Bleeding with resultant hemophilic arthropathy constitutes the largest cause of morbidity in patients with hemophilia. It results from repeated bleeding episodes in the joint and is characterized by synovial hypertrophy and cartilage and bony destruction. Hemophilic arthropathy assessment is a continually evolving process and is particularly challenging in children and young adults in whom joint disease may be missed or underestimated as obtaining serial “baseline” magnetic resonance imaging scans of multiple clinically asymptomatic or nearly asymptomatic joints may be unjustifiable and cost-ineffective. Musculoskeletal ultrasound—particularly, point-of-care musculoskeletal ultrasound—has emerged as a promising imaging modality for the early detection and management of hemophilic arthropathy, and for the evaluation of hemarthrosis and painful musculoskeletal episodes in patients with hemophilia. This review summarizes currently available data on the emerging role of this new imaging modality, its limitations, and gaps in knowledge. The review also raises unanswered questions, highlights the need for consolidated research efforts, and delineates future directions expected to advance this technology and optimize its use in this patient population. The musculoskeletal consequences of hemophilia constitute the largest cause of morbidity in persons with hemophilia (PwH)2; musculoskeletal disease may be minimized by early initiation of prophylaxis yet has continued to occur9, and a single episode of hemarthrosis can impact long-term joint outcomes11. There is evidence that the key processes involved in the pathogenesis of HA include intra-articular inflammation, mediated by hemoglobin release, iron deposition, cytokines, hydroxyl radicals, and the “inflammasome”, and neo-angiogenesis, mediated by pro-angiogenic factors such as vascular endothelial growth factor13. However, the pathophysiology of HA remains incompletely understood, and the pathways are yet to be fully elucidated. HA remains a critical focus of research and study as this debilitating disease negatively impacts quality of life, physical activity, and bone health of the aging PwH population, whose life span is now comparable to that of the normal population16. A gap continues to exist in our ability to detect and impact this sequela at the preclinical or asymptomatic phase when the disease process is early and potentially reversible.Hemophilia A and B are X-linked congenital bleeding disorders characterized by reduced or absent levels of coagulation factors VIII or IX, respectively. The disease is classified, on the basis of residual coagulation factor activity level, as severe (<1%), moderate (1–5%), or mild (5–40%). Spontaneous hemarthrosis is the hallmark of severe hemophilia, and hemophilic arthropathy (HA), characterized by synovial hypertrophy and cartilage and bony destruction, which ultimately results from repeated joint bleeding episodes19, and a number of imaging modalities21. X-ray grading systems/scores include the Arnold–Hilgartner score 22 and the World Federation of Hemophilia–recommended and widely used Pettersson score 23. X-rays are widely available and may capture advanced joint changes but are insensitive to early change and unreliable for cartilage and soft tissue evaluation1. These limitations necessitated the development of a number of HA scoring systems using joint magnetic resonance imaging (MRI)26. The most widely used, developed in 2012 by the International Prophylactic Study Group (IPSG), merges two scales of a combined MRI scoring scheme into a single scale27. This scale, which includes soft tissue and osteochondral subscores, proved useful for the analysis of both early and moderate stages of arthropathy, comparison among different studies and patient populations, and the evaluation of prophylactic factor infusion regimens. To date, joint MRI remains the gold standard for the evaluation of HA, providing a comprehensive multi-tissue evaluation with sensitivity to detect early joint pathology and changes over time29. However, MRI is time-consuming, expensive, not consistently readily accessible, and may require sedation in young children. MRI also detects a large number of minor changes over time, not all of which are necessarily clinically meaningful, and their impact on joint outcomes has yet to be determined30. Therefore, obtaining serial “baseline” MRI scans of multiple clinically asymptomatic or nearly asymptomatic joints of children and young adults, may be unjustifiable and rather cost-ineffective. Conversely, if joint evaluation is restricted to clinically manifest joint disease, early arthropathic changes in this patient population will be missed without consistent sensitive monitoring.Evaluation of HA is a dynamic, multi-dimensional process which employs clinical outcome assessment tools, including the World Federation of Hemophilia Physical Examination Score (Gilbert score), the hemophilia joint health score (HJHS)28—that musculoskeletal ultrasound (MSKUS), particularly point-of-care MSKUS (POC-MSKUS), has emerged as a promising imaging modality for the early detection and management of HA36 and for the evaluation of hemarthrosis and painful musculoskeletal episodes in PwH40. MSKUS can provide a detailed and dynamic assessment of synovial joints and periarticular structures, including tendons, ligaments, and muscles, using high-resolution, high-frequency transducers (3.5–15 MHz) with power Doppler (PD) imaging. As this imaging modality gained attention, so did the debate and controversy about its reliability, diagnostic accuracy, and ultimately impact on joint outcomes in PwH42, but an important distinction to make prior to critically reviewing the role of MSKUS in the evaluation of HA is that between full-joint MSKUS and POC-MSKUS. Full-joint MSKUS is a 360° assessment of the joint, using all standard scanning planes, and is performed by expert radiologists and ultrasonographers. POC-MSKUS is a focused evaluation of the joint, using limited scanning planes or an abbreviated scanning protocol, and is usually carried out by trained clinicians at the patient’s bedside to answer specific yes–no questions about the status of the joint.It is not surprising—given its accessibility, wide availability, safety, efficiency, low cost of examination, and lack of interference of susceptibility artifacts on gradient-echo MRI sequences in joints with hemosiderin deposition, including the “blooming” effect43, making it useful in determining the etiology of acute painful musculoskeletal episodes in PwH as arthritis-mediated or bleed-related38. Radiographic biomarkers of HA include both soft tissue changes and osteochondral abnormalities, both of which can be detected with variable accuracy by MSKUS45. In 2014, in an observational descriptive study, Sierra Aisaet al. showed that MSKUS is useful in detecting joint bleeds, synovial hyperplasia, and joint erosions, and the results were comparable to those of joint MRI, but MRI better detected bone cysts and cartilage loss42. Similarly, in 2015, Doriaet al. conducted a cross-sectional study to assess the reliability of interpretation of MSKUS findings according to data blinding in maturing hemophilic joints (knees and ankles) and to determine the diagnostic accuracy of MSKUS compared with MRI for assessing joint components41. The authors concluded that, if performed by experienced radiologists using a standardized protocol, MSKUS is highly reliable to assess soft tissue abnormalities  in ankles and knees, and substantially to highly reliable to assess osteochondral changes in these joints, with high sensitivity to diagnose erosions and cartilage loss, regardless of the severity of arthropathy, and poor sensitivity and negative predictive values for depicting subchondral cysts in advanced disease.Compared with conventional MRI in the evaluation of hemarthrosis, MSKUS was found to be extremely sensitive in detecting low concentrations of intra-articular blood (as low as 5%) and in discriminating between bloody and non-bloody (serous/simple) fluid46. PD imaging is valuable as vascular remodeling and angiogenesis are known to contribute significantly to bleed propagation and development of HA as shown in human and mouse studies50. There is evidence that increased PD signals at baseline correlate with acute pain and more bleeding episodes and that PD signals significantly increase at the time of acute bleeding38.Unique to MSKUS, PD imaging allows the evaluation of synovitis by measuring synovial thickness and hypervascularity, which correlates well with the significantly more expensive dynamic contrast–enhanced MRI in hemophilic jointset al. in 201751. The review included 20 articles investigating either the diagnostic accuracy of MSKUS and/or MSKUS scanning protocols and scoring systems for the assessment of HA. Two independent reviewers evaluated 14 retrospective cross-sectional studies examining the diagnostic accuracy of MSKUS in the assessment of HA in children and adults58, both for reporting quality using the Standards for Reporting of Diagnostic Accuracy (STARD) tool and for methodological quality using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool. With STARD, 1 out of 14 and 8 out of 14 studies were scored to be of high and moderate reporting quality, respectively. Assessment with QUADAS-2 reported 2 out of 14 and 6 out of 14 studies as having high and moderate methodological quality, respectively. The remainder of the studies were scored as low or very low. These studies were also assessed for outcome measures from three clinimetric constructs  as follows: (1) clinical joint assessment tools: Gilbert and IPSG scores59; (2) radiographic scores: Arnold and Hilgartner22 and Pettersson23; and (3) MSKUS measures: Muça-Perjaet al. progressive score31 and five other additive scores by Klukowskaet al.53, Querolet al.60, Melchiorreet al.10, Martinoliet al.45, and Doriaet al.41.The most comprehensive systematic review and semi-quantitative assessment of currently available evidence on the value of MSKUS in the assessment and management of HA in children and adults with hemophilia and von Willebrand