 kBq [125I]I-pHLIP (Var7), 1.2 ± 0.5%ID/g at 4 h, and 0.5 ± 0.1%ID/g at 8 h postinjection. The results of SPECT/CT imaging in small animals basically showed the same biodistribution trend, as liver uptake was the highest among all organs analyzed, and tumor uptake at 1 h was relatively high.I-pHLIP (Var7) could bind to MDA-MB-231 cells in an acidic environment. The biodistribution results showed that the probe had the highest radioactive distribution in the tumor at 1 h after injection. Tumor images were clearly observed in small animals by SPECT at 1 and 2 h after probe injection.This study found that


Structures of sterols 1–8 were determined from 1D and 2D NMR data. Among the sterols, 8α-hydroxytheonellasterol (4) spontaneously underwent an allylic 1,3-hydroxyl shift to produce 15α-hydroxytheonellasterol (9) as an artifact; this was rationalized by quantum mechanical calculations of the transition state. In addition, the 1,2-epoxy alcohol subunit of 8α-hydroxy-14,15-β-epoxytheonellasterol (5) was assigned using the Gauge-Independent Atomic Orbital (GIAO) NMR chemical shift calculations and subsequent DP4+ analysis. Finally, comparison of the 13C chemical shifts of isolated 7α-hydroxytheonellasterol (6) with the reported values revealed significant discrepancies at C-6, C-7, C-8, and C-14, leading to reassignment of the C-7 stereochemistry in the known structure.A total of eight new oxygenated 4- Theonella swinhoei has been one of the most diverse sources of natural products. Since the isolation of theonellasterol A from T. swinhoei in 1981 + .Theonellasterol-5,8-oxide (β-Hydroxytheonellasterol (2): colorless oil; + . 8β-Hydroxytheonellasterol (3): colorless oil; + .15α-Hydroxytheonellasterol (4): colorless oil; 1H NMR and 13C NMR, see 8α-Hydroxy-14,15-β-epoxy-theonellasterol (5): amorphous powder; + .8α-Hydroxytheonellasterol (6): colorless needle-shaped solid; + .7β-Hydroxy-7α-formyl-B-northeonellasterol (7): colorless oil; + .88): yellow oil; + .28-Homoswinhoeisterol (α-Hydroxytheonellasterol (9): colorless oil; + .1510): white powder; + .Theonellastrol-3,7-dimethyl ether , computed with the same level of theory . The conformers within an energy threshold of 5 kJ/mol were optimized employing DFT calculations at the mPW91PW1/6-3lG* level of theory to estimate gas phase energies and Gibbs free energies. All of the optimizations were performed at “fine” grid density and “ultrafine” accuracy level. The structure that has the lowest gas phase energy was selected, and NMR shielding constants were calculated with the mPW91PW1/6-3lG**/CPCM benzene basis set. The calculated 1–3, 5–9) and six known sterols (11–16) at various concentrations for 3 h, and then were incubated with or without 5 ng/mL of lipopolysaccharide . After 24 h of incubation, the supernatant was collected and subjected to enzyme-linked immunosorbent assay (ELISA). The levels of IL-6 were measured by using mouse IL-6 Quantikine ELISA kit  according to the manufacturer’s instructions. The murine macrophage RAW264.7 cells were obtained from Dong Hyun Sohn, Pusan National University, Yangsan, South Korea. The RAW264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. For determining interleukin-6 (IL-6) production, RAW264.7 cells were pre-treated with eight new sterols (exo-methylene sterols 1–8 and six known sterols (11–16) were isolated from T. swinhoei. The C-7 stereochemistry of the reported 7α-hydroxytheonellasterol has been revised based on the outcome of a series of chemical modifications and the X-ray crystallography data of 6. The stereo and regiochemistry of the 1,2-epoxyalcohol moiety in 8α-hydroxy-14,15-β-epoxy-theonellasterol (5) was determined by GIAO chemical shift calculations. The reaction pathway for the 1,3-hydroxyl migration of 4 was calculated using quantum mechanical calculations to explain the observed reaction spontaneity. In addition, the unusual downfield shifts observed for H-5 in compounds 4 and 5 were rationalized through geometry optimizations, which indicated the presence of an 8α-hydroxyl group in 6/6/6/5-fused cyclic sterols.A total of eight new oxygenated 4-


Compared to Control, HG+PA treatment significantly reduced field stimulation-induced calcium transient amplitudes  and the levels of caffeine-induced calcium transients , however significantly increased spontaneous Ca2+ sparks firing levels in single cardiomyocytes  and ROS production , which suggest that HG+PA treatment increases the Spontaneity Ca2+ spark frequency, and then induced partial reduction of SR Ca2+ content and subsequently weaken systolic Ca2+ transient in cardiomyocyte. Remarkably, these impairments in calcium signaling and ROS production were largely prevented by pre-treatment of the cells with CaD. Therefore, CaD may contribute to a good protective effect on patients with calcium mishandling and contractile dysfunction in cardiomyocytes associated with diabetic cardiomyopathy.Calcium dobesilate (CaD) is used effectively in patients with diabetic microvascular disorder, retinopathy, and nephropathy. Here we sought to determine whether it has an effect on cardiomyocytes calcium mishandling that is characteristic of diabetic cardiomyopathy. Cardiomyocytes were sterile isolated and cultured from 1 to 3 days neonatal rats and treated with vehicle (Control), 25 mM glucose+300 μM Palmitic acid (HG+PA), 100 μM CaD (CaD), or HG+PA+CaD to test the effects on calcium signaling (Ca The worldwide prevalence of diabetes mellitus increases markedly to cause various cardiovascular complications, which are the major cause of morbidity and mortality in this population , 2. Amon2+ plays an essential role in cardiac EC-coupling. Ca2+ major stores in sarcoplasmic reticulum (SR) in the mammalian heart. At systole, instantaneous high concentration of Ca2+ is released from SR trigged by Ca2+ influx trough L-type Ca2+ channel, consequently Ca2+ transient that triggers myofilaments contraction in heart  is a vascular protective agent, highly effective