Postoperative non-union of femoral neck fracture often needs secondary operation. We report a case of a postoperative non-union of femoral neck fracture treated with teriparatide.A young male patient with Garden IV femoral neck fracture who showed no obvious signs of healing 3 months after percutaneous hollow nail fixation in which the fracture line was enlarged and the hollow nail was withdrawn. Bone non-union healed after 6 months of continuous subcutaneous injection of teriparatide at a dosage of 20 mg/day after the patient refused a secondary surgery. As far as we know, there have been no relevant reports on this type of fracture yet.Teriparatide is expected to be beneficial in treating young patients with a displaced femoral neck fracture who have difficulty in healing from non-union and who are keen on avoiding secondary surgery. Femoral neck fractures are uncommon in young adults 3%–10%) and are often caused by high-energy trauma. Anatomical reduction and stable internal fixation are the key to the current treatment for young patients with higher activity and functional requirements 0% and ar. HoweverTeriparatide is a recombinant preparation derived from the first 34 amino acids of the human parathyroid hormone (rhPTH). It has been approved by the United States Food and Drug Administration (USFDA) for the treatment of male osteoporosis , 6. Ther2D3 within the normal ranges, were performed to exclude possible metabolic disorders. A dual-energy x-ray bone mineral density test indicated normal bone mineral density. Finally, the patient was diagnosed with non-union and he consented to empirical, off-label therapy with the approved dose of teriparatide (20 μg/day for osteoporosis) instead of a second surgery. Trabeculae bridging through fracture sites and fracture space reduction were seen on the radiological images (A 45-year-old male patient sustained a Garden IV fracture of the left femoral neck  caused bl images  after 3 l images . X-ray (l images  and CT fl images  showed tl images  when theArthroplasty is generally considered to be the most effective treatment for displaced femoral neck fractures in elderly patients; however, for younger patients, open reduction and internal fixation is considered the gold standard approach . PostopeThe native hip joint should be preserved as far as possible in the treatment of femoral neck non-union for physiologically young patients . There aSome cases related to the treatment of fracture non-union with teriparatide are now being reported , 21, 22.In summary, teriparatide could be used in the treatment of non-union of displaced femoral neck fractures in young patients who are keen on avoiding secondary surgery on the basis of our case and treatment experience. Therefore, additional randomized clinical studies are necessary to clarify the effectiveness and scope of application of the drug.


Family-based therapy (FBT) has been proven effective in treating eating disorders among children and adolescents. However, many families have difficulties implementing the measures recommended in outpatient therapy.This study examines the effectiveness of add-on home treatment (HT) to family based therapy (FBT) in adolescents with anorexia nervosa (AN). The HT intervention is delivered by specialized nurses and aims at supporting patients and parents to re-establish family meals in the home environment.We performed an case-control study in AN patients comparing 44  adolescents receiving FBT augmented with HT compared to 22  participants receiving FBT alone. Eating disorder diagnosis, psychopathology and severity of clinical symptoms were assessed using  and clinical parameters  at baseline and after 3-months.After 3 months both treatment groups showed a significant early weight gain, a reduction in the rate of AN diagnoses assessed with the EDE interview and a reduction in EDI-2 total scores. The combined HT/FBT group showed a significantly greater increase in BMI than the FBT-only group. In the combined HT/FBT group none of the patients had to be admitted to hospital while 13.6% of the FBT-only group had to be referred to inpatient treatment. Treatment satisfaction in the combined HT/FBT group was high in both patients and parents.Our results suggest that HT augmented FBT is superior compared to FBT alone in terms of early weight gain and might reduce the risk of hospital admission in adolescent AN.No significant relationships.


In this work, we test the effectiveness of using highly transparent holographic phase reflection and transmission volume gratings based on multifunctional acrylates as linear compression and rotation sensors. The gratings are recorded in a holographic mixture based on multi-reticulated acrylate and haloalkanes. To activate the photo-polymerization process, we used a mixture of 6-oxocamphore and rhodamine 6G. The mixture is a simplified version of the mixture used in previous works and shows some interesting features mainly in connection with the different roles played by the rhodamine 6G dye at different writing wavelengths  In recent years, the well-established use of holographic gratings as sensors has led to many forms being developed in this research field. Surface relief gratings, inverse opals, metal nanoparticle and nanoparticle-free holographic sensors are used in medical and biological applications  or as huDipentaerythritol-hydroxy-penta/hexa-acrylate (DPHA), halo-alkanes (hexyl-bromide (BH) and butyl-bromide (BB)), 6-Oxocamphor (6OC), rhodamine 6G (RH6G) were purchased from Merck, Darmstadt, Germany.w/w), 1.9% 6OC, 0.05% RH6G are blended together until a homogeneous pale orange colour is observed. The mixture is stirred at room temperature, under dark and aerobic conditions for 6 days and then stored for 90 days, in the same environment before being used.DPHA (≈68%), BH (10%), BB  while an external compression is applied to the cell using a clamp (C). Changes in the reflected signal are monitored by using an optical fiber (OFD) connected to a real-time spectrometer (SP). Detection of the diffracted signal on free-standing samples is achieved by applying rotation to the film containing a transmission grating while a white Xe-lamp-generated light illuminates the sample. The signal transmitted by the grating is acquired through an optical fiber connected to a real-time spectrometer. For each rotation angle The calculated average refractive index of the grating is n = 1.49. The grating pitch at the end of the writing and post-polymerization process for the reflection holograms is rocesses ). A typi= 530 nm . It is rValues around ≈50% were obtained showing a remarkable FWHM of 5 nm, using incoherent white light. This measured FWHM value shrinks by 0.7 nm when full compression is applied to the sample. The corresponding high value of angular selectivity represents an optimal feature for the detection of small-wavelength shifts due to the application of compression on both sides of the cell and near the grating area. As reported in At the same time, the grating diffraction efficiency decreases to a minimum value as shown in By completely removing the applied force, the system gradually returns to the initial condition as reported in Unfortunately, the time needed to restore the initial condition limits the use of our halo-alkane/acrylate-based mixture as compression sensors. As a consequence, we decided to test the device in reflection mode and in a free-standing configuration. The free-standing films are prepared by following the procedures given in The free-standing film containing the transmission grating is clamped on the two edges and subjected to an external linear displacement (L) by using a small vertical rod, the section of which is represented in The changes in diffracted wavelengths are monitored in real time by acquiring the transmission spectra of the sample at different rotation angles. A typical result is reported in Finally t system . In prinIn conclusion, we investigated the use of a haloalkane–acrylate mixture as a compression and rotation sensor. The mixture is a simplified version of a mixture used in previous works and in different applications. Reflection and transmission gratings were recorded and employed to detect small compressions and rotations. Transmission volume holographic gratings are used in a free-standing configuration producing sensitivity values up to 159 nm/rad. We believe that these specific gratings can find application in fields requiring detection of small compressions and rotations.