disease were provided by Ligocki60 were included in the review and the authors divided the parameters of the ultrasound scoring systems into soft tissue changes  and osteochondral abnormalities . The only parameter proposed in all scoring systems was synovial hypertrophy.Six studies that described MSKUS scoring systems63. With the exception of Martinoli’s POC protocol64, PD evaluation of soft tissue vascularity was suggested by all remaining full-joint diagnostic protocols. The only other POC protocol evaluated in this review was that of Ceponiset al.38. The protocols of both Martinoliet al. and Ceponiset al. aim to simplify the scanning technique for elbows, ankles, and knees so they can be applied by clinical providers at the bedside without requiring radiologists or radiology technicians. Items included in a number of MSKUS scanning protocols and scoring systems used in the assessment of HA are shown in33. The score proposed by Doriaet al.41, not shown in the table but included in the systematic review, is an ultrasound-based imaging scale with an additive score (0–14) that was adjusted to the subscores of the IPSG MRI scale (0–17)27. To enable correspondence between ultrasound and IPSG MRI scores, items that are not technically feasible by ultrasound, including surface erosions, subchondral cysts, and full-thickness loss of joint cartilage in at least half of the joint surface were excluded65.Five grey-scale ultrasound scanning protocols were included in this review51 concluded that there is currently fair evidence (grade B) to recommend MSKUS as an accurate technique for the early diagnosis of HA , to demonstrate that MSKUS scores correlate with clinical/ultrasound constructs, and to prove an association between MSKUS findings and functional joint status. However, there is still insufficient evidence (grade I) to conclude that MSKUS-detectable findings are responsive or sensitive to changes in prophylactic therapy in children or adults with HA. The evidence was insufficient in both quality and quantity.The authors of this very valuable systematic review67. One group, Ceponiset al., demonstrated that hemarthrosis was present in only one third of acute painful joints38 and that disappointingly about two thirds of painful musculoskeletal episodes were judged incorrectly by either the patient or physician. POC-MSKUS findings changed the management in more than 70% of episodes, which resulted in symptom improvement in more than two thirds of the cases. Wider use of POC-MSKUS may serve to substantiate these data. Furthermore, with appropriate training, POC-MSKUS has been increasingly used as part of the bedside evaluation of muscle hematomas in PwH69 and for ultrasound-guided joint aspiration and intra-articular corticosteroid injection for symptomatic relief and pain control in PwH and chronic joint pain secondary to advanced HA71. A very recent article by De la Corte-Rodríguezet al. showed that arthrocentesis carried out immediately after the diagnosis of acute hemarthrosis in strictly aseptic conditions and under hemostatic coverage was well tolerated and accelerated joint recovery72. Lastly, Rezendeet al. demonstrated that joint lavage followed by injections of triamcinolone and Hylan G-F 20 (viscosupplement) improved balance, function, and bleeding events in patients with severe HA73, and Liet al. suggest that a single intra-articular platelet-rich plasma injection results in better improvement in pain relief, joint function, and reduced synovial hyperemia when compared with five weekly intra-articular injections of hyaluronic acid in patients with HA of the knee74.Over the last decade, POC-MSKUS has emerged as a critical tool in the evaluation of hemarthrosis and painful musculoskeletal episodes in PwHet al.51, the “Joint Activity and Damage Exam” (JADE) POC scanning and scoring protocol has been developed and validated by Vollandet al.75 as the first quantitative POC-MSKUS algorithm for precise measurements in hemophilic joints of adult PwH. The protocol evaluates for effusions and measures osteochondral surface defects, cartilage thickness, soft tissue expansion, and microvascular perfusion abnormalities on PD for ankles, elbows, and knees. Validation studies showed high intra-/inter-rater reliability, which the authors hope would allow easy quantification of the progression of HA. To date, a similar quantitative POC-MSKUS protocol for children with hemophilia has not been proposed, and the semi-quantitative Hemophilia Early Arthropathy Detection with UltraSound (HEAD-US) POC protocol of Martinoliet al.64 remains the most widely used in this patient population for the early detection of HA76. The protocol has an additive score to a maximum score of 8 for synovial hypertrophy and cartilage and bony changes. Joint effusions are excluded from the HEAD-US score, and PD imaging is not carried out as part of the scanning protocol; validation studies of the additive HEAD-US score have not yet been published. Although the protocol has its limitations, its few to no measurements, short learning curve, and proposed examination times of less than 2 to 5 minutes per joint are attractive to busy clinicians and physical therapists in the outpatient setting. The authors propose that a six-joint baseline scan of bilateral elbows, knees, and ankles can be completed within 30 minutes and this protocol has demonstrated good reliability when performed by clinicians and non-radiologists after only limited training79. Furthermore, HEAD-US scores correlated well with HJHS for the elbows, knees, and ankles, and the correlation coefficient was as high as r = 0.70 (P <0.01) in one study56. In a more recent series by De la Corte-Rodríguezet al.80, 14% of patients exhibited HEAD-US signs of early HA in joints without reported bleeds and an HJHS 2.1 score of 0. The authors concluded that, in their experience, the HEAD-US scanning and scoring protocol was superior to bleeding history and HJHS 2.1 score in the early detection of HA. The right ankle was the most severely involved joint in this report. These findings were supported by several other studies highlighting the sensitivity and utility of HEAD-US score in the early detection of HA as compared with HJHS or physical exam alone83.Since the systematic review by Ligockiet al.84. HEAD-US was found to be able to accurately assess the presence/absence of synovial hypertrophy in joints of PwH with a positive predictive value of 94%  73–100%) and a negative predictive value of 97% (CI 91–100%). The HEAD-US and IPSG MRI scores had a strong correlation of r = 0.90 (P <0.01) for the severity of synovial hypertrophy as well. These findings are very promising since synovial hypertrophy is associated with an increased risk of bleeding85 and may be responsive to treatment86; early detection with POC-MSKUS may allow customization and personalization of treatment regimens for PwH in the clinic as they actively participate in the scanning process and review findings in “real time” with their clinical providers.The diagnostic accuracy of the HEAD-US, POC-MSKUS protocol for the detection of HA biomarkers, compared with joint MRI, was recently evaluated by Foppen42. Limited penetration power of the ultrasound beam means that most of it is reflected over the bony surfaces and the evaluation is limited primarily to peripheral joint surfaces; POC-MSKUS can provide only a general idea of the osteochondral surface and whether cartilage and bony alterations are present or absent. MRI is still the best imaging modality for the detailed evaluation of osteochondral derangements in HA, particularly those pertaining to the central aspect of the cartilage and subchondral bone evaluation87.The overall diagnostic accuracy of HEAD-US for synovial hypertrophy detection was 97% but was not as high for osteochondral surface abnormalities and this is similar to what was described for full-joint MSKUS in the past75 have become available, but they have not been globally standardized or validated, limiting their widespread use and implementation for that purpose. To achieve this goal, we need to reach a consensus regarding which sonographic biomarkers of HA should be included in the POC-MSKUS score(s) and what the POC-MSKUS definitions of pathologic conditions are90. Well-designed longitudinal cohort studies to evaluate the value of ultrasound in the early detection of HA and its clinical impact on treatment strategies and prophylactic regimens are urgently needed51, as are guidelines and recommendations for necessary training and appropriate use and implementation of POC-MSKUS as a diagnostic tool in PwH91.POC-MSKUS promises to improve our workflow, eliminating the need for sedation in children and allowing the early detection of HA in its subclinical stage; several gaps in knowledge continue to exist and questions remain unanswered. A number of scanning/scoring protocols91. Currently, formal certification in MSKUS scanning is not required to implement POC-MSKUS in clinical practice, but resources are available and could be used to achieve this end. The need for additional and/or special training in POC-MSKUS scanning becomes evident when evaluating pediatric patients, muscle injuries, and other joints (hips and shoulders). When a pediatric joint is scanned, a sophisticated understanding of the immature skeleton, appearance of growth plates, secondary ossification centers, and areas with abundant normal periarticular fat is key65. The paucity of available literature coupled with the need for specialized training and challenges in image acquisition in the more active and often less cooperative pediatric patient contributed to the underutilization of this technique for assessing pediatric joints in both research and clinical practice. Atlases on expected soft tissue and epiphyseal cartilage thickness and vascularization, and normal ossification of the joints of maturing healthy children are needed95. Awareness of normal variation and contralateral comparison with the other joint, if normal, are helpful. If POC-MSKUS does not provide an answer, referral for a full-joint MSKUS or other imaging modalities such as MRI and plain x-ray should