Conditional Localization Pharmacology Manipulates the Cell Cycle with Spatiotemporal Precision.

Traditional pharmacology has limited control of drug activity and localization in space and time. Herein, we described an approach for kinase regulation using conditional localization pharmacology (CLP), where an inactive caged inhibitor is localized to a site of interest in a dormant state using intracellular protein tethering. The activity of the inhibitor can be regulated with spatial and temporal precision in a live cellular environment using light. As a proof of concept, a photocaged MPS1 kinase inhibitor (reversine) bearing a Halo-tag ligand tether was designed to manipulate the cell cycle. We demonstrate that this new caged reversine halo probe (CRH) strategy is capable of efficient localization and exceptional spatiotemporal control over spindle assembly checkpoint (SAC) silencing and mitotic exit.

Effect of a context-specific health education intervention on knowledge of health risk behaviors among adolescents in Nigerian public and private secondary schools: a comparative study.

OBJECTIVES: Health education programmes that utilizes multicomponent classroom teaching strategies have the potential to enhance knowledge, attitudes, and actions of adolescents concerning health risk behaviors (HRBs). This study assessed the effect of a school-based context-specific health education intervention (CsHEI) on knowledge of HRBs among in-school adolescents in public- and private-owned secondary schools in Nigeria. METHODS: In-school adolescents were recruited into this quasi-experimental study using stratified random sampling from secondary schools in Ile-Ife, Osun-State, Nigeria. Knowledge of HRBs was assessed pre- and post-implementation of a CsHEI. The intervention was a four-week instructor-led health education class on HRBs, delivered over a 2-h class activity per week. Data was summarized using descriptive statistics as well as Inferential statistics. RESULTS: With a mean age of 14.9 ± 1.56 years, a total of 145 females (54.3 %) and 122 males (45.7 %) participated in this study. Over 70 % of the participants had good knowledge of HRBs prior to the intervention with the pre-test mean general knowledge of HRB (t=-3.13, p=0.002) and knowledge of personal safety (t=-2.99, p=0.003) being significantly lower between students in public-schools compared to their private-school counterparts. Post-intervention, a significant improvement in participants' knowledge was observed in all HRB domains with over 90 % of participants having a good knowledge of HRBs. CONCLUSIONS: The CsHEI improved knowledge of in-school adolescents in public- and private-owned secondary schools in Osun state, Nigeria in all domains of HRBs. Integrating the CsHEI with other health behavioral change strategies can mitigate prevalence of HRBs among adolescents, thereby improving health-outcomes and well-being of adolescents.

A Multicenter Training and Interrater Reliability Study of the BASED Score for Infantile Epileptic Spasms Syndrome.

PURPOSE: The best possible outcomes in infantile epileptic spasms syndrome require electroclinical remission; however, determining electrographic remission is not straightforward. Although the determination of hypsarrhythmia has inadequate interrater reliability (IRR), the Burden of AmplitudeS and Epileptiform Discharges (BASED) score has shown promise for the reliable interictal assessment of infantile epileptic spasms syndrome. Our aim was to develop a BASED training program and assess the IRR among learners. We hypothesized moderate or better IRR for the final BASED score and the presence or absence of epileptic encephalopathy (+/-EE). METHODS: Using a web-based application, 31 learners assessed 12 unmarked EEGs (length 1-6 hours) from children with infantile epileptic spasms syndrome. RESULTS: For all readers, the IRR was good for the final BASED score (intraclass correlation coefficient 0.86) and +/-EE (Marginal Multirater Kappa 0.63). For all readers, the IRR was fair to good for all individual BASED score elements. CONCLUSIONS: These findings support the use of our training program to quickly learn the BASED scoring method. The BASED score may be a valuable clinical and research tool. Given that the IRR for the determination of epileptic encephalopathy is not perfect, clinical acumen remains paramount. Additional experience with the BASED scoring technique among learners and advances in collaborative EEG evaluation platforms may improve IRR.