Conclusion: The geometry of the thoracic aorta showed no influence on the positioning of self-expanding TAVI valve types.Background: The impact of transcatheter heart valve (THV) position on the occurrence of paravalvular leakage and permanent pacemaker implantation caused by new-onset conduction disturbances is well described. The purpose of this study was to investigate the influence of the geometry of the thoracic aorta on the implantation depth after TAVI  using self-expanding valve (SEV) types. Methods: We evaluated three-dimensional geometry of the thoracic aorta based on computed tomography angiography (CTA) in 104 subsequently patients receiving TAVI with SEV devices (Evolut R). Prosthesis position was determined using the fusion imaging method of pre- and post-procedural CTA. An implantation depth of ≥4 mm was defined as the cut-off value for low prosthesis position. Results: The mean implantation depth of the THV in the whole cohort was 4.3 ± 3.0 mm below annulus plane. THV position was low in 66 (63.5%) patients and high in 38 (36.5%) patients. After multivariate adjustment none of the aortic geometry characteristics showed an independent influence on the prosthesis position—neither the Sinus of Valsalva area ( As the number of TAVI procedures continues to grow worldwide, increased knowledge about risk factors for possible complications is crucial for any specific valve design. The impact of the position of the transcatheter heart valve (THV) on the occurrence of paravalvular leakage (PVL) and permanent pacemaker implantation caused by new-onset conduction disturbances (CD) is well described .The fusion imaging method of pre- and post-procedural computed tomography angiography (CTA), published by our group, allows a three-dimensional visualization of the THV within the native annulus plane post TAVI . Using tA reduced calcification of the device landing zone is known as predictor of a low prosthesis position in balloon-expandable valves (BEV) . HoweverTherefore, we aimed to examine the influence of the thoracic aorta geometry on the implantation depth after TAVI using SEV devices.Candidates for inclusion in this study were all patients with implanted newer generation SEV  at our institution between January 2015 and June 2020 and an evaluable post-TAVI CTA. As previously described, we perform routine post-TAVI CTA in all eligible patients in our institution ..7].The aortic angle was assessed as the angle between annular plane and ideal horizontal plane A 5]. Ad. Ad5]. AThe angle of ascending aortic curvature is defined as the angle between the perpendicular line to the annular plane and the perpendicular line to the cross-sectional plane of the aorta at the level of the brachiocephalic artery ,9.For the aortic apex angle we first determined the center points of the ascending (A) and descending aorta (D) at the mid-level of the right pulmonary artery flow ,11. The Additionally, we measured the axial distance between A and D (= ascending descending distance) and between the central points of the annular plane and the descending aorta at the transverse level of the annular plane (= annulus descending distance).t-test  or the Mann–Whitney-U Test  to test differences between the groups with high and low THV position. Univariate and multivariate logistic regression models were performed to assess possible predictors for a low prosthesis position. All variables with a p-value < 0.05 in the group comparison were included in the univariate and—if significant—in the multivariate logistic regression models. When multiple variables were directly related , we chose the variable with the lowest p-value. A two-sided p-value of <0.05 was defined as statistically significant in all tests.Statistical analysis was performed using SPSS software, Version 25.0  and MedCalc, Version 19.4 . We used the χ2-test , the Student’s In total, 118 consecutive patients received newer generation SEV and post-TAVI CTA within the study period. The image quality of post-TAVI CTAs was too poor in 2 patients; 12 further patients were excluded due to a valve-in-valve procedure or a surgical revision caused by a THV dislocation. Finally, we included 104 patients. Details of the study population were described elsewhere [p = 0.013), ascending aorta  and proximal aortic arch . Additionally, we found a longer distance from annulus to descending aorta  and longer distance from ascending to descending aorta  in this group.Using an implantation depth of ≥4 mm as cut-off value, THV position was low in 66 (63.5%) patients and high in 38 (36.5%) patients. All aortic geometry characteristics are presented in p = 0.668). Furthermore, the aortic angle (p = 0.733), length of ascending aorta (p = 0.150) or aortic apex arch angle (p = 0.334) were similar between patients with high and low prosthesis position.Most patients showed an aortic arch type II (78.8%) without any influence on the implantation depth (We included the Sinus of Valsalva area, maximum diameter of ascending aorta, mean diameter of proximal aortic arch, as well as the annulus and ascending descending distance in the logistic regression models. After multivariate adjustment none of the aortic geometry characteristics showed an independent influence on the prosthesis position .In this investigation we examined the influence of aortic geometry on positioning of the self-expanding Evolut R TAVI prostheses using 3D fusion imaging of pre- and post-TAVI CTA. Our results demonstrate that none of aortic geometric features is associated with a low prosthesis position, possibly generating new conduction disturbances. Hence, a detailed assessment of the aortic anatomy seems not to be mandatory before implantation of these prostheses.While the association between THV implantation depth and the occurrence of TAVI complications is well described ,2,12,13,Up to now, only the impact of aortic angle on THV position was assessed . ContrarTo the best of our knowledge, we are the first group to investigate the influence of the complex geometry of the thoracic aorta on THV using SEV. Using fusion imaging with its 3D capacity we were able to demonstrate that THV positioning in SEV is unaffected by specific aortic geometric features. Therefore, we conclude that a separate assessment of aortic geometry is not crucial when planning the procedure in SEV.The limited sample size of our cohort limits the power to identify minor anatomic predictors for a low prosthesis position. Nevertheless, the statistical analysis seems sufficient to conclude that the aortic geometry has no major influence on the THV implantation depth. Moreover, our study was limited to patients with one specific SEV type. It remains speculative, if these results are transferable to other SEV types or BEV.The geometry of the thoracic aorta showed no influence on the positioning of self-expanding TAVI valve types.Prevention of a low prosthesis position after TAVI is desirable to reduce the occurrence of new conduction disturbances. The results of our current study imply that a separate analysis of the thoracic aortic geometry is not mandatory when planning the TAVI-procedure using SEV.


Background: Many internal medicine residency programs have incorporated ultrasonography into their curriculum; however, its integration with physical examination skills teaching at a graduate medical level is scarce. The program’s aim is to create a reproducible elective that combines physical exam and bedside ultrasound as a method for augmenting residents’ knowledge and competence in these techniques with the ultimate goal of improving patient care. Methods: We designed and implemented a two-week elective rotation for senior internal medicine residents, combining evidence-based physical examination with diagnostic bedside ultrasonography. The rotation took place in an inpatient setting at Denver Health Hospital. Program evaluation data was collected data between February 2016 to March 2019. IRB approval was waived. Results: Since its inception in 2016, 19 residents completed the rotation. Residents performed a pre-test and a post-test under direct observation by course faculty. Each resident was measured on the ability to perform pre-determined physical exam and point-of-care ultrasound (POCUS) skills. In the pre-test, participants correctly performed an average of 40% of expected physical exam maneuvers and 32% of expected POCUS skills. At elective conclusion, all participants were effectively able to demonstrate the highest yield physical exam and ultrasound maneuvers. Discussion and Conclusion: An elective designed specifically to integrate POCUS and physical exam modalities improves the ability of resident physicians to utilize both diagnostic modalities.  Medical schools and training programs in the United States have shifted emphasis away from physical exam teaching A review by Oliver et. al. Additional motivators for our elective include: 1) the expansion of POCUS into inpatient medicine; 2) a national trend to incorporate ultrasound Internal Medicine residency training programs Acknowledging the persistent value of the physical exam while recognizing the need to teach ultrasound skills to future physicians, we sought to synthesize the subjects into a two-week “Master Clinician” elective. We designed and implemented a two-week elective, offered yearly since 2016, combining teaching of EBPE skills with POCUS. In order to perform the program evaluation, we collected and analyzed data between February 2016 and March 2019. Six faculty members from the division of Hospital Medicine at Denver Health Hospital Authority (DHHA), an Internal Medicine Residency Program (IMRP) affiliated site for the University of Colorado, School of Medicine (CUSOM) facilitated the curriculum and taught the content at DHHA. The elective was offered for up to six PGY-2 and PGY-3 categorical Internal Medicine residents in the CUSOM IMRP per course iteration. Since inception, 19 residents have participated. On average, 20 were on the waitlist each year.The program’s main objective was to create a reproducible and effective elective rotation combining evidence-based physical exam (EBPE) and POCUS as a method for cultivating resident’s knowledge and competence in these techniques, ultimately leading to improved quality of care and patient safety. The program’s aims included: 1) use of case-based-learning to identify gaps in residents’ skills; 2) employ direct observation to provide learners real-time, targeted feedback; 3) analyze published literature on discussed topics; 4) correlate ultrasound with physical examination findings; 5) provide guidance on how to integrate this information into clinical practice 6) test newly acquired knowledge and skills through near-peer teaching.By the end of the elective rotation, participants were able to: 1) understand the utility, importance, and evidence behind common and uncommon physical exam maneuvers; 2) demonstrate the ability to apply likelihood ratios (LR) to modify diagnostic probability; 3) exhibit the aptitude to implement an EBPE by correctly performing high-yield physical exam maneuvers, accurately identifying the presence of pathology during examination, and synthesizing signs with clinical decision making; 4) confirm or challenge clinical diagnosis by skillfully performing high yield POCUS maneuvers; 5) utilize POCUS to increase success rates and decrease complications of medical bedside procedures. Institutional Review Board approval was not required for this study.To ensure integration, each instructional day provided EBPE teaching and POCUS training sessions focused on a single content area. We organized each day as follows: one hour of independent reading and literature review, followed by one-hour long interactive EBPE didactic. This was followed by two hours of EBPE bedside-rounding led by faculty on prescreened patients with pathology relating to that day’s topic. In the afternoon, interactive pedagogies continued with a one-hour POCUS didactic session, followed by two and a half hours of POCUS rounding, led and supervised a trained faculty. Session topics (Table 1) and clinical cases were selected based on the relevance to hospital medicine (commonly encountered diagnosis) and on published literature in EBPE and POCUS (Table 2). During EBPE didactics faculty highlighted the limited diagnostic value of commonly performed exam maneuvers, subsequently discussing and demonstrating specific exam maneuvers with the highest diagnostic yield. Course participants practiced these maneuvers under direct observation of faculty while receiving feedback. POCUS content focused on: image acquisition, identification of pathologic findings, and integration into clinical reasoning. Image acquisition was taught systematically for each application and can be deconstructed as follows: 1) visualization during a short didactic lecture; 2) hands-on guided scanning with one of the two POCUS content faculty; 3) practicing during unguided free-scanning; 4) quality assessment guided by POCUS faculty. Integration of POCUS into clinical reasoning aimed to supplement the EBPE using systematic approaches to scanning in each content area and body region. Course participants then demonstrated POCUS knowledge acquisition at course end by preparing and presenting key concepts to their peers during a noon conference, with POCUS faculty on-hand to guide discussion and ensure accuracy. Enrollment was capped to optimize the participant to attending ratio, which was maintained at a 5:1 ratio.To overcome a common challenge of identifying patients with relevant pathology, our faculty developed reporting tools within our Electronic Medical Record (EMR- Epic) to identify patients with diagnoses relevant to the course. We