be made91.The highly operator-dependent nature of ultrasound justifies the initial skepticism it was met with and necessitates rigorous operator training to avoid misuse and misdiagnosis43, MSKUS remains less sensitive in soft tissue discrimination (fat versus synovium) and in detecting hemosiderin, which remains debatable and controversial96; some studies describe distinctive echotextural features distinguishing hemosiderin from synovium61 and others do not find a difference in the sonographic appearance of hemosiderin-laden and hemosiderin-free synovium97. MSKUS unreliability/inability to detect hemosiderin deposition in the synovium of PwH will compromise its ability to detect “microbleeds”, an almost decade-old concept that refers to asymptomatic or subclinical leakage of microscopic amounts of blood into a joint hypothesized to contribute to deterioration of the joint without clinical evidence of hemarthrosis98. Although we do not currently have direct evidence that joint microbleeding occurs in PwH, this does not mean that it does not exist! In a very recent review, Puetz98 sheds some light on this topic and argues that medical imaging findings in joints free of clinically evident bleeds and symptoms (MIFFS) constitute a significant clinical issue that deserves appropriate investigation; assuming that MIFFS in PwH are directly related to or caused by microbleeds (or vice versa) can lead to unnecessary, or ineffective medical treatment.Additionally, it is important to note that although MSKUS can readily distinguish between fluid and soft tissueLastly, the value of three-dimensional ultrasound and the newer advances in ultrasound technology in the evaluation of HA has yet to be determined. Currently, most scoring protocols are semi-quantitative. Do we foresee a future where artificial intelligence algorithms automate the quantification and assessment of soft tissue proliferation, cartilage thickness loss, degree of bony derangement, and hypervascularity making the process less operator-dependent and time-consuming?101.Continued innovative development and widespread utilization of POC-MSKUS are expected over the next few years. The dynamic, timely, beneath-the-surface evaluation of hemophilic joints at the bedside using POC-MSKUS as an adjunct to physical examination is invaluable and ultimately will not only result in a more refined understanding of the pathophysiology of HA and natural course of the disease but also enhance patient/parent compliance and adherence to treatment regimens for both acute injuries and prophylaxis. POC-MSKUS can provide visual evidence of joint health which can be used as a motivational tool for prophylaxis-reluctant or non-adherent patients at visits to their hemophilia treatment centers102, POC-MSKUS was most often performed by a physiotherapist (53%) or a hematologist (43%) and was perceived as most useful to confirm an acute joint bleed and the presence of synovial hypertrophy. The greatest perceived barriers to the implementation were lack of trained health-care professionals and the overall time commitment required to perform the exam102.In the recently published IPSG survey of the use of ultrasound for assessment of musculoskeletal disease in PwHet al. explored the potential of a pocket-sized handheld ultrasound device for the evaluation of joint effusions and hemarthrosis in hemophilia103. The image quality of the handheld ultrasound device was deemed sufficient to identify joint landmarks necessary to accurately localize effusions, and the device was promising to be both feasible and reliable even when performed by non-experts  after a brief training period103. Perhaps it is possible to envision a future where patients are equipped with handheld ultrasound devices, but important questions remain to be resolved. Would this allow more efficient use of factor products and improved adherence to prophylactic regimens or false reassurance by false-negative scans and subsequent under-treatment? Will POC-MSKUS finally provide us with an objective rather than subjective annualized bleeding rate, allowing a more accurate comparison of the hemostatic effect of different factor products and newer non-factor therapies, or will it leave our patients feeling unheard? Is POC-MSKUS going to fuel the microbleed debate? Can POC-MSKUS accurately detect microbleeding and differentiate acute on top of chronic bleeding episodes in persistently hemorrhagic target joints? Where do POC-MSKUS and pocket handheld devices fit into personalized treatment strategies and outcome measures105? Finally, will ultrasounds bridge the physical distance between patients and their hemophilia treatment centers, allowing them to have a more objective evaluation of their acute musculoskeletal painful episodes remotely, or will it result in non-compliance with poor follow-up when the home scans reassure against progressive joint disease and the perceived role of the clinical exam is lost?Recently, Zhou91. International consensus guidelines and recommendations for use should be established to allow the effective implementation and utilization of this imaging modality in hemophilia. Studies to evaluate the feasibility of implementation and time needed to complete scans for both the evaluation of acute musculoskeletal concerns and longitudinal joint disease monitoring are needed. Frequency of six-joint surveillance ultrasound needs to be established for asymptomatic, near-asymptomatic, and target joints. Objective evidence highlighting the limitations of POC-MSKUS should be provided. What POC-MSKUS can and cannot do should be clearly disclosed to patients/parents, and when the question posed is not answered, the referral to a diagnostic imaging department and an experienced musculoskeletal radiologist for either a full-joint MSKUS or a different imaging modality should be made. As beautifully stated by Lawsonet al., “point-of-care ultrasound should be respected for the complexity of the technology and the skill required to interpret it”91. Only then will we avoid misdiagnosis and misuse.POC-MSKUS is very promising, yet we should acknowledge the need for formal training, attaining and maintaining competence, quality assurance, and understanding its appropriate use


The SWI/SNF chromatin remodeling complex has been found mutated in a wide range of human cancers, causing alterations in gene expression patterns, proliferation and DNA damage response that have been linked to poor clinical prognosis. Here, we investigated weather knockdown of ARID1B, one of two mutually exclusive subunits within the SWI/SNF complex, can sensitize colorectal cancer cell lines mutated in the other subunit, ARID1A, to ionizing radiation (IR). ARID1A-mutated colorectal cancer (CRC) cell lines are selectively sensitized to IR after siRNA mediated ARID1B depletion, as measured by clonogenic survival. This is characterized by a decrease in the surviving cell fraction to 87.3% ± 2.1%, 86.0% ± 1.1% and 77.2% ± 1.5% per 1 Gy compared with control siRNA exposed cells in the dose range of 0–6 Gy for the LS180, RKO and SW48 lines, respectively . The magnitude of this dose modifying effect was significantly larger in ARID1A mutated than in non-mutated cell lines . Furthermore, initial formation of RAD51 foci at 4 h after IR, as a measure for homologous recombination repair, was significantly reduced in ARID1A-mutant CRC cell lines but not in the majority of wildtype lines nor in fibroblasts. These findings open up perspectives for targeting ARID1B in combination with radiotherapy to improve outcomes of patients with ARID1A-mutant CRC. ARID1A and ARID1B are two mutually exclusive regulatory subunits of the SWI/SNF complex. Notably, mutations of ARID1A were found in several cancers. Wei and colleagues described loss of ARID1A in colorectal cancer being associated with late TNM stage, poor pathological classification and distant metastasis, and concluded that patients with ARID1A-mutated cancers may benefit from therapy targeting chromatin modifying enzymes2. Recent studies identified ARID1B as a potentially lethal target in ARID1A-mutant tumors, where depletion of ARID1B selectively impaired proliferation and caused destabilization of the SWI/SNF complex4.The multiprotein chromatin remodeling complex SWI/SNF plays a key role in the dynamic regulation of gene expression, and mutations of its subunits have repeatedly been linked to human malignancies. Literature findings suggest a wide-spread role of alterations in the expression of SWI/SNF subunit genes in human cancer susceptibility and patient survival times5. As a key member of the chromatin remodeling family, increasing significance is attributed to the SWI/SNF complex in the processing of double strand breaks (DSB) especially during homologous recombination repair (HRR)6. Additionally to its involvement in HRR, the SWI/SNF complex was also linked to the recruitment of several factors involved in non-homologous end joining (NHEJ)7. Targeting of chromatin remodeling, particularly of the SWI/SNF complex8, may therefore prove to be a successful strategy for the sensitization of cancer cells to DNA-damaging therapy such as irradiation.Various publications have emphasized the importance of chromatin structure for DNA damage repair, particularly highlighting the process of chromatin remodeling required to facilitate access of repair enzymes to DNA endsHere, we investigated the effect of ARID1B knockdown on radiosensitivity in colorectal cancer (CRC) cell lines by measuring clonogenic survival after irradiation of cells with and without ARID1A mutation. Additionally, formation of DSB repair foci such as RAD51 and 53BP1 was evaluated to determine the mechanism of radiosensitization in ARID1A-mutant CRC cells.et al.9. Treatment with ARID1B targeted siRNA reduced the expression levels to about 26%, 38%, 50%, 22%, 37% and 55% in HCT15, HCT116, Colo320DM, LS180, RKO, and SW48, respectively  and mutant ARID1A  were investigated. As shown in Fig. ely Fig. .Figure 1To evaluate the impact ARID1B knockdown has on radiation sensitivity, colony formation assays were performed in ARID1A-proficient and ARID1A-deficient CRC cell