Continuous Manufacturing of Solvent-Free Cyclodextrin Inclusion Complexes for Enhanced Drug Solubility via Hot-Melt Extrusion: A Quality by Design Approach.

Conventional cyclodextrin complexation enhances the solubility of poorly soluble drugs but is solvent-intensive and environmentally unfavorable. This study evaluated solvent-free hot-melt extrusion (HME) for forming cyclodextrin inclusion complexes to improve the solubility and dissolution of ibuprofen (IBU). Molecular docking confirmed IBU's hosting in Hydroxypropyl-ß-cyclodextrin (HPß-CD), while phase solubility revealed its complex stoichiometry and stability. In addition, an 11 mm twin-screw co-rotating extruder with PVP VA-64 as an auxiliary substance aided the complex formation and extrusion. Using QbD and the Box-Behnken design, we studied variables (barrel temperature, screw speed, and polymer concentration) and their impact on solubility and dissolution. The high polymer concentration and high screw speeds positively affected the dependent variables. However, higher temperatures had a negative effect. The lowest barrel temperature set near the Tg of the polymer, when combined with high polymer concentrations, resulted in high torques in HME and halted the extrusion process. Therefore, the temperature and polymer concentration should be selected to provide sufficient melt viscosities to aid the complex formation and extrusion process. Studies such as DSC and XRD revealed the amorphous conversion of IBU, while the inclusion complex formation was demonstrated by ATR and NMR studies. The dissolution of ternary inclusion complexes (TIC) produced from HME was found to be ≥85% released within 30 min. This finding implied the high solubility of IBU, according to the US FDA 2018 guidance for highly soluble compounds containing immediate-release solid oral dosage forms. Overall, the studies revealed the effect of various process parameters on the formation of CD inclusion complexes via HME.

Acute periocular haemorrhage associated with severe proptosis as a presenting sign of orbital tumours.

Periocular haemorrhage without a preceding history of trauma, and in the absence of vascular malformations is rare. While acute proptosis is well described in the setting of orbital pathology, accompanying periocular haemorrhage has been rarely reported. We describe three cases with these concomitant presenting signs in orbital malignancies - metastatic small cell bladder carcinoma, haemangiopericytoma (solitary fibrous tumour) and myeloma. Clinical photographs and radiology are presented with review of the current literature. All cases had an associated rapid onset of severe proptosis and co-existing periocular bruising on the same side. The presence of ecchymosis of the eyelids in addition to proptosis without a history of trauma warrants thorough investigation to rule out underlying potential sight and life threatening illness.

Diversity, equity and inclusion: a survey of pediatric radiology fellowship graduates from 1996 to 2020.

BACKGROUND: Understanding the sociodemographic diversity and the experiences of trainees is imperative for developing an inclusive pediatric radiology workforce and improving patient outcomes. OBJECTIVE: To describe the sociodemographics of a pediatric radiology fellowship program over 25 years and examine potential associations with (a) a trainee's sense of inclusion and belonging during training and (b) graduates' ultimate choice/type of practice, progression to positions of leadership and mentorship of others. MATERIALS AND METHODS: We designed and administered an online survey of sociodemographic diversity to graduates of one of the largest pediatric radiology fellowship programs in North America who graduated over the last 25 years. Respondents answered questions regarding (a) racial/ethnic identity, gender identity, sexual orientation, disability and other sociodemographics; (b) their sense of inclusion and belonging during fellowship training; and (c) their current type of practice, leadership and mentorship of others. Univariate analysis of variance (ANOVA) tests determined whether sociodemographic background was associated with inclusion/belonging during training, and with current type of radiology practice, leadership and mentorship. We assessed associations with covariates of interest and current leadership and mentorship roles using chi-square or Fisher exact tests. A heat map assessed individual and overall Likert scores across covariates. RESULTS: Seventy-six graduates completed the survey (46% response rate). Three percent of respondents identified as Black/African American, 7% as Latino/a/x, 45% as female, 4% as LGBTQ (lesbian, gay, bisexual, transgender, queer/questioning) and 5% as living with a disability at the time of fellowship. Graduates of international medical schools (P = 0.09) and osteopathic medical (DO) schools (P = 0.09), first-generation college graduates (P = 0.023), those with a first language other than English (P = 0.02) and U.S. military veterans (P = 0.048) reported significantly lower feelings of inclusion and belonging during training. Gender identity and sexual orientation did not correlate with feelings of exclusion or inequity. Graduates between 1996 and 2010 were significantly more likely to have filled a leadership role than those graduating after 2011 (P = 0.004) and were also significantly more likely to have mentored individuals underrepresented in medicine/pediatric radiology than those who graduated after 2011 (P = 0.04). None of the other sociodemographic variables demonstrated an association with leadership or mentorship, although 92.3% of those with a first language other than English served as a mentor compared to 68.2% of native English speakers (P = 0.097). CONCLUSION: Efforts to promote a more diverse and inclusive workforce in pediatric radiology should be directed toward increasing numbers of fellows who are underrepresented in medicine (Black/African American, Hispanic/Latino/a/x) and providing a more inclusive training environment for first-generation college graduates, graduates of international and DO medical schools, fellows whose first language is not English, and veterans of the U.S. Armed Forces.