combined quantitative and qualitative measures. Beginning in 2016, all participants underwent a pre-test and post-test which involved direct observation of learners conducting the physical exam, followed by real-time feedback with the goal of motivating them to improve their physical examination skills. For the pre-test, participants were asked to perform a comprehensive cardiac examination for a patient with suspected heart failure, a comprehensive pulmonary exam for a patient with suspected pneumonia, a targeted exam for cirrhotic patients, and a focused neurologic examination. They were directly observed using a standardized checklist  to assess whether they correctly perform physical exam maneuvers recognized in the literature to have good predictive value for heart failure, pneumonia, and complications of cirrhosis.In 2019, the three participants that completed both pre- and post-tests —correctly performed an average of 3.3 of 9 (range 0-7) components related to visual assessment of JVP in the pre-test and 4.7 in the post-post (range 4-6). Only 1 of 3 assessed the point of maximal impulse (PMI) in the pre-test; all 3 assessed PMI in the post-test. In the pre-test, no participant correctly assessed for egophony or asymmetric pulmonary expansion; all 3 assessed both in the post-test. Cirrhosis inspection and palpation scores improved from 0.67(range 0 -2) to 4.67 (range 3-6) (of 6) and 2 (range 1-3) to 4 (range 3-5) (of 5) pre- to post-test, respectively (Table 3). A twenty-six point checklist created by the POCUS faculty was used to perform a pre-elective hands-on cardiac and lung POCUS skills assessment. The US skill checklist is shown in Figure 2. This assessment tested knowledge and performance of ultrasound probe and machine functions, relevant anatomy focused on standard views of the heart and lungs, and basic diagnostic POCUS assessments of these organs. The median pre-test score was 9 out of 26 possible points. At the conclusion of the elective, participants prepared and delivered small group teaching sessions to junior learners, discussing the evidence-based elements of the cardiac and pulmonary examinations. They also demonstrated and led learners in the proper examination techniques on actual patients. Their teaching was directly observed, and they received feedback on their teaching techniques. All participants were effectively able to describe the pathophysiology underlying abnormal exam findings, demonstrate the highest yield physical exam maneuvers, and explain the method of acquisition and clinical implications of basic cardiac and pulmonary POCUS. At the end of the elective the POCUS hands-on skills assessment was repeated with an improvement of the median to 26 of 26 points. We also included components not covered in the course (neurologic examination) to serve as a control measure for our pre- and post-test exams. The average score on the neurologic pre-assessment was 3.67 (range 3-5) and the post course assessment average score was 2.67 (range 2-3).The Master Clinician Elective underwent continuous comprehensive evaluation following the Plan-Do-Check-Act (PDCA) format, widely known as a strategic planning modality. Learners were asked to provide constructive feedback on rotation structure, individual didactic sessions, and faculty at course end. In the era of high value care, we believe it imperative for all future practitioners to master EBPE and POCUS skills. Because faculty and ultrasound resources are limited, we are working to expand our ability to teach this important content through both internal faculty development and partnering with other IMRP clinical sites. Our data clearly shows a marked improvement of learners in both their physical exam and POCUS skills.While our elective is a collaboration of four physical exam-focused providers and two POCUS providers, it could feasibly be run with as few as 1 of each with enrollment limited to four participants. One of the challenges for our faculty and for faculty at other programs is limited protected time for POCUS education. Our study demonstrates the value of this elective, which should support decisions regarding allocation of protected time for faculty to dedicate to POCUS education. This elective requires the availability of bedside ultrasound technology and faculty with a level of expertise to accurately teach bedside ultrasound applications. In our experience, learners require approximately 1 machine for every 2 to 3 learners to have adequate scanning time to achieve course ultrasound goals. Our POCUS expert faculty collaborated in the curriculum design and reflected their experience as hands-on educators at a national level. They are invited speakers and faculty at annual conferences for the American College of Physicians, Society of Hospital Medicine (SHM), American College of Chest Physicians (CHEST), and Society of General Internal Medicine. A low number of learners per machine is a design tool used at the national level to maintain learner engagement and maximize individual teachable moments for this hand-eye skill. The incorporation of unguided scanning time with post-image acquisition quality review mimics on a small-scale the portfolio building processes used by both CHEST and SHM certification programs.Our study is single-center and, by design, small in size. We believe it would be easily reproduced at other institutions, with limitations. The limitations are, in order of anticipated impact, a requirement of faculty skilled and capable of teaching POCUS and assessing skill and image acquisition of novice learners, access to ultrasound devices, faculty capable and skilled in teaching EBPE. While there are standards for what constitutes quality image acquisition and interpretation, there is inherent subjectivity in a bedside assessment of the learner. Furthermore, the assessment of learners was not blinded and was conducted by course faculty, leading to a possible bias of skills assessment and is a limitation of our findings.Other considerations include the fact that we did not assess skill decay. The optimal frequency of “refresher” courses that would allow knowledge and skill retention is yet to be determined. We recognize that many GME programs offer POCUS training An elective designed specifically to integrate POCUS and physical exam modalities improves the ability of resident physicians to utilize both diagnostic modalities. This elective enhances clinical reasoning by weaving traditional EBPE with novel POCUS, however little is known regarding the clinical impact of this training paradigm. How would this change medical imaging ordering practices? Will the positive yield of these orders increase? Will this process decrease or increase length of stay and overall hospital cost? These are amongst some of our high priority questions.None declared.


Citrus tristeza virus was employed successfully to create an infectious clone that was used to silence endogenous citrus genes. Phytoene desaturase (PDS) and delta (δ)-aminolevulinic acid dehydratase (ALAD) were targeted successfully in citrus. Silencing PDS usually results in a photo-bleached leaf phenotype while silencing ALAD causes discrete yellow spots in leaves. Silencing two or more genes simultaneously using the same infectious clone could be difficult due to the capacity of the plasmid and subsequent cloning. On the other hand, inoculating a new construct into a citrus plant pre-infected with another construct fails due to the superinfection exclusion phenomenon. Herein, I report our successful trials whereby we simultaneously graft-inoculate constructs targeting PDS and ALAD. The budwoods were graft-inoculated into the same tree but on two different branches. Interestingly, a new phenotype was produced because of the silencing of the two genes, which we called “color-breaking”. The phenotype was observed in both branches. Gene expression analysis showed a significant reduction of PDS and ALAD transcripts. This finding suggests the possibility of targeting more than one gene using different constructs, however, the graft-inoculation must be at the same time.Virus-induced gene silencing is a promising technique for functional genomics studies.  The complex recognizes and guides the degradation of the homologous regions in the viral RNA.5Virus-induced gene silencing technique (VIGS) has been commonly employed to study the functional genomics of genes in plants.6 The efficiency of the VIGS technique depends on the movement and the distribution of viral particles within the plants.1 Therefore, it is suggested to use an extra reporter gene such as phytoene desaturase (PDS) to measure the amount of silencing of the targeted gene.7 Silencing the reporter gene usually causes a specific phenotype, photobleaching, in the case of PDS.7The VIGS vectors are created by cloning the entire genome of a plant virus into a backbone plasmid. By integrating a truncated sequence of the host gene, the defense machinery will target the corresponding mRNAs and cause downregulation of the gene expression.Citrus tristeza virus (CTV), a member of the genus Closterovirus, to knock down endogenous genes. The use of this infectious clone provides many advantages: (i) it does not provoke any symptoms in inoculated citrus; (ii) it can express exogenous genes in citrus and remains for years;8 (iii) it can be used in the field to protect citrus from pests or diseases;9 and (iv) more than one exogenous gene could be inserted into the infectious clone simultaneously.10 Previously, we demonstrated that the wild-type CTV-T-36 (CTV-wt) does not cause any dramatic effects on the phloem sap composition and released volatiles of Citrus macrophylla.11In citrus, VIGS is accomplished using the mild strain T-36 of PDS), a key plant enzyme involved in carotenoid biosynthesis.12 Silencing the PDS gene in citrus causes “photo-bleached leaf” phenotype (12 Silencing the citrus PDS gene reduces the production of carotenoids and accumulates the precursor phytoene (PDS-as) causes a stronger photobleached-leaf phenotype compared to the sense orientation (CTV-tPDS).12 Although the expression of PDS gene was down-regulated when sense and antisense orientations of truncated sequences were used to build the construct, superior silencing of PDS and stronger phenotype were achieved with the antisense orientation due to the production of subgenomic RNAs that complemented the small interfering RNAs.12Among the genes targeted using CTV is the reporter gene, phytoene desaturase -aminolevulinic acid dehydratase (ALAD), which is a key enzyme in tetrapyrrole synthesis, and an important intermediate for chlorophyll biosynthesis using CTV.islands” . In addinic acid .13One major challenge in the VIGS technique is targeting more than a single gene at once due to the limited capacity of the infectious clone plasmid to contain long inserts. This fact may lead us to think about using two different constructs each has a single truncated gene sequence. However, the phenomenon of homologous interference, also known as superinfection exclusion, may occur and one of the constructs may be excluded. The phenomenon of superinfection exclusion is well known in viral diseases, wherein the first established viral infection blocks a secondary infection with the same or a closely related virus.14 It was shown that superinfection exclusion happened only between isolates of the same strain. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided perfect exclusion of the second isolate. For example, primary infection with CTV-T36 prevented superinfection with the infectious clone T36-based GFP-expressing CTV (CTV-T36-GFP).14 This phenomenon has been well employed for the cross protection as a strategy to prevent the second infection in the field.14For CTV, the phenomenon has been reported and well-studied.C. macrophylla with both CTV-tALAD-as and CTV-tPDS-as to test if we could repress two genes using two different constructs of the CTV-T-36 based infectious clone. The source trees infected with CTV vectors were produced in our previous work.3 Briefly, the CTV constructs were agroinfiltrated into Nicotiana benthamiana followed by virion purification using a sucrose cushion gradient, and purified virions were bark-flap inoculated into C. macrophylla . In these trees, we did not observe the newly grafted phenotype, only the original phenotype for each group. Therefore, we decided to perform another trial grafting both constructs onto the same healthy plant simultaneously. We created “Y-shaped” trees by selectively trimming branches to form two distinct main branches. On these trees, both branches were side-grafted, one branch with budwood harboring the CTV-tPDS-as and the other with budwood harboring CTV-tALAD-as (In the first trials, we grafted budwood from tALAD-as . After atALAD-as . The dev15 The CTV populations were very comparable in all treatments (PDS, ALAD, and genes implicated in their pathways was carried out as described in previous studies.3 The expressions of PDS and ALAD in the double-inoculated plants were reduced confirming the RNAi effect (CTV titers were measured using quantitative PCR as described by Harper et al.eatments , suggesti effect . In facti effect .Figure In summary, this work suggests that simultaneous inoculation with two constructs of the same infectious clone containing two different genes allows repression of the expression of both and causes effective RNA interference. The work provides a simple solution when a virus infectious clone plasmid cannot carry more than one truncated gene, and when the goal of the study is to knockdown two genes. This solution will help in functional genomic studies, especially those exploring the relationships between two biosynthetic pathways.