lines. ARID1B knockdown lead to a significant reduction of the surviving fraction in CRC cells harboring ARID1A mutations Fig. . RadiatiTo elucidate the mechanistic background of radiosensitization in ARID1A-mutated cell lines, RAD51 foci formation as a marker of HRR was measured. Following ARID1B knockdown, cells were irradiated with 0 and 4 Gy and stained at 4 and 24 hours after irradiation. RAD51 foci formation was studied in cells counterstained with cyclin B1 as a marker for G2-phase cells, to only measure cells proficient in HRR as a pathway for DNA damage repair Fig. . DepletiAdditionally, we investigated 53BP1 foci formation as a marker of non-homologous end-joining (NHEJ) activity following DNA damage. ARID1B knockdown did not affect 53BP1 foci formation in either the ARID1A-mutated or the ARID1A-wildtype cell lines at 1, 4 and 24 hours following irradiation with 0, 0.5 and 4 Gy Fig. . FurtherTo evaluate the effect of ARID1B knockdown on proliferation, we compared the short-term proliferative potential of ARID1A-proficient and ARID1A-deficient cell lines after depletion of ARID1B. Knockdown of ARID1B significantly inhibited proliferation of ARID1A-proficient, but not in ARID1A-deficient cell lines Fig. . Colony Identifying new cancer-specific vulnerabilities arising in the context of mutations within the SWI/SNF chromatin remodeling complex can be of pronounced clinical significance, and can potentially give rise to more effective use of radiotherapy through combination with chromatin-targeted therapy. In the present study, ARID1B knockdown in the context of ARID1A deficiency was evaluated for the effects on the radiosensitivity of CRC cell lines.Our results demonstrate a novel approach to selectively increase radiation sensitivity in ARID1A-mutated cancers. The data from the colony formation assay reveal that ARID1B knockdown sensitizes ARID1A-deficient cells without considerably affecting proliferation or plating efficiency, highlighting the specific effect on radiosensitivity. In comparison, downregulation of ARID1B showed no sensitizing effect in the clonogenic assay for fibroblast cell lines and CRC lines with wild-type ARID1A, except for a minor sensitization of HCT116. While alterations in SWI/SNF composition have wide-spread roles in tumor biology, these findings describe a very confined and specific vulnerability of the residual SWI/SNF complex in cancers containing inactivating ARID1A mutations.10. In addition, ARID1B also accumulates at DNA-double strand breaks. Suppression of ARID1A or ARID1B could lead to reduced non-homologous end-joining4. While we did not find an effect of ARID1B knockdown on 53BP1 foci formation, a marker of NHEJ, in any of the CRC cell line, this does not rule out a role of NEHJ on the observed radiosensitizing effect of ARID1B knock down in ARID1A mutated CRC cell lines. Park et al. pointed out that in ARID1A deficient tumors, decreased accessibility of 53BP1 to DNA lesions leads to reduced NHEJ activity11. Comparing ARID1A -proficient and -deficient cell lines, we found a broad overlap in the surviving fraction at 4 Gy as a measure for radiosensitivity. Taken together, evidence exists from different studies that both NHEJ and HR can be affected by ARID1B knockdown. In ARID1A mutated cell lines, ARID1B knockdown led to a marked dose dependent radiosensitization, that was not dependent on an anti-proliferative effect. Growth inhibition or reduction in platting efficiency was minor or not present in these cell lines and this highlights the selectivity of the radiosensitizing effect. This radiosensitizing effect on the surviving fraction has the potential to be raised to high power during fractionated irradiation of colorectal carcinomas. In ARID1A-proficient CRC cells, as well as in fibroblasts, depletion of ARID1B led to a significant inhibition of proliferation without DNA damage, but not to a dose dependent radiosensitization. However, several of radiation dose-limiting normal tissues around a tumor are non-proliferating. Furthermore, the anti-proliferative effect of ARID1B knockdown in ARID1A proficient cells is consistent with previous findings describing opposing roles of ARID1A and ARID1B in tumor biology12. Depletion of ARID1B may result in an overbalance of the tumor suppressing properties of ARID1A within the SWI/SNF complexes, resulting in decreased proliferation of un-irradiated cells with wild-type ARID1A. Consistent with our study, Wang and colleagues demonstrated that ARID1B depletion inhibits proliferation in bladder cancer cell lines without ARID1A mutation13. Additionally, high levels of ARID1B expression have been linked to poor clinical outcomes in bladder urothelial carcinoma and breast carcinoma, while low ARID1A levels have been linked to poor clinical outcomes17. On the other hand, Helming and colleagues found a synthetic lethal relationship of ARID1A and ARID1B in a subgroup but not in all of the analyzed ARID1A mutated cancer cell lines, describing ARID1B as a specific vulnerability in ARID1A-mutant cancers3.In order to further investigate the mechanisms of radiosensitization, we investigated whether ARID1B knockdown affects formation of DSB repair foci. Consistently with the data from the clonogenic survival assay, depletion of ARID1B had a significant impact on the radiation induced RAD51 foci formation in ARID1A-mutated cell lines. RAD51 foci formation in ARID1A-proficient colorectal carcinoma (CRC) cell lines was not affected, except for HTC116. In addition, siARID1B had no effect on RAD51 foci in two different normal skin fibroblast cells. Rad51 foci were measured in cyclin B1-positive cells alone, since HRR is present in S/G2 phase cells only, further refining this assay to specifically represent HRR activity. An altered interaction between members of the SWI/SNF complex and HRR enzymes following depletion of ARID1B might ultimately contribute to an increased radiosensitivity in ARID1A-mutated cell lines. However, much of these possible interactions between SWI/SNF subunits and DSB repair enzymes remain elusive. ARID1A is recruited to double strand breaks mediated by ATR19. ARID1B is potentially drugable through its E3 ubiquitin ligase interaction20, and therefore similar approaches may be used in targeting ARID1B.Here, we showed that independent from growth inhibition ARID1B knockdown can sensitize ARID1A mutated CRC cells to ionizing irradiation paralleled by a reduction of RAD51 foci induction indicating reduced homologous recombination. Effective treatment with small molecular inhibitors targeting EZH2, ATR and PARP has already been demonstrated in ARID1A-deficient tumorsIn conclusion, our study shows that radiosensitivity in ARID1A mutant CRC cell lines can selectively be increased through depletion of ARID1B, suggesting ARID1B as a potential therapeutic target to increase radiosensitivity in ARID1A-deficient tumors.2. Irradiation was done using the RS320 X-Ray machine by XStrahl Ltd. at 300 kV, 10 mA, dose rate 0,9 Gy/min.The human CRC cell lines LS180, RKO, SW48, HCT15, HCT116 and Colo320DM were obtained from ATCC . LS180, RKO and SW48 were kept in MEM (Invitrogen) supplemented with 15% FBS, 1% essential amino acids and antibiotics. HCT15, HCT116 and Colo320DM were kept in RPMI (Invitrogen) supplemented with 10% FBS plus antibiotics. Adult dermal fibroblasts  and neonatal fibroblasts  were obtained from ATCC. Fibroblasts were cultured with fibroblast basal medium  supplemented with low serum fibroblast growth kit . Cells were cultivated at 37 °C in 5% CO2O. After 4 hours of incubation with the transfection reagent, 500 µl of culture medium with double FBS was added and cells incubated for 48 hours until harvesting for further experiments. Expression of targeted proteins was regularly checked by Western blots.Cells were seeded in 6-well plates and incubated for 24 hours, obtaining a 70–80% confluent monolayer. Cells were then washed in HBSS and OptiMEM (both Gibco), and subsequently incubated with transfection reagent for 4 hours. We used 500 µl OptiMEM with 80 nM siRNA and 6 µl Lipofectamine RNAiMAX (Invitrogen) for transfection reagent. To downregulate ARID1B, we established a mix of two siRNAs  at 40 nM each for optimal efficiency. As controls, we used non-targeting siRNA (Ambion 4390843) as well as HWestern blots were performed with anti-ARID1A , anti-ARID1B , anti-RAD51  and anti-GAPDH . The secondary antibodies were HRP-linked antibodies raised against mouse or rabbit IgG  and Alexa Fluor 488-linked antibodies against mouse (Invitrogen A11029).6 cells were seeded per 25 cm2 flask. Cells were harvested and counted by automatic counting system Luna (Logos Biosystems) at day 1, 2, 3, 4, 7 after seeding.48 hours after transfection, 0,5 × 102 culture dishes. After 4–6 hours in culture, cells were irradiated and subsequently incubated for 10–14 days at 37 °C in 5% CO2. Cells were fixed and stained with 96% ethanol, 15% Giemsa and destained with distilled water. Colonies consisting of at least 50 cells were counted. Surviving fractions after the indicated treatments are presented as a fraction of the growth of non-irradiated colonies8.48 hours after transfection, cells were harvested and plated in triplicates in 9,6 cm48 hours after siRNA transfection, cells were harvested, reseeded in chamber slides and irradiated 4 hours later. At indicated times after IR, cells were washed, fixed, permeabilized and incubated with blocking buffer. Primary antibodies anti-RAD51 , anti-cyclin B1  and anti-53BP1  were added to the cells and incubated overnight. Secondary antibodies anti-mouse Alexa Fluor 488  and anti-rabbit Cyanine Cy3  were applied together with 1 µg/ml DAPI (Invitrogen) nuclear counterstain for 1,5 hours at room temperature. Slides were examined on the fluorescent microscope imager Z1 (Zeiss).Colony data were analyzed using a linear-quadratic model describing the dependence of the logarithm of cellular survival on dose. The interaction between ARID1B knock down and the radiation dose response was described as a slope modifying effect of the linear term of the linear-quadratic model . Higher order effects were considered if significant at alpha = 0.01.


