On October 29, 2024, a virtual meeting, brought together chairs and vice chairs from several research-oriented U.S. Schools of Pharmacy to discuss the current landscape of pharmaceutical sciences, advocacy strategies, and best practices in graduate education. Key topics included alumni engagement, and the relatively low number of trainees interested in an academic career path. Dr. Dawn Beraud, the Executive Director of the American Institute for Medical and Biological Engineering (AIMBE) emphasized the importance of science advocacy, including increasing science funding and linking evidence-based decision making to policy. Discussions about graduate education highlighted best practices in using large language models, financial support challenges, and the significant role of industrial professionals in teaching. The proposed NextProf PharmSci program was discussed, which aims to address the imbalance of trainees leaning towards industry by preparing them for academic roles through a multi-day workshop. Participants expressed strong support for this initiative, emphasizing the need for a training pipeline and mentoring junior faculty. Participants agreed the event should be held annually, as it was found to be useful, and discussed future topics including sharing graduate and PharmD curricula as well as a shared summer school program.

Non-degradative histone ubiquitylation plays a myriad of well-defined roles in the regulation of gene expression and choreographing DNA damage repair pathways. In contrast, the contributions of degradative histone ubiquitylation on genomic processes has remained elusive. Recently, the APC/C has been shown to ubiquitylate histones to regulate gene expression in pluripotent cells, but the molecular mechanism is unclear. Here we show that despite directly binding to the nucleosome through subunit APC3, the APC/C is unable to ubiquitylate nucleosomal histones. In contrast, extranucleosomal H2A/H2B and H3/H4 complexes are broadly ubiquitylated by the APC/C in an unexpected manner. Using a combination of cryo-electron microscopy (cryo-EM) and biophysical and enzymatic assays, we demonstrate that APC8 and histone tails direct APC/C-mediated polyubiquitylation of core histones in the absence of traditional APC/C substrate degron sequences. Taken together, our work implicates APC/C-nucleosome tethering in the degradation of diverse chromatin-associated proteins and extranucleosomal histones for the regulation of transcription and the cell cycle and for preventing toxicity due to excess histone levels.

Red blood cells (RBCs) have been employed to convey and deliver a variety of therapeutic agents, from small molecules to proteins. The therapeutics are typically installed within the RBC interior via a pore-forming process that results in membrane disruption and a partial loss of hemoglobin. An alternative approach, namely appending therapeutics to the RBC surface, has received significantly less attention. Here we focus on the characterization of an array of membrane anchoring modalities (noncovalent, reversible covalent, and covalent). Surface modification is experimentally simpler and structurally less invasive than its membrane disruptive counterpart. This panel is designed, synthesized and assessed with respect to RBC loading capacity, retention, and rate of transfer to other cell populations. The cell surface anchors are appended to a structural scaffold (cobalamin) that can house and deliver therapeutic agents. Imaging studies for a series of representative derivatives reveal that these species are not internalized by the RBCs, consistent with the absence of an active endocytic pathway in mature RBCs. Furthermore, enzymatic digestion of the glycocalyx failed to impair loading or retention, suggesting that the derivatives are likely anchored to the RBC membrane. The structural motifs identified in this study provide a template for the development of membrane tethered therapeutics that are specifically designed to be transported to diseased sites by RBCs.

Abstract ID 87610Poster Board 262Aim: Cardiac cAMP signaling is coordinated in distinct intracellular domains where PKA protein kinase A (PKA) localization occurs. This signaling axis plays a functional role in cardiac homeostasis and disease progression. However, the use of traditional pharmacological tools that stimulate global activation fails to capture the unique contribution of spatially specific PKA signaling. Photoactivatable adenylyl cyclase is a potential means to study cAMP-dependent PKA signaling with spatiotemporal resolution. This study tests the hypothesis that compartmentalized generation of cAMP by photoactivatable adenylyl cyclase will achieve spatially specific PKA signaling in cardiac cells.Methods: Cytosolically diffuse (bPACCy), plasma membrane (bPACPM), and outer mitochondrial membrane (bPACOMM) anchored photoactivatable adenylyl cyclase from beggiotoa (bPAC) were transfected into H9c2 cardiac myoblast cells. The cells were exposed to blue light stimulus and cAMP signaling was assessed by the protein kinase A phosphorylation of a genetically encoded sensor derived from vasodilator-stimulated phosphoprotein (VASP). The phosphorylation of endogenous VASP was used to validate cAMP generation in response to light. PM, and OMM anchored VASP reporters were used to map light induced cAMP signaling at distinct intracellular domains. Western blot analysis was conducted to determine the extent of protein phosphorylation.Results: Pharmacological cAMP stimulation using forskolin (100 uM, 30 m) leads to the phosphorylation of endogenous VASP as well as the PM and OMM VASP reporters. Cytosolic bPACCy increases phosphorylation of OMM VASP reporter in response to light, while having no impact on PM localized VASP reporter or endogenous VASP. bPACPM increased endogenous VASP phosphorylation in response to light but had no effect on OMM VASP reporter. bPACOMM did not phosphorylate endogenous VASP, but increased phosphorylation of PM VASP reporters.Conclusion: bPAC can be used as a tool to differentially produce cAMP in response to light. The optogenetic constructs activated either endogenous, PM, and OMM-anchored VASP preferentially in light, highlighting the use of bPAC to study PKA stimulation with spatiotemporal control. The optogenetic control of cAMP production allows for the ability to study microdomain specific signaling that characterizes cardiac signaling in health and disease.This work is supported by NIH grant R01 HL159194 (L.H.) and the Morehead-Cain Foundation.