The Renaissance of Clinical Proteomics: Biomarkers, Imaging and Therapeutics”. This time, the second AOHUPO Online Education Series was hosted by the UKM Medical Molecular Biology Institute (UMBI) affiliated to the National University of Malaysia (UKM) in Kuala Lumpur, Malaysia on three consecutive Fridays . More than 300 participants coming from 29 countries/regions registered for this 3-days event. This event provided an amalgamation of six prominent speakers and all participants whose interests lay mainly in applying MS-based and non-MS–based proteomics for clinical investigation.In 2021, the Asia-Oceania Human Proteome Organization (AOHUPO) initiated a new endeavor named the AOHUPO Online Education Series with the aim to promote scientific education and collaboration, exchange of ideas and culture among the young scientists in the AO region. Following the warm participation, the AOHUPO organized the second series in 2022, with the theme “ •The AOHUPO organized the second AOHUPO Online Education Series in March 2022.•An amalgamation of speakers and participants in clinical proteomics was provided.•The AOHUPO continues this event online that brings AO researchers together. In 2021, the Asia-Oceania Human Proteome Organization (AOHUPO) initiated the AOHUPO Online Education Series (AOHUPO OES) with the aim to promote scientific education and collaboration and exchange of ideas and culture among the young scientists in the AO region. Following the warm participation, the AOHUPO organized the second series in 2022, with the theme “The Renaissance of Clinical Proteomics: Biomarkers, Imaging and Therapeutics.” This event provided an amalgamation of six prominent speakers and all participants whose interests lay mainly in applying MS-based and non-MS-based proteomics for clinical investigation. The organizing committee received registrations from 334 participants who came from 29 countries/regions . Almost The Challenge of Hypothesis Building from Multi-Omics: The Case of Alzheimer's Disease”.The second AOHUPO OES was jointly open by Professor Ho Jeong Kwon, the President of AOHUPO, and Associate Professor Nor Azian Abdul Murad, the director of UKM Medical Molecular Biology Institute; both also acted as cochairs for Professor Yoshiya Oda (University of Tokyo)’s plenary talk entitled “Professor Oda first introduced proximity extension assay (PEA), which is more sensitive in accessing the low-abundance portion of the plasma proteome (<1 ng/ml) than LC-MS , 3. He dMany participants were intrigued. Among the questions raised include how such multi-omics study can assist treatment/prevention of AD, and if existing drugs for treating blood vessel leakage would be effective for treating AD. Another panellist, Professor Maxey Chung wondered if the little overlap between LC-MS and PEA datasets would affect final data integration. Meanwhile, a young scientist from Malaysia was interested to know if a longitudinal study was available. Further, since blood vessel leakage is often linked to inflammation, this might point the likely association of AD with inflammation. Prof. Oda concluded that multi-omics integration of AD is still at an early stage, whereby the main bottleneck arises from sample availability which constraints longitudinal. Also, PEA is specifically designed as panels that target a certain selection of low-abundance proteins; therefore, it can be biased. On the other hand, LC-MS adopts a discovery approach that tends to detect higher abundance proteins. Therefore, both methods should be viewed as complementary.Exploring the Uncharted Proteome”. Proteins are the closest surrogates to functional phenotypes, though contemporary proteomics research falls short of proteome-wide investigation of the proteoforms and protein-protein interaction (PPI) networks , which mines existing bottom-up DIA datasets to reveal the alternative spliceforms among different conditions (Professor Ruedi Aebersold (ETH Zurich) delivered the second talk - “networks . Furthernetworks . With thnetworks . Applyinnditions . It doesnditions  Fig. 2)ExploringAmong the interesting questions from the audience include (i) how proteolytic digestion and its effects on protein coverage affect the detection of proteoforms and whether (ii) digestion with multiple proteases would improve the COPF algorithms. Prof. Aebersold explained that while it would be ideal (but rarely) to achieve 100% protein coverage, the use of DIA improves the reproducibility of peptide detection such that if a missing peptide is detected in a given sample, it can be used to formulate a new hypothesis and to test the presence of a new proteoform—be it phosphorylated, spliced, out or carrying a mutation.The Human Protein Atlas – spatial proteomics in health and disease”, introducing The Human Protein Atlas . She focused on the human Tissue Atlas, which contains immunohistochemistry images of tissue microarrays in single cell resolution, as well as quantitative transcriptomics data for each protein-coding gene  presented “ing gene . Currenting gene . Bulk tring gene .via the upper airway including the nose and bronchus before it enters the lungs. Meanwhile, investigation of the microbiome is still not a part of the HPA. Another audience inquired if there are highly expressed mRNAs or proteins that do not correlate with the corresponding tissue or organ functions. Dr Lindskog concurred and informed that HPA did find mRNAs and proteins that do not agree with their functions as documented in the UniProt; and research is still underway to resolve this discrepancy. The President of HUPO, Prof. Yu-Ju Chen commented that HPA is a useful resource used by many colleagues for their research and publication. She hoped that Dr Lindskog can organize a HPA training course for this region, which Dr Lindskog gladly agreed.The session cochair Prof. Hsueh-Fen Juan was particularly surprised that ACE2 was expressed only at low levels in the lungs although it is a receptor for SARS-CoV2. She further questioned if Dr Lindskog had plan to also investigate tissue-specific microbiome. According to Dr Lindskog, SARS-CoV2 had been shown to first enter human body The Spatial Organization of the Cell”. Proteins and RNAs must traffic to the correct cellular localities for proper functions; whereas aberrant localizations may end up in diseases. Prof. Lilley studies how proteins/RNAs end up at the right addresses; how biomolecular features affect their localities, and whether proteins/RNAs carry out the same functions at different locations. Her lab develops methods to characterize the localizations of proteins and RNA in a system-wide manner. To assign the proteome to correct subcellular addresses, an ultracentrifugation-based subcellular fractionation method called LOPIT  is used, where similar correlation profiles of proteins among the consecutive ultracentrifuged fractions indicate organelle colocalization (Professor Kathryn Lilley (University of Cambridge) presented “lization . LOPIT clization , 14. Funlization . Intereslization . Both LOlization .The discussion panel and audience raised several questions to this thought-provoking topic, including (i) how LOPIT and LoRNA can be used to study neurodegenerative diseases; (ii) how RNA binding can contribute to the relocalization of proteins; and (iii) the possibility of RNAs acting as a molecular chaperone to assist protein folding. Prof. Lilley explained that RNA–protein interaction is a widely observed phenomenon, including many metabolic enzymes. However, it is still early to tell if the functions of such RNA-protein are linked or that they serve other moonlighting purposes.Exploring Drug-Target-Phenotype Interaction with MS-based Proteomics Approaches and its Translational Impacts”, Professor Ho Jeong Kwon (Yonsei University) described how he chose small molecules as tools to explore the functional proteome for therapeutic application. The key strategy in his lab involves in a step-wise manner: (i) cell-based phenotypic screen with chemical libraries to identify target small molecules that can perturb a selected phenotype; (ii) chemical proteomics to identify protein targets that bind to these target small molecules; (iii) validation of drug-protein binding with structural bioinformatics and activity and functional assays; (iv) designing better drugs and establishing platform to expedite drug discovery; thus providing (v) feedback to discover new drugs targeting new biological process ExploringProf. Shamshad from Pakistan commented that Prof. Kwon workflow was unique as most similar studies would start with computational simulation such as molecular docking before wet lab. Prof. Maxey Chung questioned about the chemical libraries used. According to Prof. Kwon, he prefers to first use wet lab approach to identify real drug targets using proteomics before validating them with computational approaches, if protein structures are available. He also mentioned that the quality of a chemical library is essential. In his case, he prefers a knowledge-driven approach in selecting lead compounds that have well-known traditional efficacy and are sourced from the local environment but with unknown protein targets.Identification of HLA ligands through MS-based Immunopeptidomics for Development of Personalized Cancer Immunotherapy”, Dr Michal Bassani-Sternberg (Ludwig Institute of Cancer Research in Lausanne) introduced MS-based immunopeptidomics for tumor antigen discovery, in unraveling the tumor-associated antigens  of the purification method that relies on pan-HLA antibodies. As of now, noncanonical peptide sequences have not been included for clinical studies and Dr Bassani-Sternberg also found little immunogenicity among them; and she added that the three most important pieces of information to consider in developing the AI algorithm are the detection of mutation and expression levels with RNA-seq and the binding affinities of HLA peptides.https://www.aohupo-aoapo-2023.org/). We are looking forward to the first successful face-to-face AOHUPO Congress since the COVID-19 pandemic.The second AOHUPO OES was very well-received especially among the early career researchers. During the postmortem of the event, several proposals have been put forward, including: (i) to encourage AO young scientists to organize the events themselves, (ii) to split the event into separate theoretical and lab demo sessions, as well as (iii) to rotate the organizers among the AO regions. The AOHUPO decided that this event should be continued and should remain online even though the threat from COVID-19 had subsided, since it is an excellent platform that brings AO researchers together for knowledge sharing. Meanwhile, the AOHUPO also organizes a biennial on-site event called the AOHUPO Congress. In 2023, the Joint 11th AOHUPO and seventh AOAPO  will be co-organized by the Singapore Society of Mass Spectrometry (SSMS) and the Malaysian Proteomics Society (MAPS). The Congress will take place in Singapore on 8th-10th May 2023 (The authors declare that they have no conflict of interest with the contents of this article.