Corneal Complications Secondary to Involutional Entropion at Presentation.

PURPOSE: To determine the frequency and significance of corneal complications at presentation of involutional entropion in an Australian population. Patient demographics and duration of symptoms were collated to assess how these factors related to presentations. METHODS: Case records were retrospectively interrogated at an Oculoplastic Ophthalmology practice over a 15-year period to identify patients with lower eyelid involutional entropion. All patients had ophthalmic examination by an ophthalmologist. Patients meeting inclusion criteria had data collected including detailed assessment of corneal examination findings. RESULTS: The final cohort included 203 patients of which 50.7% were male. The mean age was 75.1 years. Cornea findings at presentation included superficial punctate keratopathy (69%), no change (25.6%), corneal ulcer (4%), and other (1%). One percent of patients had vision loss attributable to complications of entropion. Range of duration of symptoms was 2 weeks to 6 years with a mean of 11.5 months. CONCLUSIONS: At presentation, involutional entropion has mild corneal findings with 95.1% of patients demonstrating superficial punctate keratopathy or normal corneal epithelium. Vision loss is a rare complication and was observed in 1% of patients in this study, all with preexisting corneal pathology.

Examining the initial prescription leading to long-term opioid use at a military medical treatment facility.

OBJECTIVE: Chronic pain is common in the military, and over a quarter of active-duty military members have received a prescription for opioids. This study reviewed the initial opioid prescription among those who became future long-term users at an Air Force base in the United States and reports the characteristics of the provider and patient. METHODS: Our single-center retrospective study evaluated initial opioid prescriptions leading to long-term use within the military's electronic medical record at a large military medical treatment facility including active-duty patients and veterans. Of the 3,701 charts reviewed, 348 patients met the inclusion criteria for the long-term opioid use. RESULTS: Older patient groups received a higher initial amount of opioids than younger groups (p = 0.007). Primary care outpatient clinics started 43 percent of initial long-term opioid users, while surgical specialties contributed to 34 percent of the sample. In our study, 35.9 percent of the long-term opioid users were given their first opioid prescription within 30 days of an operation. Veterans or those separated from the military were less likely to have a behavioral disorder than active duty or family members. CONCLUSIONS: Our sample mirrored the civilian population in terms of age, gender, and most common pain diagnosis. We found that older patients initially received a higher dispensed amount compared to our younger patients. There was a concerning trend for surgical patients to develop into chronic opioid users.

Bilateral Enophthalmos as an Unusual Presenting Feature of Non-Hodgkin's Orbital Lymphoma.