Demographically, aging of the world’s population is a fact that requires the implementation and development of integrated, active policies for the inclusion of older people in economic and social life. In response to this challenge, an international UN plan for active aging was adopted in Madrid in 2002. By adopting this plan, all EU member states are committed to contribute to active aging by integrating the rights and needs of older people into national economic and social policies. The European Commission formulates the concept of active aging as adopting a healthy lifestyle, longer labor market participation, later retirement, and retention after retirement. In recent years managers of higher education institutions in Bulgaria have been pursuing a policy of preserving the labor and intellectual potential of retired research and teaching staff. There are different practices and activities aimed at preserving this valuable resource built for decades. This paper outlines the main activities and evaluation of the effectiveness of the Mentoring Program for Shared Experience and Knowledge. This pilot program was implemented over a five-year period from 2012 to 2017. It involved teachers in pre-retirement and retirement age working in higher education institutions. Monitoring and evaluation of the different forms of activities included in the program were made. The benefits for all participants have been derived. The results and the analysis made support the initial suggestion that the program is an opportunity for gradual withdrawal from work activity, preserving social well-being and successful adaptation to retirement from active labor activity.


Embedding healthy eating and physical activity best practices in early care and education settings is important for instilling healthy behaviors early in life. A collaborative partnership between Nemours Children’s Health System and the Centers for Disease Control and Prevention was created to implement the National Early Care and Education Learning Collaboratives Project (ECELC) in childcare settings in 10 states. We measured improvement at the program level by the self-reported number of best practices implemented related to healthy eating and physical activity.The ECELC implemented a collaborative model with state-level partners  and early care and education programs. Intervention components received by program directors and lead teachers included 1) self-assessment, 2) in-person learning and training sessions, 3) action planning and implementation, 4) technical assistance, and 5) post-reassessment.A pre–post design assessed self-reported policies and practices related to breastfeeding and infant feeding, child nutrition, infant and child physical activity, screen time, and outdoor play and learning as measured by the validated Nutrition and Physical Activity Self-Assessment for Child Care (NAP SACC) best practices instrument. The sample included 1,173 early care and education programs.P < .001). Almost all increases occurred regardless of participation in the Child and Adult Care Food Program, Quality Rating Improvement System, Head Start/Early Head Start, and/or accreditation status.The number of best practices met for each of the 5 NAP SACC areas increased from pre-assessment to post-assessment approximately 6 months later and ranged from 1.5 to 4.7 best practices (The innovative and collaborative partnerships led to broad implementation of healthy eating and physical activity–based practices in early care and education settings. Development, implementation, and evaluation of policy and practice-based partnerships to promote healthy eating and physical activity among children attending early care and education programs may contribute to obesity prevention in the United States. Incorporating healthy eating and physical activity best practices in early care and education settings is important for instilling healthy behaviors early in life.A collaborative intervention among a health care system, state-level partners, and early care and education programs increased the number of best practices being met related to breastfeeding and infant feeding, child nutrition, infant and child physical activity, screen time, and outdoor play and learning in early care and education settings.Development, implementation, and evaluation of policy and practice-based partnerships to promote healthy eating and physical activity among children attending early care and education programs may help prevent obesity in the United States. More than 1 in 8 children 14%) aged 2 to 5 years were obese in 2016 . Possibl% aged 2 Promotion of healthy eating and physical activity (HEPA) behaviors in early care and education (ECE) settings can reduce the risk of obesity among the nearly 13 million children aged 5 years or younger who spend some portion of their week in this setting . Given tNemours Children’s Health System (Nemours) collaborated with the Centers for Disease Control and Prevention (CDC) to implement the National Early Care and Education Learning Collaboratives (ECELC) Project in 10 states. In 2007, Nemours developed and implemented an intervention in Delaware to promote HEPA among children in various settings, including ECE settings. A key part of the initiative was the establishment of learning collaboratives using a “train-the-trainer” model with ECE programs to identify and implement healthier policies and practices . The NutThe ECELC recently ended its sixth and final year of implementation. The project established and implemented learning communities with teams of ECE providers to promote peer learning and to support and improve their HEPA policies and practices. The ECELC’s learning collaborative design is an adaptation of the Institute for Healthcare Improvement’s Breakthrough Series model . The ECEInputs. Inputs included the national team , state partners, program materials, and financial resources. State partners included a state implementing partner (statewide organization serving as the implementer), a state project coordinator (a staff member employed by the state implementing partner), and trainers to facilitate learning sessions and provide technical assistance. Program materials included the ECELC curriculum and training materials , as well as self-assessments . Lastly, each ECE program received a $500 stipend to support staff time and purchasing of equipment.Activities. Although Nemours provided guidance and direction on implementation, the state implementing partner had flexibility for the purposes of ownership and buy-in. It was anticipated that ECE program involvement in other state-level programs and initiatives had potential for impact on the effectiveness of the ECELC, so the model was intended to be tailorable at the state, local, and ECE program levels. ECE programs were recruited by state project coordinators through various informal methods, including personal telephone calls, online recruitment, and connections with groups such as Head Start/Early Head Start. A strength of this recruitment method was many state implementing partner agencies had existing relationships with programs and were providing them with support in other, nonhealth areas of program improvement. To be eligible to participate, ECE programs initially had to be operating in a center-based physical facility and designate a team of up to 3 people  to attend 5 in-person learning sessions. During the first 5 years of the ECELC, 2,266 ECE programs were enrolled and 1,910 completed the intervention (84%).ECE program engagement. The ECELC implementation cycle spanned approximately 10 months and consisted of 5 main strategies: 1) self-assessment; 2) in-person peer learning sessions, 3) action planning and implementation, 4) technical assistance, and 5) re-assessment.In-person peer learning. Leadership teams and other staff participated in 5 approximately 6-hour in-person learning sessions led by trainers over a 10-month period. These sessions included didactic presentations on HEPA-based content, interactive activities, and peer sharing and support. Of the 572 programs enrolled in the first year of this project, the average number of learning sessions attended per program was 3.4 sessions .Action planning. After each of the first 4 learning sessions, teams were tasked to return to their ECE programs and share what they learned. This type of peer sharing aimed to help build program-wide staff support for implementing best practices across the 5 topic areas. Each program created improvement goals with corresponding objectives based on their self-determined need (using what they learned from their self-assessment as a guide), interest, and capacity. Programs were not required to set goals for each of the 5 topic areas. Using a social ecological approach , what NAP SACC topic area the technical assistance was related to, and if the technical assistance was related to the program’s action plan.A pre–post design assessed the self-reported changes in policies and practices related to breastfeeding and infant feeding, child nutrition, infant and child physical activity, screen time, and outdoor play and learning for programs participating in the ECELC. The primary outcome data were derived from the NAP SACC instrument . Other dParticipating ECE programs completed the NAP SACC instrument after the first learning session (pre-assessment). A previous study demonstrated that 89% of NAP SACC items showed at least moderate agreement for test-retest reliability, 100% of items showed at least moderate agreement for inter-rater reliability, and 52% of items showed at least moderate agreement for validity when tested against the Environment and Policy Assessment and Observation (κ ≥ 0.20) . The NAPThe inclusion criteria for this evaluation included center-based ECE programs that participated in the ECELC through June 2017. Family child care homes were excluded from this analysis because of the heterogeneity of that setting compared with center-based ECE programs. The eligible pool of ECE programs was 1,879. Programs were further excluded from analysis if they served any combination of age groups other than preschoolers only; toddlers and preschoolers; or infants, toddlers, and preschoolers, the 3 most common configurations of age groups served in this sample. To align with the self-determined, pre–post design of this evaluation, programs