Background:To measure the impact of an intensive eight-week postgraduate year one (PGY-1) otolaryngology bootcamp on the acquisition and retention of otolaryngology residents’ procedural skills compared to the traditional method of skill acquisition through clinical exposure.Methods: Residents at our institution were evaluated on their performance of flexible laryngoscopy, suture ligature, and rigid bronchoscopy setup at three time points: pre-bootcamp, one-week post-bootcamp, and one-year post-bootcamp. Video recordings were scored by two blinded faculty reviewers using a multipoint rating system. A control group of rising postgraduate year two (PGY-2) residents who did not participate in bootcamp were recorded performing these same skills. Scores in the three skills were compared between groups viat-tests. The eight-week bootcamp curriculum for PGY-1s was held at the Montefiore Einstein Center for Innovation in Simulation at Albert Einstein College of Medicine/Montefiore Medical Center. The participants were two classes of PGY-1 residents (n=8) at our institution who participated in a bootcamp at the beginning of residency, and one class of rising PGY-2 residents (n=3) who did not participate in a bootcamp (control group).Results: A comparison of pre-bootcamp scores to one-week post-bootcamp scores showed significant improvement in suture ligature (P<0.05) and rigid bronchoscopy (P<0.05), but no difference in flexible laryngoscopy (P=0.54). Suture ligature (P=0.09) and rigid bronchoscopy (P=0.25) skills were not significantly different from one-week post-bootcamp to one-year post-bootcamp; however, a significant skill improvement was observed in flexible laryngoscopy (P<0.05). By June of PGY1 year, the two bootcamp cohorts were similar to controls in all three skills: flexible laryngoscopy (P=0.05), rigid bronchoscopy (P=0.26), and suture ligature (P=0.10).Conclusions: Participation in PGY-1 bootcamp was associated with improved acquisition and short-term retention of basic procedural skills, suggesting that bootcamps can be an effective arena to teach basic skills in otolaryngology. PGY-1 bootcamp is a promising arena for multi-institutional development. Simulation is widely used in otolaryngology, with 82% of otolaryngology residency programs reporting some use of simulation at their institution. The immediate purpose of bootcamp is to increase residents’ comfort and competence with basic knowledge and technical skills relevant to the field of otolaryngology with the goal of improving patient safety. Bootcamps have demonstrated success in increasing resident confidence and training efficiency, though their impact on the development of technical skills is underexplored.In the field of otolaryngology, several institutions have developed simulation-based bootcamps for resident education. Bootcamps are defined as short, intensive training sessions , which teach or reinforce technical skills, clinical knowledge, and team dynamicsOur institution developed a unique eight-week bootcamp curriculum for postgraduate year one’s (PGY-1) to jumpstart their education at the beginning of residency. It is novel in the extended timeline of eight weeks and the evaluation of task-based skills as an outcome metric. We designed and implemented the extended bootcamp experience with goals of (1) increasing competency in basic procedural skills, (2) improving proficiency in ENT emergencies, and (3) expanding basic otolaryngology knowledge. We sought to objectively evaluate resident technical ability before and after participation, hypothesizing that scores in flexible laryngoscopy, suture ligature, and rigid bronchoscopy set-up would increase after bootcamp and be retained one year later. We also hypothesized that residents at the beginning of postgraduate year two (PGY-2) who had participated in bootcamp as PGY-1s would demonstrate better technical skills than a control group of early PGY-2 residents who had not participated in bootcamp.Meeting information: This work was presented at the Triological Society Combined Otolaryngology Spring Meeting, Austin, Texas, May 4, 2019.The study was approved by the Einstein Institutional Review Board (2018-9818). Resident participation in the study was voluntary and all participants provided written informed consent.. Here we focus on the assessment of procedural skill acquisition and retention as evaluated with three task trainer modules: flexible laryngoscopy, suture ligature, and rigid bronchoscopy set-up. A total of eight PGY-1 members from two separate years participated. The study started in June 2016 and ran through August 2019.Faculty and senior residents in the Otolaryngology—Head and Neck Surgery Department at Montefiore Medical Center contributed to curriculum design. Bootcamp took place over eight weeks during the first summer of residency at the Montefiore Einstein Center for Innovation in Simulation at Albert Einstein College of Medicine/Montefiore Medical Center in Bronx, NY. Modules held daily from 8am to 12pm incorporated multimodal learning techniques including lectures, task trainer stations, simulated scenarios, and debriefing sessions. The last two days were dedicated to evaluation in the form of a written test and skills assessment. The bootcamp has since been redesigned into weekly sessions held on Friday mornings during the first two months of the year. The entire curriculum of bootcamp is beyond the scope of this paper and has been previously described by our groupTwo faculty members  taught the majority of modules, with assistance from senior (postgraduate year four [PGY-4] and postgraduate year five [PGY-5]) residents.We chose three tasks which incorporate fundamental skills in otolaryngology including airway evaluation (flexible laryngoscopy), hemorrhage control (suture ligature), and airway intervention (rigid bronchoscopy). We chose these procedures because they are fundamental to our field and incorporate a variety of skills including visuospatial ability, dexterity, and memorization.Flexible laryngoscopy. In this module, participants performed flexible laryngoscopy on a mannequin using a 3.5 mm flexible laryngoscope with an attached Stryker HD camera and light source. The image was displayed on a Storz camera tower during the procedure.Suture ligature. Participants were provided a variety of instruments with which to perform suture ligature of a mock blood vessel represented by a 16 French red rubber catheter.Rigid bronchoscopy setup. This module challenged participants to assemble an array of rigid bronchoscope parts into proper functioning format. Bootcamp participants were filmed at three time points: before beginning bootcamp (in the first week of intern year), one week after completing bootcamp (in September of intern year), and one year after bootcamp (in the first weeks of PGY-2 year). During filming sessions, one resident at a time entered the room and was filmed with an iPad while they performed each task.. Items which were not applicable to a mannequin (as opposed to a human patient) were removed, including obtaining consent, patient positioning, and application of local anesthesia  reviewed and rated videos of the residents’ performance in each task. To ensure that faculty were properly blinded, identification badges were removed prior to filming, residents wore identical gloves and hospital-issued scrubs, and the video recordings restricted the field to the participant’s hands and removed sound. To limit bias, pre-, post-, and one-year recordings were randomized for rating in a single batch. The rating scale for flexible laryngoscopy was based on a previously published assessment toolsthesia . Assessmsthesia . The scoThree PGY-2 residents who had not participated in bootcamp as PGY-1s composed the control group. (The fourth resident in that class was completing an off-site rotation and was unable to participate.) The control group’s only evaluation and the study group’s last evaluation both occurred in the first week of PGY-2 year such that they had completed the same amount of residency training time.t-tests. Unpairedt-tests were used to compare the intervention group with the control group. GraphPad Prism 9.3.1  was used for statistical analysis. In all cases, significance was defined as alpha = 0.05.Bootcamp participants’ performance in flexible laryngoscopy, suture ligature, and rigid bronchoscopy at the various time points were compared using pairedEight PGY-1 residents who participated in this bootcamp were evaluated in flexible laryngoscopy, suture ligature, and rigid bronchoscopy setup at three time points: pre-bootcamp, one-week post-bootcamp, and one-year post-bootcamp. One resident was not evaluated in flexible laryngoscopy one week after participation in bootcamp and thus was excluded from the analysis for that task. Each individual’s scores over time are displayed inP<0.05) and rigid bronchoscopy (P<0.05), but no difference in flexible laryngoscopy (P=0.54). Suture ligature (P=0.09) and rigid bronchoscopy (P=0.25) skills were not significantly different from one-week post-bootcamp to one-year post-bootcamp; however, a significant skill improvement was observed in flexible laryngoscopy (P<0.05). By June of PGY1 year, the two bootcamp cohorts were similar to controls in all three skills: flexible laryngoscopy (P=0.05), rigid bronchoscopy (P=0.26), and suture ligature (P=0.10) (A comparison of pre-bootcamp scores to one-week post-bootcamp scores showed significant improvement in suture ligature (P=0.10) ., they are limited by their reliance on survey-based self-reporting over objective, blinded metrics. Of note, some studies have started to provide objective evidence that bootcamps increase knowledge as demonstrated by improved scores on multiple-choice exams and better performance in simulated scenarios. Our study is unique in its focus on the objective evaluation of the impact of an otolaryngology bootcamp on procedural skill acquisition and retention via the use of blinded faculty rating.Although previous studies have demonstrated improvement in participants’ confidence and knowledge after