in treating patients with various diabetes-associated microvascular disorders, retinopathy, nephropathy, and cataract –18. CaD Sprague–Dawley rats of 1–3-days-old were purchased from the Animal center of Guangdong Experimental Animal Center and handled according to a protocol approved by the Institutional Care and Use Committee of Shenzhen University, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . NRVMs were isolated and cultured as previously described  in a recording chamber. Confocal line-scan imaging was carried out in resting cells at 488 nm excitation and 505 nm collection with a Zeiss 780 inverted confocal microscope (Carl Zeiss) with a 40x oil immersion lens for detecting Ca2+ sparks as described previously  for 10 min at 37°C and then perfused with Tyrode's solution  remove the dye and complete de-esterification. After the cardiomyocytes reached a steady-state with field stimulation (1 Hz), line-scan imaging was acquired along the longitudinal axis of the cells.2+ loading. SR Ca2+ content was assessed by detecting the amplitude of the Ca2+ transient. Cells were superfused with normal external solution.Short puffs of caffeine (20 mM) were applied to completely empty the SR, after a train of 1-Hz field stimulation to achieve steady-state SR CaCells were loaded with the probe 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate  for 10 min at 37°C before the experiment. The 2′,7′-dichlorodihydrofluorescein (DCF) fluorescent intensity was measured by using LSM 780 (Zeiss) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm .t-test between 2 groups was performed, one-way analysis of variance (ANOVA) followed by the Dunnett's post-hoc test was used for comparisons among four groups. In all tests, values of p < 0.05 were considered be statistically significant.All values are given as mean ± SE. Unpaired Student's 2+ sparks in NRVMs. HG+PA increased the frequency of Ca2+ sparks by 1.54-fold    . In addim (FDHM) . Remarkaand FWHM . These r2+ content affects the frequency and amplitude of Ca2+ sparks in cardiomyocytes; the higher of SR Ca2+ content results in the higher frequency of spontaneous Ca2+sparks (2+ transient (ΔF/F0) indicating SR Ca2+ content. High concentration of Caffeine (20 mM) was used to stimulate the change of calcium storage capacity in each group. We found that HG+PA treatment significantly decreased SR Ca2+ content , however, ameliorated HG+PA–induced impairment of SR Ca2+ release  have been well-known, we study for first-time documents that CaD protects cardiomyocytes from HG+PA induced injury and Ca2+ mishandling.In this study, we found that exposure of neonatal rat ventricular cardiomyocytes to high glucose and high lipid (HG+PA) results in a marked increase in the intracellular ROS and CaOur findings have a high translational value for the treatment of diabetic cardiomyopathy. Since the 1970's, CaD has been front-line medication for the prevention and treatment of diabetic retinopathy with an excellent safety profile. Our data suggest that it may have a direct protective effect on cardiomyocytes in diabetes and that its use may be extended to diabetic heart disease. This is particularly important because currently there is no drug to specifically target high-glucose and high-fat induced cardiomyocyte injury. Notably, our results indicate that CaD is effective after HG+PA treatment and does not affect cardiomyocyte function at baseline. Therefore, it is probably safe and consistent with its safety profile in the clinic.2+ spark firing frequency in cardiomyocytes, and that inhibition of RAGE significantly reduces the calcium spark frequency and protects heart function (Diabetes mellitus is a metabolic disorder characterized by abnormal glucose and lipid metabolism. Hyperglycemia and high fat can cause cardiac damage, leading to diabetic cardiomyopathy , 4, 25. function . Additiofunction , 28.2+ sparks in the cardiomyocytes (2+ leak has been suggested to be a potential cause of reduction of SR Ca2+ content (2+ concentration determines cardiac contractility. In rodent heart, Ca2+ influx through LCC accounts for no more than 30% while SR Ca2+ release contributes to over 70% of Ca2+ transient in systolic (2+ content, which is consistent with the alteration of the magnitude of the systolic Ca2+ transient. Our finding indicates that HG+PA decreases cardiac contractile function, which may be an important mechanism for cardiac dysfunction in diabetic cardiomyopathy.Recently, a series of studies found that SR calcium leakage increases in the cardiomyocytes of heart failure patients and contributes to the pathogenesis of heart failure , 30. Notmyocytes , 32. Enh content , and thesystolic . Our datin vitro and in vivo (CaD, as an oxygen free radical scavenger, have been shown inhibits free radical production both  in vivo –37. In tin vivo diabetic cardiomyopathy models. Thus, cautions should be taken when translating these findings into potential clinic use of the drug. In addition, the molecular targets of CaD that mediates its effects on ROS reduction and consequent Ca2+ signaling in cardiomyocytes are yet to be identified, which remains a subject of our future studies.One limitation of this study is that our experimental system was primarily based on the cultured neonatal rat cardiomyocytes, which may have electrophysiological features different from the adult cells or the In conclusion, CaD protects cardiomyocytes from HG and PA-induced elevation in oxidative stress and spontaneous spark frequency and restores sarcoplasmic reticulum calcium levels. Thus, it may be utilized for the treatment of cardiac contractile dysfunction in diabetic cardiomyopathy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Laboratory Animal Science Research Committee of Shenzhen University Health Science Center.JD and DY conceptualized the study and contributed to resources. XC, ZC, MH, YL, HF, HC, HL, and JL collected data. JD and GQ wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.