were further excluded from topic area-specific analyses if they did not respond to at least one item in both the pre-assessment and post-assessment for that topic area of the NAP SACC instrument.t tests to assess overall effects of program characteristics on change scores without controlling for covariates. Significance was set at a 2-sided α level of P < .05.Primary comparisons of NAP SACC instrument change scores were conducted by using a longitudinal linear mixed model where the outcome variables were the 5 NAP SACC topic area scores measured for each ECE program at pre-assessment and post-assessment. Covariates contained in every model were: wave (denoting time point), age-groups served , implementation cycle, wave-by-age-groups-served interaction (except for breastfeeding and infant feeding), and wave-by-implementation-cycle interaction. Models for specific program characteristics  also included the identified characteristic and a wave-by-characteristic interaction. The interrelatedness of a program’s pre-assessment and post-assessment scores was captured by using a first-order autoregressive structure covariance pattern . We usedThe final analytic sample included 1,173 ECE programs (62% of eligible programs), of which 260 served preschoolers only; 229 served toddlers and preschoolers; and 684 served infants, toddlers, and preschoolers . Almost P < .001)  . The perP < .001). Additionally, accredited programs improved with regard to screen time, but by a smaller amount  when compared with nonaccredited programs.This evaluation focused on the potential for 4 program characteristics to influence NAP SACC scores: CACFP, QRIS, Head Start/Early Head Start, and accreditation . Of thesWe found that the ECELC was an effective multisector approach to promote important changes to policies and practices in ECE programs related to breastfeeding and infant feeding, child nutrition, infant and child physical activity, outdoor play and learning, and screen time. Findings suggest that the ECELC fulfilled a key, short-term outcome, in that ECE programs made changes to policies and practices that evidence suggests may lead to improved food and physical activity environments for young children in ECE settings –15. An evaluation conducted after the ECELC was initially implemented found that a subsample of ECE programs that had participated in the ECELC maintained improvements in NAP SACC best practices for all topic areas one year later , suggestA key part of the ECELC included building collaborations across ECE programs and with community partners . NationaIn 2017, Richter et al urged scaling up of effective interventions for early childhood development by integrating into systems of health, education, and social and child protection, expressing that health and nutrition services are ideal starting points because of the relative affordability . OverallAlthough these findings are unsurprising, they help illustrate how the ECELC can operate synergistically among other HEPA-based efforts among ECE settings. Conversely, differences in change scores by program characteristics were reported only in the topic areas of child nutrition and screen time for Head Start/Early Head Start and accredited programs, respectively, and participation in these external initiatives for these topic areas was associated with lesser improvement. When programs did not improve at the same rate, it may have been because they were already meeting more best practices at the start of the ECELC. Regardless, an opportunity exists to explore why seemingly high-quality programs voluntarily enroll in HEPA-based interventions, as well as how to reach ECE programs in greater need for improvement. That programs usually improved at the same rate whether they participated in external initiatives or not suggests that the ECELC may help fill a gap in resources, educational materials, and/or setting standards among all ECE programs. The multisector learning collaborative model also helped facilitate ECE programs to be more ready to meet standards, a need that exists among CACFP programs ,29. A neAlthough currently no federal nutrition or physical activity standards are enforced in ECE programs and most states lack meaningful regulations related to HEPA , state-lThe US Departments of Health and Human Services and the US Department of Education have stated that ECE programs and providers must receive proper education and training, as well as fair compensation, to promote high-quality experiences for all children in these settings . HoweverThese findings should be interpreted with caution because of the contextual differences across locations as well as measurement-based limitations. Although a strength of this intervention is that strategies were consistent throughout the 6 years of the intervention, the degree to which ECE programs received intervention components  varied. In addition, the intervention was supported via funding  and staff support. Reasons for programs enrolling or dropping out of the intervention were not tracked consistently, and participating programs may have been motivated to change. Despite these limitations, results from annual evaluations were used to inform the development and improvement of the ECELC, contributing to the spread and scaling of the intervention across multiple states with consistent results . The repThis evaluation demonstrated that the innovative and collaborative partnership led to broad implementation of best practices related to breastfeeding and infant feeding, child nutrition, infant and child physical activity, outdoor play and learning, and screen time in ECE settings. The ECELC model was found to be complementary and not duplicative to existing programs and initiatives . Findings also suggest that the multisector learning collaborative model may be generalizable to both well-resourced and poorly resourced ECE programs and that an opportunity exists to engage additional sectors  to collaborate and work toward shared goals, such as developing a healthy workforce through fostering early development. By implementing policies and practices in these settings, there is potential for reaching approximately 1 in 4 children aged 5 years or younger and their families. Development, implementation, and evaluation of policy and practice-based partnerships to promote HEPA among children attending ECE programs may contribute to obesity-prevention in the United States.


Hghly stable, reusable, and multi-functional biocatalytic microparticles with Laccase (Lac) enzyme (Lac/particles) were synthesized for bisphenol A (BPA) removal from aqueous solution. The Lac/particles were prepared by encapsulating Lac enzymes into poly ethylene glycol (PEG) hydrogel via the UV assisted emulsion polymerization method followed by cross linking with glutaraldehyde (GA). The obtained Lac/particles were spherical and micron sized (137–535 μm), presenting high enzyme entrapment efficiency of 100%, high activity recovery of 18.9%, and great stability at various pHs (3–7) than the free Lac. The Lac/particles could adsorb the BPA into the catalytic particles in a short time, promoting contact between BPA and enzyme, and further enzymatically degrade them without the shaking process and independent surrounding buffer solution. The Lac/particles could be reused for another round BPA adsorption and biotranformation by maintaining over 90% of BPA removal efficiency after seven times reuse. The synergistic effects of adsorption and biocatalytical reaction of Lac/particles have significant values in high efficient and cost-effective BPA removal. Bisphenol A (BPA) has attracted public concern, because its hormone-like effect on the endocrine system disrupts physiological functions. As an important industrial material, small amounts of BPA inevitably enter the environment during production and processing . Low dosVarious methods have been utilized to eliminate BPA in polluted water, including photocatalysis , adsorptHydrogels are well known for providing biocompatible environments for immobilizing biosubstances in a desired composite structure. It was utilized in various medical and industrial applications, such as drug delivery ,16, tissIn view of facilitating easy mass transfer and efficient biocatalytic reaction for pollutants removal, the production of hydrogel biocatalysts with micro size and spherical shaped particles, accompanied with an adsorption property, is of great benefit. In this study, we synthesized Lac entrapped polyethylene glycol (PEG) hydrogel microparticles (Lac/particles) consisting of poly (ethylene glycol) diacrylate (PEGDA) and poly (ethylene glycol) methacrylate (PEGMA) by using a UV assisted emulsion polymerization method. PEG-based hydrogels are particularly attractive because of their excellent biocompatibility, tunable network structure, good toughness, and readily modifiable properties . The emuPoly (ethylene glycol) diacrylate , poly (ethylene glycol) methacrylate , 2-hydro-xy-2-methylpropiophenone (HMPP), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), mineral light oil, Span 80, Tween 20, BPA, glutaraldehyde (GA), fluorescein isothiocyanate (FITC) assay kit, and Pluronic F-127 were purchased from Sigma-Aldrich . Laccase  from Trametes versicolor was also purchased from Sigma-Aldrich and was used without further purification. Bradford reagent was purchased from Thermo fisher . Other agents were at least of analytical grade. Citric phosphate buffer (50 mM) and ultrapure water were used in all experiments. Lac entrapped in hydrogel microparticles (Lac/particles) were prepared using reverse emulsion assisted by UV polymerization, as shown in In this study, 200 µL of the precursor solution was added to 1 mL of light mineral oil with 1% Span 80 and 1% Tween 20 and vortexed for 10 s to achieve a well homogenized emulsion. Then, the emulsion was polymerized by exposure to UV light  for 3 min and separated from the oil by repeated washing using 0.1 wt% Pluronic F-127 and citric phosphate buffer (pH 7). Subsequently, GA was added to the Lac/particles with the final concentration of 0.5% (v/v), and chemical cross-linking was performed at room temperature under shaking for 30 min. The excessive aldehyde groups were capped by incubation in the Tris-HCl buffer for 30 min. The produced Lac/particles were stored at 4 °C until used. The surface