participation in otolaryngology bootcampsWe found that residents who participated in bootcamp significantly improved in suture ligature and rigid bronchoscopy setup skills immediately after participation in the bootcamp, which suggests that bootcamps can improve objectively measured technical skills. Additionally, residents demonstrated retention of these skills at the one-year mark, and scores were similar to controls. Bootcamps participants’ flexible laryngoscopy skills started at a higher level at baseline, which might in part explain the lack of significant improvement immediately after bootcamp. It is likely that PGY-1 residents were more commonly exposed to flexible laryngoscopy than suture ligature and rigid bronchoscopy setup in their usual clinical work over the course of their PGY-1 year, which might explain why flexible laryngoscopy skills continued to improve from immediate post-bootcamp to the one-year mark compared to the other skills.. As residents learn increasingly complex operative skills and techniques over the course of residency, simulation-based learning for senior residents should incorporate more advanced skills and focus on the non-technical aspects of patient management and teamwork. Our experience suggests that, beyond increasing resident self-confidence and knowledge, participation in bootcamp can accelerate acquisition of technical skills in otolaryngology compared to clinical experience alone. This finding supports the increased use of simulation-based bootcamps for resident education in the field of otolaryngology. This simulation education study targeted to junior residents focused on fundamental manual skills in otolaryngology, consistent with junior residents’ preference for procedural skills and didactic teaching over non-technical skills such as communication and leadership at this stage in their trainingThe main limitation of our study is the small sample size inherent in studying single institution surgical specialty residency programs. Additionally, the control group was comprised of rising PGY-2s who spent three months on a dedicated otolaryngology service whereas the PGY-1s who participated in bootcamp spent six months on a dedicated otolaryngology service, which may account for some of the improvement in their scores of the course of the year but would be unlikely to influence the immediate post-bootcamp results. Furthermore, familiarity prior to training or clinical experience with the skills tested could not be controlled for within each cohort. The fidelity of mannequins in lieu of patients for flexible laryngoscopy and suture ligature skills, though standard for training, was also a limitation. The rigidity of the rubber substituting for mucosa may have limited the ability of participants to display proper flexible laryngoscopy technique and could partially explain the greater variability in performance observed for that task, although it is unlikely to be different by observation timepoint. As a resident-driven program, our institution’s bootcamp evolves each year as we tailor it to our residents’ perceived areas of need. More focused study of the skills valued by residents and program directors will allow us to further develop the program to address areas for further growth and attention.Future investigation is needed to explore whether task acquisition during bootcamp correlates to improved resident performance and patient safety, overall speed of surgical skill acquisition and fluency, and improved patient care.Simulation-based bootcamp can be an effective method to teach fundamental procedural skills in the field of otolaryngology and is a promising arena for multi-institutional developments.B2Share: Impact of PGY-1 Otolaryngology Bootcamp on Procedural Skill Developmenthttp://doi.org/10.23728/b2share.c6b965776b5844fb80046f1b07da971bThis project contains the following underlying data:Flexible Laryngoscopy A-E.csv (raw data of participants)Rigi Bronchoscopy A-C.csv (raw data of participants)Suture Ligature A-C.csv (raw data of participants)Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Simulation in technical education and Bootcamps have gained traction in resident education.  The efficacy of such teaching methods are still unknown and the authors address an important issue in an organized and thoughtful manner.  There is clear acknowledgment of the limitations of the study, appropriate analysis of the data, clear and organized methods. There are no significant concerns regarding this submission.Have any limitations of the research been acknowledged?YesIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Simulation EducationI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This article attempts to provide something that has been lacking in simulation education, a demonstration that the training actually accelerates the acquisition of skills without endangering patients. The authors should be acknowledged for their dedication to advancing ORL resident education through a very thorough training program and their time spent researching the topic.  The methods are sound and reproducible. The number of participants was limited as this is a single institution study.  The authors acknowledge that there was heterogeneity in the PGY-2 control group and the PGY-1 experience due to RRC changes.  We have no major criticisms and recommend this submission for passing peer review.Have any limitations of the research been acknowledged?YesIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Simulation training for resident educationWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


A hybrid blood–brain barrier (BBB)-on-chip cell culture device is proposed in this study by integrating microcontact printing and perfusion co-culture to facilitate the study of BBB function under high biological fidelity. This is achieved by crosslinking brain extracellular matrix (ECM) proteins to the transwell membrane at the luminal surface and adapting inlet–outlet perfusion on the porous transwell wall. While investigating the anatomical hallmarks of the BBB, tight junction proteins revealed tortuous zonula occludens (ZO-1), and claudin expressions with increased interdigitation in the presence of astrocytes were recorded. Enhanced adherent junctions were also observed. This junctional phenotype reflects in-vivo-like features related to the jamming of cell borders to prevent paracellular transport. Biochemical regulation of BBB function by astrocytes was noted by the transient intracellular calcium effluxes induced into endothelial cells. Geometry-force control of astrocyte–endothelial cell interactions was studied utilizing traction force microscopy (TFM) with fluorescent beads incorporated into a micropatterned polyacrylamide gel (PAG). We observed the directionality and enhanced magnitude in the traction forces in the presence of astrocytes. In the future, we envisage studying transendothelial electrical resistance (TEER) and the effect of chemomechanical stimulations on drug/ligand permeability and transport. The BBB-on-chip model presented in this proposal should serve as an in vitro surrogate to recapitulate the complexities of the native BBB cellular milieus. Current state-of-the-art blood–brain barrier (BBB) in vitro models utilize transwell assays or microfluidic channels to culture brain cells. While transwells are mostly static cultures, they fail to replicate shear-stress-induced BBB properties in vitro . BiochemWith the advent of lithographic techniques in cell culture, there was a corresponding increase in research to understand the effects of confinement on cellular morphology and, in turn, on cellular structure and function . HoweverThe primary objective of this study was to decode the effects of micropatterning-based endothelial cells as ring and linear geometries to exhibit in-vivo-like BBB features in terms of tight junction expression, paracellular permeability, and overall barrier properties  (PCS-100-013) were from American Type Culture Collection (ATCC), and human astrocytes from Lonza (CC 2565) were cultured as per the manufacturer’s instructions and used for experiments at a passage of 3–5. The hUVECs were cultured in media containing vascular basal medium (ATCC PCS-100-030) and endothelial cell growth kit-BBE (ATCC PCS-100-040) prepared as per the manufacturer’s instructions. Similarly, the astrocytes’ growth medium, AGMTM (CC3186), was used for culturing the astrocytes. The cells were cultured at 37 °C and 5% CO2)11-EG3, Prochimia) for 3 h, which created cell-adhesive ring/line patterns  from the previous step 1. Before surface sterilization, the inlet and outlet channels were defined by punching holes (diameter: 0.5 mm) under mild force and flushed with ethanol to remove debris. Lastly, the patterned surfaces were surface sterilized using ethanol and incubated with ECM protein, 25 µg/mL fibronectin , for 30 min prior to seeding (The whole procedure can be grouped into three steps: (a) elastomeric polydimethylsiloxane (PDMS) stamp design: (b) protein microcontact printing; (c) hUVECs’ culture on the cell-adhesive line/ring patterns in step 1, and the initial mold was obtained using a negative SU-8 2050 photoresist (Micro-Chem Corp.) and chromium photo mask containing line and ring geometries by UV exposure. PDMS monomer and Dow Corning curing agent (SYLGARD™ 170 Silicone Elastomer Artikel-Nr.0721SY17010) at a ratio of 10:1 were poured onto the developed mold and cured for 4 h at 70 °C to obtain PDMS stamps. The PDMS stamps were used to transfer the octadecanethiol , as self-assembled monolayers (SAMs), onto the gold-sputtered nanocoating at the transwell membrane. These SAMs acted as surface-active molecules for immobilizing functional anchoring units similar to proteins in the extracellular matrix, such as collagen and fibronectin, in step 2 of the protocol. The transwell membrane was then immersed in ethylene-glycol-terminated SAM  were cultured on the apical side of the transwell insert. After 24 h, the transwell inserts were placed in 6-well culture plates facing the abluminal surface, where the astrocytes