Lymphoma is the commonest orbital malignancy. The typical presentation is proptosis or swelling, which warrants imaging and confirmation by tissue biopsy. Enophthalmos is a much rarer clinical sign and if bilateral and symmetrical can often present late. We describe a patient who presented with bilateral enophthalmos and symptomatic, secondary entropion due to bilateral non-Hodgkin's lymphoma in which orbital fat was replaced by a monoclonal proliferation of small B cells. Low-dose orbital radiotherapy and entropion surgery relieved the patient's symptoms.

Current practice trends for lacrimal gland neurotoxin in the management of epiphora-a BOPSS survey.

PURPOSE: Injection of botulinum neurotoxin A (BoNTA) to the lacrimal gland (LG) offers a simple and effective treatment in the management of epiphora. However, there is little data on current practice trends or uptake as an alternative to surgery. This study assesses current practice trends of such treatment amongst BOPSS (British Oculoplastic Surgery Society) members. METHODS: All consultant BOPSS members were invited to participate in a web-based survey which consisted of 5 questions, with a reminder invitation to participate. The role, dose, potential side effects, use as an alternative to surgical intervention, and impact on service delivery were assessed. RESULTS: Fifty-one BOPSS consultants (43% uptake) completed the survey. Ninety percent of respondents were regularly using LG BoNTA in their management of epiphora. The main indicators for considering BoNTA use were medical comorbidities and elderly patients. The mean first treatment dose of Botox® was 3.6 units (SD 1.5). Diplopia and ptosis complications were always discussed in the consent for treatment in addition to dry eye. Twenty-five percent of surgeons reported doing less conjunctivo-dacryocystorhinostomies (cDCR) due to the availability of LG BoNTA. No respondents felt that the requirement for repeated BoNTA treatments was impacting on their service delivery. CONCLUSION: Uptake of LG BoNTA in the management of epiphora is at a similar rate to all other available treatments. As a result, respondents are performing less surgical procedures, particularly cDCR in patients at higher surgical morbidity.

Cutaneous pleomorphic adenoma of the periocular region - a case series.

Mixed tumour of the skin is a rare entity also known as chondroid syringoma and pleomorphic adenoma. These usually present as slow-growing skin nodules with a smooth surface, clear boundaries, and no ulceration. Case series exist describing pleomorphic adenomas in the periocular region including the lids and orbit, separate to the more familiar lacrimal gland pleomorphic adenoma. These may arise from accessory or ectopic lacrimal gland tissue but in the eyelids are more likely to arise from sweat glands in the skin. Histopathological analysis of these lesions is important to identify complete excision, minimising recurrences and in identifying rare but potential malignant transformation. We describe the clinical features and outcomes in three cases of pleomorphic adenoma with two at the medial canthus (including one recurrence) and one in the brow region.

Lacrimal gland pleomorphic adenoma with extensive necrosis.

Lacrimal gland pleomorphic adenomas (LGPA) are benign mixed tumors. Diagnosis is based on clinical and radiological findings which usually prompts complete excision of the lesion to minimise recurrence and a cumulative risk of malignant transformation. Necrosis in pleomorphic adenoma has been rarely reported in salivary gland PA, either spontaneously or due to iatrogenic interventions. Necrosis is suggestive of a malignant process and makes interpretation of histology specimens difficult. A 23 year old woman, while awaiting biopsy for a mass in the left lacrimal gland, which had been symptomatic for only several months, presented with acute pain and swelling of the left lateral lid. An incisional biopsy showed an inflamed lacrimal gland with focal necrosis and atypia of adjacent cytology and gland architecture. Subsequent excisional biopsy confirmed an LGPA with some inflammation but no necrosis. Necrosis may occur as an atypical presentation in LGPA.

A protocol for rapid and parallel isolation of myocytes and non-myocytes from multiple mouse hearts.