morphologies and size distribution of Lac/particles were analyzed using scanning force microscopy  and laser particle size distribution analyzer , respectively. The fluorescence of the Lac/particles was observed using a fluorescence microscope . The absorbance for activity assay was determined using a UV-vis spectrophotometer .The enzymes to be trapped in the hydrogel were initially fluorescently labeled to monitor the formation of the Lac/particles. Free amino groups of Lac were linked with isothiocyanate reactive groups of FITC, forming a stable thiourea bond. Then, 5 mg/mL Lac was reacted with 50 mg/mL FITC in a 0.1 M carbonate-bicarbonate buffer (pH 9.0) for 2 h in a dark room at room temperature. Then, the solution was isolated by a gel filtration column to separate Lac, which has been labeled. The conjugation of FITC to the Lac was confirmed by an UV-vis spectrophotometer .m0 and tm represent the initial and residual amounts of Lac, respectively. The amount of the entrapped Lac was estimated by measuring the concentration of enzymes in the initial solution or supernatant by Bradford assay. The enzyme loading yield was calculated as follows: +) at 420 nm  in 50 mM citric phosphate buffer (pH 3.5) with shaking under 150 r/min at 25 °C. One unit (U) of Lac activity was defined as the amount of enzyme forming 1 μmol of ABTS+ per minute. All spectrophotometer measurements were performed using a Shimadzu UV-2450 .Lac activity was determined by monitoring the oxidation rate of ABTS to its cation radical  in citric phosphate buffer  at 25 °C. The mK and maxV of the free and Lac/particles were calculated from the Lineweaver-Burk plot as follows:mK and maxV are the kinetic constants, and [S] is the concentration of the substrate.The kinetic constants The pH stabilities of free Lac and Lac/particles were tested by measuring the activity after incubation with different citric phosphate buffer solutions (pH 3–7) under continuous shaking (150 r/min). Enzymes were statically stored at 4 °C, and the residual activity of both free Lac and Lac/particles were assayed to determine the storage stability. The residual activity was expressed as a percentage relative to the initial enzymatic activity. The biotransformation of BPA was performed by addition of Lac/particles (30 mg) into 0.5 mL of BPA solution (25 mg/L) and incubating at room temperature with shaking at 150 r/min to enable a homogeneous and sufficient enzymatic reaction. Solvent extraction was conducted to extract residual BPA retained in the Lac/particles at the end of the incubation period. Lac/particles thoroughly mixed with 0.5 mL of methanol was sonicated for 20 min, and the supernatant was collected. This process was performed twice to extract completely. The extracted BPA in the solution was measured by HPLC analysis.C0 and tC represents the initial and residual concentrations of BPA in the reaction, respectively.For BPA quantification, the HPLC system was equipped with a UV-vis detector at 278 nm and an Eclipse Plus C18 column . The mobile phase was composed of acetonitrile and deionized water with a volume ratio of 50:50. The flow rate was set to 1.0 mL/ min at 30 °C. The BPA transformation efficiency was assessed as follows: The reusability of Lac/particles was studied using Lac/particles and deactived Lac/particles (deLac/particles). BPA removal was performed at 14 consecutive experiments with shaking at 150 r/min. In each cycle, 30 mg of Lac/particles or deLac/particles were reacted with 0.5 mL of 25 mg/L of BPA (pH 5) at room temperature. The supernatant was decanted after 1 h incubation, and then the fresh BPA solution was added for the next round reaction. The Lac/particles were prepared by emulsion of the PEGDA/PEGMA mixture containing Lac  in mineral oil phase followed by UV induced polymerization . Under oTo visualize the distribution of Lac inside the hydrogel droplets, Lac was labeled with FITC fluorescent dye in advance, and the fluorescence was imaged using a fluorescence microscope. As shown in The emulsion method produced micron spherical particles (2.33–535 μm diameter), and the particles size was mostly in the range of 137–535 μm, accounting for 73.54% of the total e particlmK and maxV were obtained from the Lineweaver-Burk plot using ABTS as substrate. The mK values of Lac/particles and free Lac were 1649.35 and 20.32 µM, respectively. The increase in mK for Lac/particles was due to the reduced affinity to substrate after enzyme immobilization, which was caused by the diffusion limitation given the loss of enzyme flexibility for substrate binding. The maxV of Lac/particles (0.8342 μM/min) was increased by approximately 18.5 fold compared to free Lac (0.045 μM/min). In general, most enzyme carrier materials increase mK, accompanied by a decrease in the maximum rate of reaction. However, in this study, the mK and maxV were increased simultaneously. The possible reason is that the adsorption capacity of Lac/particles guaranteed the substrate agglomeration on the surface of the Lac/particles and entrance to the internal to contact with the enzyme. The result was consistent with that obtained by Sun et al. [maxV was improved, accompanied by increasing mK.Kinetic parameters n et al.  who entrAny changes in the chemical and physical properties of wastewater can lead to the inactivation of Lac. Thus, the stability is a crucial factor when the Lac/particles were intended to treat polluted water. In this sense, the stability profiles of Lac/particles at various pH conditions were investigated. The stability profiles of entrapped enzymes in the citric phosphate buffer at various pH levels  at 25 °C with shaking at 150 r/min were evaluated by measuring the residual activities at specific time intervals. After 2 h shaking, the remaining enzymatic activities of Lac/particles were still 100% at all pH levels a, and evThe enzymatic biotransformation of BPA at various entrapped Lac concentrations was accessed. The increment of Lac loading amounts from 0.01 to 0.32 mg Lac per milligram particles accelerated BPA biotransformation efficiency by showing 15.48%, 37.19%, 66.6%, 65.76%, and 62.56% within 10 h of reaction, respectively a. A highThe biotransformation of BPA (25 mg/L) at various pH conditions was investigated by changing pH levels from 3 to 7 at 25 °C. The data presented in The enzymatic biotransformation of BPA at different temperatures was also accessed by changing reaction temperatures from 10 °C to 40 °C. As shown in Lac/particles (30 mg) were added into BPA solution , and the resulting solution was incubated at room temperature with shaking at 150 r/min to make a homogeneous and sufficient enzymatic reaction. The increment of incubation time accelerated BPA biotransformation efficiency during the first 12 h, indicating almost 85.6% BPA biotransformation d. Then, To understand the detailed mechanism of BPA removal process by the Lac/particles, a series of control experiments were conducted by treating BPA (25 mg/L) with the free Lac and the deLac/particles. The fates of the BPA, i.e., residual BPA in the liquid phase, adsorbed BPA in Lac/particles, and enzymatically biotransformed BPA, were accessed, respectively, after 24 h of reaction at 25 °C with shaking (150 r/min) condition.As shown in As free Lac was utilized for BPA removal, 20.41% of BPA was biotranformed that was much lower than with the Lac/particles. Inadequate contact between Lac and the substrate is usually one of the obstacles for efficient micropollutants biotransformation. The reason for the enhancement of BPA biotransformation efficiency by the Lac/particle in this study was firstly due to its great adsorption capability that can concentrate BPA inside the Lac/particles, allowing easy and fast mass transfer, promoting the interaction between BPA and active sites of enzymes, and facilitating efficient catalytic reaction. Another reason was that the Lac enzyme molecules entrapped in the PEG hydrogel microparticles may be less affected by the environmental changes compared to the free one, and the activity loss during reaction was avoided. To investigate whether the mixing process is necessary during catalytic degradation of BPA by using Lac/particles, the BPA biotranformation profiles at continuous shaking and no-shaking conditions were compared. It was found that the Lac/particles (30 mg) can adsorb all of the BPA in the solution within 30 min, and no BPA could be detected in the supernatant. This phenomenon confirmed that all BPA had been adsorbed into the Lac/particles. Thus, for the no-shaking experiment, the supernatant solution was decanted after 30 min, and the Lac/particle that was enriched with BPA was statically incubated at 25 °C for catalytic reaction. For continuous shaking experiment, BPA solution containing Lac/particles were continuously shaken (150 r/min) during the whole reaction process without decanting the supernatant. As shown in The reusability of Lac should be an essential concern when it was intended to be applied for polluted water remediation. In this study, the removal efficiency of BPA by Lac/particles and deLac/particles were investigated by repeated usage for treating fresh BPA solution for 14 cycles. The BPA removal efficiencies were both 100% for the first cycle, and the enzymatic biotransformation functioned in the following cycles a. The diIn all experiments, the Lac/particles maintained their shape during the whole reaction. We also evaluated the release of Lac from the particles under all reaction conditions, including all pH levels, and no leakage of Lac from the particles was detected by Bradford method. Spherical and micron sized Lac/particles were prepared via UV assisted emulsion polymerization, which was suitable for mass production of enzyme entrapped hydrogel miroparticles with high enzyme loading, advantageous mass transfer, and convenient recovery. The Lac/particle was much more stable and presented fast adsorption and efficient biotransformation profiles toward BPA. External shaking was not a necessary factor due to the conducting of the enzymatic BPA removal inside the micro reacting environment of the Lac/particles. The Lac/particle could be reused more than 10 times with little decrease of removal efficiency. The combination of adsorption and enzymatic biotransformation of the Lac/particle is promising for fast and cost-effective BPA remediation.