were cultured. The cells were allowed to grow in co-culture under perfused conditions for a total of 72 h (unless mentioned otherwise) before different characterization techniques were used as described subsequently.In the step 3, the final step, the cultures were seeded in 6-well corning transwell plates with a growth area of 4.5 cmR) generated in response. A background control of the microprinted membrane coated with fibronectin was also measured (mR) and subtracted. The TEER value was calculated for the unit area normalization using the cell growth area (A) of the transwell microfluidics system with the following formula:The transendothelial electrical resistance (TEER) was measured using a Millicell ERS-2  with an STX2 electrode  to characterize the barrier function of the hUVEC–astrocytes transwell microfluidics . The ERSPrior to the TEER measurements, functionality checks on the Millicell ERS-2 m were conducted by connecting the STX04 test electrode through an inserting plug. The “R Adj” setting on the meter was adjusted until the meter displayed 1000 Ω. Care was taken during the TEER measurements to insert the electrode in such a way that the shorter tip was dipped in the culture plate insert, and the longer tip was immersed in the outer well. It was made sure that the shorter tip did not contact the cells growing on the membrane, and the longer tip only slightly touched the bottom of the outer well. The electrodes were held steady and at a 90° angle to the plate insert. The TEER measurements were performed for the unpatterned hUVEC microfluidic system, patterned with only hUVECs and patterned with co-culture subsequently on days 1, 3, and 7. For the pathology studies with nanoparticle exposure, the microfluidic cell culture system was initially exposed to nanoparticles for 24 h subsequently followed by TEER measurements. To ensure cell survival, the cells were supplemented with fresh media on both the transwell as well as the basolateral well every day until the seventh day of the TEER measurements.n = 3). Using the deviation from the circumferential angle of each subregion, the orientation was converted into an angle bias for each subregion.We acquired high-resolution phase contrast images of the live patterned hUVECs, with each pixel representing 0.645 μm. Based on the automated detection of the intensity gradient and circular statistics, the L-R asymmetry was quantified using a custom-written code in MATLAB (MathWorks R2021a). A Gaussian differential filter was utilized in the algorithm to determine the intensity gradient pixel by pixel in each image portioned into subregions for analysis. An accumulator scheme in the algorithm was adopted to determine the dominant local direction base on the orientation of each pixel utilizing the von Mises distribution. All subregions were analyzed using circular statistics to determine the mean angle and standard deviation of the biased alignment. More than 50 rings were analyzed for each geometry in at least three independent experiments  on the endothelial cells was assessed by staining in the absence (0A) and presence (EA) of astrocytes  expression by following immunostaining protocols. In brief, cells were seeded at a density of 5 × 10trocytes . The von2, 2 mM EGTA, and 0.32 M sucrose) for 30 min. Actin staining for the cytoskeleton was performed using Phalloidin TRITC  for 1 h. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). The phase contrast and fluorescence images were obtained using an LSM 700, and a Zeiss microscope was for the morphology and alignment analyses. Image analysis was conducted by merging the stained actin (red) and DAPI images using ImageJ (NIH) i) amplitude was quantified by measuring the average image intensity of the fluo-4-positive cells in all time-lapse fluorescence images. The ImageJ-based plugin, spark master, was used to analyze the calcium oscillations and their morphology [Live cell imaging occurred every 3 s using a cooled CCD camera (Zeiss Z-observer). The intracellular calcium concentration ([Carphology .2+]i) amplitude was also quantified utilizing a method described previously [2+]i = Kd × (F − Fb)/(Fmax − F), where F is the fluorescence intensity inside the cell; Fmax and Fb are the fluorescence intensity when saturated with and in the absence of Ca2+; Kd is the ion dissociation constant, and it was assumed to be 345 nM i signaling at the microfluidics BBB, calcium signaling was measured in astrocytes with a fluo-4 AM calcium probe. In co-cultures (EA), spontaneous calcium sparks were observed analogous to that excitation–contraction , metal and metal oxide  overload is well established in contributing to the disease pathophysiology ,36 as haWe developed a unique BBB model that aimed to mimic three crucial aspects of actual BBB vasculature viz. shear-induced barrier properties, co-culture based barrier properties, and traverse and longitudinal cross-sections of vessels. This allowed for the realistic investigation of tight junction expression. Moreover, the developed model provided a platform for studying the influence of micromechanical forces on the biochemical responses in an actual BBB environment. Typically, the reconstitution of actual BBB vasculature with respective micromechanical forces and multicellular environment is a challenging feat. To this end, the model proposed in our study easily integrated a perfused system with micropatterned transwells, thereby replicating the flow-induced stress and cross-sectional effects of BBB cells on the barrier properties. It was expected that cellular interactions and communication from an endothelial–astrocyte co-culture would benefit the secretion of adhesion molecules and junctional protein expression, thus maintaining better barrier properties with improved cell growth and survival. This can be attributed to the synergy among the different cell types as mentioned previously in the literature . SimilarCellular alignment in BBB is critical for several functions including enhanced barrier properties, secretion of junctional molecules with adhesion properties, nucleus gene expression of tight junctions, and ECM remodeling. Reports suggest that the spatial orientation of cells plays a crucial role in pattern formation in embryogenesis and neuron maturation ,42. For Further characterization of the BBB properties using conventional endothelial and astrocytes marker indicated a differentiated but continuous layer of the barrier. Studies on the permeability and transport across BBB are a required part of BBB characterization for any in vitro model. Typical resistance offered by BBB in vivo can be studied effectively by co-culturing BBB relevant cells like endothelium and astrocytes as demonstrated in the current study. The existing in vitro BBB models are commonly assessed by the resistance offered by the cells for transport or permeability of molecules. However, the barrier properties contributed by micropatterning can shed light on the importance and relevance of BBB architecture towards resistance. The high permeability of Na-fluorescein in control (unpatterned) noticed in the current study can be considered as a poor representation of BBB in vitro. Interestingly, the paracellular permeability of Na-F was relatively higher in patterned co-culture conditions relative to the patterned monoculture of endothelial cells which is due to the higher expression of transport proteins that are used for the transport of bioactive compounds across BBB. A decrease/increase in Na-F transport across the BBB depends on organic anion transporter (OAT)-3 and multidrug resistance protein (MRP)-2 expression , and fur2+]i is involved in the regulation of blood flow; this is supported by the elevation of [Ca2+]i at the astrocyte end-feet when neurons are stimulated. However, the role of calcium signaling at BBB ‘neurovascular units’ has not been systematically explored in relation to their linear or radial architecture [2+]i which is further linked with the tight junction opening i changes that are also known to spatially spread across boundaries as intercellular calcium waves [2+]i signaling could be centrally involved in the dynamic force control exercised by astrocytes in the regulation of barrier morphology via astrocyte-to-endothelial communication. We also observed enhanced cadherin expression in patterned cells. Occludin and cadherin expression is dependent on calcium activity [Astrocytes form contact with microvessels and the synapses between neurons in vivo; this is important for matching oxygen and glucose transport to neural activity through regulation of local blood flow . While citecture . Transfo opening . It is cum waves . Amplituactivity  and is bactivity  and alloactivity . HoweverIn the present study, we successfully developed a hybrid BBB model by micropatterning with ECM proteins and perfusion system integration into a transwell. The difference in barrier properties for the unpatterned monoculture and patterned mono- and co-culture were studied. Micropatterning demonstrated that mechanical stresses play a major role in determining the cellular microenvironment and in the integrity of the barrier properties. Micropatterning further influenced the elongation and orientation of the endothelial cells as an alternate mechanism of stress-induced TJ expression. This provides knowledge on the ability of endothelial cells to respond to stress created by spatial confinement in addition to shear forces. Additionally, this study further adds to the understanding that co-culture with astrocytes improves the barrier properties as well as calcium signaling. Further, the developed BBB model was characterized with desirable BBB-like properties, thus mimicking the in vivo characteristics of appropriate traction forces and calcium signaling. The model also provides a platform to study disease pathology by iron oxide-induced MS-like pathological characteristics in terms of TJ expression and calcium signaling. We envision that this model can pave the way toward high-throughput screening and pathological studies for a variety of neurovascular disorders.