This protocol features parallel isolation of myocytes and non-myocytes from murine hearts. It was designed with considerations for (1) time required to extract cardiac cells, (2) cell viability, and (3) protocol scalability. Here, a peristaltic pump and 3D-printed elements are combined to perfuse the heart with enzymes to dissociate cells. Myocytes and non-myocytes extracted using this protocol are separated by centrifugation and/or fluorescence-activated cell sorting for use in downstream applications including single-cell omics or other bio-molecular analyses. For complete details on the use and execution of this protocol, please refer to McLellan et al. (2020).

COVID-19 diagnosis and testing in pediatric heart transplant recipients.

Pediatric heart transplant recipients have been expected to be at higher risk of adverse events from developing COVID-19 infection. COVID-19 RNA PCR and antibody testing has been performed in our cohort of patients since March 15, 2020 and outcomes were reviewed. COVID-19 infection in our population of pediatric heart transplant recipients is common (21%), despite recommendations to avoid contact with others. Asymptomatic COVID-19 infection is common as well (55%). Despite the frequency of infection, COVID-19 is well tolerated in this population (5% admission from home; 0% mortality). A suppressed immune system does not significantly inhibit an antibody response in pediatric heart transplant recipients (>70% antibody seroconversion) and appears to persist, similar to those without transplantation (>90 days). Routine testing for COVID-19 via PCR and antibody testing enhances the ability to detect COVID-19 infection in asymptomatic patients and may help reduce unintended transmission to more susceptible individuals.

A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics.

Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.

Quantification of popliteal artery narrowing with QISS MRA during active ankle plantarflexion in healthy, asymptomatic volunteers and its potential application in the diagnosis of popliteal artery entrapment syndrome (PAES).

OBJECTIVE: To assess the degree of narrowing of the popliteal artery during active ankle plantar flexion in healthy volunteers using a non-contrast quiescent-interval single-shot (QISS) magnetic resonance angiography (MRA) technique. MATERIALS AND METHODS: Following IRB approval, 10 healthy volunteers were recruited and following informed consent underwent QISS MRA of the lower extremity at rest and during ankle plantarflexion. Two pediatric musculoskeletal radiologists independently reviewed MR images in random order and recorded a number of subjective and objective anatomic variables including branch pattern, proximity of vessel to bony structures, gastrocnemius bulk, and presence of accessory muscle. Arterial narrowing with plantarflexion was recorded by a subjective assessment of 3D reconstructions (negligible or non-negligible) and objectively by measuring the narrowest diameter during plantarflexion and at rest. Agreement between reader scores was assessed using the concordance correlation coefficient (CCC) for continuous variables, and kappa and the proportion of agreement for categorical variables. RESULTS: Mean reduction in arterial diameter during plantar flexion was 17.1% (min 1.9%, max 64.1%, SD 16.7%) for reader 1 and 17.2% (min 1.7%, max 50.0%, SD 14.3%.) for reader 2 with high agreement between readers: CCC = 0.92 and CI = 0.82, 0.96. Arterial narrowing was described subjectively as "non-negligible" in 7/20 legs by reader 1 and 5/20 legs by reader 2 with proportion of agreement = 0.90, CI (0.77, 1.00). CONCLUSION: We observed a wide range of popliteal arterial narrowing with plantarflexion in asymptomatic volunteers. Larger studies, for which QISS is well suited, may be invaluable for distinguishing physiologic from pathologic arterial narrowing in patients with suspected popliteal artery entrapment syndrome (PAES).

Ex uno, plures-From One Tissue to Many Cells: A Review of Single-Cell Transcriptomics in Cardiovascular Biology.

Recent technological advances have revolutionized the study of tissue biology and garnered a greater appreciation for tissue complexity. In order to understand cardiac development, heart tissue homeostasis, and the effects of stress and injury on the cardiovascular system, it is essential to characterize the heart at high cellular resolution. Single-cell profiling provides a more precise definition of tissue composition, cell differentiation trajectories, and intercellular communication, compared to classical bulk approaches. Here, we aim to review how recent single-cell multi-omic studies have changed our understanding of cell dynamics during cardiac development, and in the healthy and diseased adult myocardium.