BCR-ABL1 transcripts are detected in less than 5% of patients diagnosed with chronic myeloid leukaemia (CML), of which e19a2 is the most frequently observed, with breakpoints in the micro breakpoint cluster region (μ-BCR) and coding for the p230 BCR-ABL1 protein. p230 CML is associated with various clinical presentations and courses with variable responses to first-line imatinib.Atypical 5.0) after treatment with single-agent ponatinib. Using CastPCR, we could trace back the presence of the T315I mutation to all the RNA samples up to the detection of T315 mutation by Sanger sequencing shortly after allogeneic hematopoietic stem cell transplantation (HSCT).Here we report a case of imatinib resistance due to an E255V mutation, followed by early post-transplant relapse with a T315I mutation that achieved a persistent negative deep molecular response  contains supplementary material, which is available to authorized users. BCR and ABL1 genes, usually as the result of the reciprocal translocation t. The exact breakpoint of the translocation and the molecular weight of the resulting fusion gene protein are variable, with most of the breakpoints on chromosome 22 falling in the major breakpoint cluster region (M-BCR), between exons 13 and 14 of the BCR gene, leading to a BCR–ABL1 mRNA with e13a2 or e14a2 junctions encoding for a p210 fusion protein [Chronic myeloid leukaemia (CML) is a myeloproliferative neoplasia caused by the fusion of the ation t9;2, low (7), and intermediate (1.639), respectively. A bone marrow aspirate showed marked hypercellularity and myelogenous hyperplasia without an increase in the blast ratio. Cytogenetic analysis showed the presence of a 46,XY,t,+der(22)t karyotype in 25 metaphases, and the presence of a BCR-ABL1 gene fusion was confirmed by FISH analysis. Molecular analysis revealed the presence of an e19a2 BCR-ABL1 transcript and the patient was diagnosed with an e19a2-positive chronic phase CML. The detection of an additional Ph+, a major route abnormality that has been reported as an adverse prognostic factor, does not mandate in daily practice a different initial treatment approach [BCR-ABL1 transcript, but no cytogenetic analysis was performed. In order to quantitate the e19a2 BCR-ABL1 transcript and monitor response to treatment, a BCR exon 19 forward primer was designed  and used with reverse primer ENR561 and probe ENP541 (7). A standard curve was established by tenfold dilutions of patient cDNA. Control ABL1 transcripts were detected as previously described [BCR-ABL1/ABL1 ratio was 40.258% . Two months later, an additional rise in BCR-ABL1 levels to 15.820%, confirmed loss of molecular response and prompted search for mutations in the ABL1 kinase domain as previously reported [BCR-ABL1 level of 0.365% and 5 months later, the patient maintained a CCyR, with a BCR-ABL1 level of 0.671%. However, in the following months, the patient showed a progressive loss of cytogenetic response: PCyR (17% Ph + metaphases), minor CyR (38% Ph + metaphases), and minor CyR (63% Ph + metaphases), 14 months, 17 months and 21 months after starting second-line dasatinib therapy, respectively. At this time, dasatinib therapy was stopped and the patient underwent allogeneic haematopoietic stem cell transplant (HSCT) from a 9/10 matched HLA unrelated donor (one HLA-B mismatch), after a non-myeloablative reduced-intensity conditioning (RIC) regimen that included busulfan, fludarabine and antithymocyte globulin (ATG). The graft-versus host disease (GVHD) prophylaxis consisted of the association of tacrolimus with mycophenolate mofetil (MMF). Neither acute GVHD nor other significant clinical complications were observed. One month after transplant, cytogenetic analysis showed persistence of disease , with a BCR-ABL1 level of 1.229%, which prompted start of nilotinib therapy (400 mg BID). Two months later, his BCR-ABL1 level rose to 8.132% with 23% Ph + metaphases (PCyR), suggesting resistant disease. Mutational analysis by Sanger sequencing revealed the presence of a T315I mutation, which led to the interruption of both nilotinib therapy and immunosuppression. Four months after transplant the patient lost his PCyR  and his BCR-ABL1 levels increased to 12.477%. The patient showed mixed chimerism predominantly donor-derived. Since ponatinib was not available at the time at our institution and approval was pending, nilotinib therapy (400 mg BID) was restarted. One month later, karyotype analysis showed a minimal CyR (97% Ph + metaphases) and the patient was submitted to donor lymphocyte infusion (DLI), which resulted only in a minor CyR (90% Ph + metaphases) that nevertheless was maintained in the following months. This was, however, accompanied by increasing BCR-ABL1 levels to a maximum of 32.879%, 10 months after HSCT. The patient also showed progressive loss of donor chimerism (mixed chimerism with < 5% donor cells). At this point ponatinib approval was granted, nilotinib therapy was interrupted and ponatinib was started at the standard dose of 45 mg QD. Before ponatinib initiation, and considering its well-known cardiovascular toxicity profile, a comprehensive cardiologic and metabolic evaluation was performed. The only cardiovascular risk-factors identified were dyslipidaemia and arterial hypertension (AHT), which were already present since the beginning of the disease and were easily manageable with lifestyle modifications and therapy , respectively. Two months later, karyotype analysis showed a PCyR (17% Ph + metaphases) and a reduction of BCR-ABL1 levels (10.940%) was observed. Subsequently, four months later, the patient achieved a CCyR associated with a BCR-ABL1 level of 0.213%. In the following months a progressive decline in BCR-ABL1 levels was documented: 0.029% at 6 months , 0.005% at 9 months , 0.001% at 12 months , and 0.000% at 15 months . The patient maintained ponatinib 45 mg QD treatment during an additional 16 months and, since a persistent and sustained MR5.0 with no detectable disease was documented, dosage was reduced to 15 mg QD. Treatment with ponatinib was well tolerated since the beginning of therapy with no evidence of toxicity. At the time of the last evaluation, 15 months after ponatinib dose reduction, the patient maintained a sustained MR5.0 with no detectable disease. No evidence of cardiovascular toxicity was observed at the last observation. Under ponatinib treatment, the patient maintained a mixed chimerism with < 5% donor cells. To evaluate if the T315I clone was present at low-levels in the earlier phases of the disease, we used CastPCR  specific for the T315I mutation in all the available RNA patient samples collected since he was admitted to our institution. For the T315I mutation detection, each sample was run in real-time PCR with the assay targeting both the mutation and the corresponding reference gene, according to the manufacturer’s instructions. After amplification, data files containing the samples Ct values were imported into Life Technologies Mutation Detector™ Software for post-PCR data analysis. CastPCR is highly specific and sensitive and can detect and quantitate rare amounts of mutated DNA/cDNA in a sample that contains large amounts of normal, i.e., non-mutated, DNA/cDNA, with a sensibility up to 0.1%. Using this approach, we could trace back the presence of the T315I mutation to all the RNA samples up to the detection of T315 mutation by Sanger sequencing shortly after HSCT.In November 2007, a 51 year-old man was admitted to the Centre Hospitalier Universitaire Vaudois in Switzerland for observation due to leucocytosis. His white blood cell (WBC) count was 232,000/μL, with 130,000/μL neutrophils 69.8%), 2300/μL basophils (1%), 7000/μL eosinophils (3%), 140 × 10%, 2300/μABL1 gene represent the most common resistance mechanism to TKI therapy in CML, occurring in 30–90% of patients who develop resistance to imatinib [p230 CML has been associated with various clinical presentations and courses , 5 with imatinib , with moimatinib . The relimatinib . Similarimatinib .5.0) after treatment with single-agent ponatinib. Recently, patients with E255K/V mutations have been described as having a particularly poor prognosis, regardless of the stage of the disease at detection, with a higher risk of transformation to advanced/blast phase, and a short survival [50 to all available TKIs, including third-generation ponatinb [BCR-ABL1 sub-clones were present at the start of therapy. This observation can be explained by differences in competitive advantage between mutant clones. Indeed, in vitro cell assays showed that selected mutant clones  have higher transformation potency and proliferation rate compared with T315I, even in the absence of BCR-ABL1 inhibitors [BCR-ABL1–positive cells is mainly shaped by TKI-selective pressure and the fitness of each ABL1 KD mutated population is the net result of the ability to survive treatment depending on the intrinsic sensitivity to the specific TKI administered and of the ability to survive the competition with all other coexisting populations [Here we report a case of imatinib resistance due to an E255V mutation, followed by early post-transplant relapse with a T315I mutation that achieved a persistent negative deep molecular response


OBJECTIVES/SPECIFIC AIMS: The NIH states, “The training of the biomedical workforce has always been an integral part of the NIH mission… It takes just one good mentor to influence the career of a new investigator; it takes a robust culture of mentorship across the research community to strengthen, sustain and diversify the entire biomedical research enterprise.” The University of Minnesota’s CTSI-Education core strives to build and maintain a strong culture of mentoring by providing CTSI KL2 scholars an opportunity to mentor an undergraduate student participating in the Pathways to Research Program (PReP). Using this mentoring model, participants gain valuable benefits and CTSI’s culture of mentoring is strengthened. METHODS/STUDY POPULATION: Participating KL2 scholars are matched with a promising PReP scholar for a 12-week mentored research project. The PReP program selects top candidates through a highly competitive application process. Students work in their mentor’s lab full-time, funded by CTSI-Ed. They engage in additional activities together including a mentor/mentee, an interview activity and 2 social events. Junior faculty scholars are asked to participate as judges at CTSI’s Poster Session and are invited to present at PReP seminars. The program culminates with the announcement of the Junior Mentor of the Year, in which scholars nominate their mentors for the award. Junior faculty mentors receive support through a training course, Optimizing the Practice of Mentoring, mentor orientation and a roundtable discussion with the program director and other mentors. The program’s infrastructure is designed to foster mentee/mentor relationships through faculty and staff support. Junior faculty receive one-on-one coaching when faced with difficult mentoring situations and are recognized for their mentoring successes. RESULTS/ANTICIPATED RESULTS: Junior faculty mentors highly rate the program on the following points; the experience was a good use of time, I am satisfied with my experience, I would recommend this program to faculty colleagues and students. Undergraduates and Professional students rated their mentoring relationship as 1 of 3 best outcomes of the program. In exit surveys, their highly rated program successes include having a network that helps move their career forward, and confidence to persist through training to become a successful researcher. DISCUSSION/SIGNIFICANCE OF IMPACT: Creating a culture of mentoring is important to the strengthen, sustain and diversify the biomedical research workforce. This mentoring model contributes to the mission while vertically integrating CTSI-Ed’s KL2 and PReP programs. On an individual level, junior faculty improve communication and management skills, develop leadership qualities, increase their network, provide a sense of fulfilment and personal growth, and reinforce their own skills and knowledge of subject. They are also provided a top undergraduate student worker fully funded by the program.