The striatum is the principal site of disease pathology in Huntington’s disease and contains neural connections to numerous cortical brain regions. Studies examining abnormalities to neural connections find that white matter integrity is compromised in HD; however, further regional, and longitudinal investigation is required. This paper is the first longitudinal investigation into region-based white-matter integrity changes in Huntington’s Disease. The aim of this study was to better understand how disease progression impacts white matter tracts connecting the striatum to the prefrontal and motor cortical regions in HD. We used existing neuroimaging data from IMAGE-HD, comprised of 25 pre-symptomatic, 27 symptomatic, and 25 healthy controls at three separate time points . Fractional anisotropy, axial diffusivity and radial diffusivity were derived as measures of white matter microstructure. The anatomical regions of interest were identified using the Desikan-Killiany brain atlas. A Group by Time repeated measures ANCOVA was conducted for each tract of interest and for each measure. We found significantly lower fractional anisotropy and significantly higher radial diffusivity in the symptomatic group, compared to both the pre-symptomatic group and controls (the latter two groups did not differ from each other), in the rostral middle frontal and superior frontal tracts; as well as significantly higher axial diffusivity in the rostral middle tracts only. We did not find a Group by Time interaction for any of the white matter integrity measures. These findings demonstrate that whilst the microstructure of white matter tracts, extending from the striatum to these regions of interest, are compromised during the symptomatic stages of Huntington’s disease, 36-month follow-up did not show progressive changes in these measures. Additionally, no correlations were found between clinical measures and tractography changes, indicating further investigations into the relationship between tractography changes and clinical symptoms in Huntington’s disease are required. Huntington’s disease (HD) is an autosomal dominant, neurodegenerative condition that causes catastrophic damage to numerous cellular structures in the brain  enables the investigation of microstructural abnormalities in vivo by characterising the diffusion of water in the brain Jones, , allowinIn HD, DTI has been used to identify microstructural damage in specific white matter tracts and networks, including striatal motor loops , in either pre-HD or in symp-HD individuals. Doing so would provide further clarity about the neuropathology of disease, particularly pertaining to how white matter microstructure degenerates over time, and how these changes are related to developing symptoms in HD.The aim of the current study was to investigate whether white matter tractography changes occur in tracts extending from the caudate to specific cortical regions of interest (ROIs) over-time. Given that previous studies have found the frontal and motor regions of the brain to be primarily impacted by white matter degeneration  was derived and as per Tabrizi et al., , gene poMRI scanning was conducted at the Murdoch Children’s Research Institute , using a Siemens 3 Tesla scanner. T1-weighted images were acquired for each participant  . The diffusion-sensitizing gradient encoding (B1) was applied in 60 directions (b = 1200 s/mm2) and 5 images acquired without diffusion weighting (b = 0 s/mm2).DTI whole brain images were acquired using double spin echo diffusion weighted EPI sequence  brain regions. This was completed using the well-validated Desikan-Killiany brain atlas, an automated labelling system . “Spherical-deconvolution Informed Filtering of Tractograms” (SIFT) was used to improve the quality of tract reconstruction outlined in The mean fractional anisotropy (FA), axial diffusivity (AD) and radial diffusivity (RD) were calculated as the mean value of all fixels (fibre bundles within each voxel), within each of the four regions of interest. The directionality of diffusivity (as measured by AD and RD) can provide more granular information into the nature of axonal damage or demyelination occurring in HD.FA is a metric that provides a simple and robust measure of the degree of anisotropic diffusion of water occurring within a region  RD is a metric which quantifies the diffusion of water perpendicular to white matter fibres. An increase in RD is indicative of axonal damage , were conducted for within groups  and between groups  using a general linear model (repeated measures ANCOVA) with Group and Time as Independent Variables (IVs) and each tractography measure  as the dependent variables (DV). Age and gender were used as covariates for all analyses.Summary statistics were calculated for the three groups on each of the white matter integrity measures and for each cortical region at each time point. Significant differences between groups and time points were calculated using t-tests. Bonferroni corrections were used to adjust for multiple comparisons across the ROIs and tractography measures.Pearson’s correlations were performed between clinical measures  and tractography measures for each region of interest. A correlation analysis between baseline tractography measures and UHDRS-TMS was conducted in the symp-HD group only. Correlations between change in mean cortical tractography measures and DBS was conducted for both HD groups.Post-hoc partial correlation analyses were conducted to investigate the correlation between caudate volume change (TP 3 minus TP1) and change in white matter tractography changes , controlling for age and gender.Using a general linear model (repeated measures ANCOVA), we sought to determine whether white matter integrity changes  occurred in specific tracts from the caudate to regions of interest , over time in pre-HD and symp-HD, compared to controls.There were no significant interaction effects of Group by Time in any of the regions of interest.F  = 3.20, p = 0.047. However, after correcting for multiple comparisons, this did not remain significant. Controls and pre-HD showed significantly higher FA than symp-HD in this area  = 9.83, p =  < 0.01. Controls and pre-HD showed significantly lower AD than symp-HD individuals  = 3.39, p =  < 0.05 and Rostral Middle Frontal Regions, F = , p =  < 0.01. In the Rostral Middle Frontal region, symp-HD individuals had significantly higher RD than controls and re-HD. In the Superior Frontal region, symp-HD differed significantly from controls, but not from pre-HD. See Fig. There were significant main effects of Group in the Precentral, There were no significant main effects of Time in any of the regions of interest.To investigate the relationship between white matter integrity and motor symptoms, partial correlations between change in UHDRS-TMS (score at 30-months minus score at baseline) and change in white matter integrity measures were conducted in the symp-HD group only. There were no significant correlations between change in UHDRS-TMS and change in white matter integrity measures in any of the eight regions of interest.Partial correlations between baseline DBS and change in white matter integrity measures  were calculated with both pre-HD and symp-HD groups combined to determine the impact of exposure to the disease across the disease continuum. There were no significant correlations between baseline DBS and change in white matter integrity measures in any of the eight regions of interest.Partial correlations between CAG repeat length and change in white matter integrity measures  were calculated with both pre-HD and symp-HD groups combined to investigate the relationship between CAG repeat length on tractography changes. There were no significant correlations between CAG repeat length and change in white matter integrity measures in any of the eight regions of interest.r (76) = -0.325, p =  < 0.01).Partial correlations between change in caudate volume and change in white matter integrity measures  were calculated in all groups to determine the relationship between caudate volumetric changes and white matter integrity. After controlling for age and gender, a significant negative correlation between change in caudate volume and RD in the rostral middle frontal region only . Tractography changes were not correlated with change in UHDRS-TMS or DBS in any of the regions of interest.The finding of significant group level tractography changes in symp-HD from the caudate to the precentral region supports previously established findings that motor circuits are particularly vulnerable in HD (Georgiou-Karistianis & Egan, Elevated AD was observed in the tracts radiating from the caudate to the rostral middle frontal region in conjunction with significantly increased RD to the precentral and rostral middle regions in symp-HD. These findings were somewhat expected as previous studies had found increased AD and RD in the corpus callosum (Phillips et al., mHTT protein may also influence a secondary pruning effect of white matter tracts projecting from it. Alternatively, decreased volume in the caudate may also occur in part due to there being fewer tightly packed axonal tracts preventing the radial diffusion of water molecules. However, previous studies have reported the effect of mHTT on cerebellar-striatal circuitry prior to the onset of disease (Tereshchenko et al., Volumetric change in the caudate is a well-established neuropathological feature in HD (Georgiou-Karistianis & Egan, There were certain limitations of the present study. Although we observed some significant group differences, we did not observe a significant effect of Time or any interaction effects of Group by Time. Additionally, numerous neuroimaging studies have reported degeneration in cortical and sub-cortical structures many years prior to clinical diagnosis, and prior to the presence of overt motor symptoms in HD (Dumas et al., Our 30-month longitudinal study remains the only longitudinal analysis of white matter microstructure changes in both pre- and symp-HD. Although we were unable to identify any interaction effects of Group by Time, which was the primary aim of the study, we did find white matter tractography differences in symp-HD in both the precentral and rostral middle frontal regions, brain regions responsible for motor control and working memory, respectively. Specifically, the symp-HD group had decreased FA in the precentral region, increased AD in the precentral region, and increased RD in the precentral and rostral middle regions compared with the pre-HD and control groups. Although these findings are suggestive of demyelination and selective neurodegeneration that occurs in specific frontal-striatal tracts during the symptomatic stages of disease, longitudinal changes were not detected in the 30-month period observed in this study..