A survey was developed and implemented to determine how pervasively safety principles and cultural expectations are included in the chemistry curriculum at the undergraduate level. The survey was completed by first-year doctoral candidates in the chemical/pharmaceutical sciences. A majority of the respondents were trained in the use of Safety Data Sheets (SDSs) and PubChem. Additionally, a majority (61%) reported frequent safety discussions prior to performing experiments in their laboratory courses. In contrast, similar discussions occurred infrequently in their research laboratories (16%). Approximately 20% of the surveyed students reported experiencing a laboratory injury, and significantly larger percentages observed near misses in their teaching and research laboratories. However, the survey did not find any correlation between the frequency of safety discussions, chemical resource training, or the delivery mode of training with the likelihood of incidents. The survey did find that a specific training method explicitly intended to preclude mishaps, namely, the RAMP risk management system, is not well integrated into the undergraduate chemistry curriculum. Finally, the survey contained a series of potentially dangerous laboratory scenarios in order to determine whether the tendency to engage in risky laboratory behavior correlates with specific demographics and/or prior experiences. The data suggest that prior experience with an injury or observation of a near miss encourages the choice of safer options when confronted with a hazardous situation.

Occlusive blood clots remain a significant global health challenge and result in emergencies that are main causes of death and disability worldwide. Thrombolytic agents (including tissue plasminogen activator, tPA) are the only pharmacological means to dissolve blood clots. However, these drugs have modest efficacy and severe safety concerns persist. We have developed light-responsive tPA-loaded red blood cells (tPA-RBCsPhoto) to target thrombolytic activity at the site of a blood clot. Herein, we describe the use of light to control the release of tPA from engineered RBCs and the subsequent degradation of a blood clot ex vivo. Furthermore, we have employed this technology to restore blood flow to an occluded mouse artery in vivo using a targeted dose that is 25 times lower than conventional systemic tPA treatment.

A variety of strategies have been introduced to tackle the issue of laboratory safety in academic settings. These strategies include innovative educational tools as students transition from the classroom into the laboratory, the introduction of policies designed to protect the researchers in the laboratory environment, and the creation of teams of peers at the departmental level that focus on creating a culture of safety. We recently developed, as a training exercise, virtual reality (VR) modules that replicate the visual and auditory complexities of authentic research laboratories (J. Chem. Educ. 2022, 99, 1982-1989). We have now used the VR environments to compare the relative abilities of individuals and of teams to identify laboratory hazards by quantifying several variables: time spent in the virtual environment, percentage of total hazards identified, percentage of hazards correctly characterized, number of attempts to identify hazards, and number of requests for assistance. In addition, an online survey was used to assess the perceptions of individual and team performances by the participants. Quantitative measures demonstrated the superior performances of teams versus that of individuals. Intriguingly, the smaller coefficient of variance associated with these metrics for teams (versus individuals) implies a group dynamic that suppresses outliers. Finally, results from the online survey reveal that the team format is perceived to be more effective than individuals in identifying hazards. However, team leaders were more subdued in their enthusiasm for the group format than were the members of their teams, which appears to be associated with subtle group dynamics.

Laboratory safety has received heightened attention due to a series ofdevastatingly tragic accidents in both academic and nonacademic settings.Consequently, chemistry departments at various academic institutions now offersome form of formal training in laboratory safety for entering graduate students.Although the extent of this training varies widely among institutions, it typicallyincludes an online assessment and/or minimal in-person classroom instruction.However, a significant gap exists between a lecture hall setting and the complexenvironment that comprises an advanced research laboratory. We've adapted thetechnological advances in virtual and augmented reality to bridge this gap. A set of360 degrees virtual reality lab experiences, highlighting safety infractions, have been createdfor a variety of subdiscipline-distinct (medicinal, organic, inorganic, physical, drugscreening) laboratory settings. Notable features include the accurate depiction of thevisual complexity associated with research settings, the opportunity for the trainee toexplore multiple laboratories in a self-paced fashion, and immediate feedback withrespect to the identification of safety hazards. The VR Lab Safety modules were very well received byfirst year graduate students,with greater than 85% of the respondents describing the VR experience as engaging and memorable, as a good supplement to safetyreading material, and as providing real world examples that are otherwise difficult to visualize

Arthritis is a leading cause of disability in adults, which can be intensely incapacitating. The location and intensity of the pain is both subjective and challenging to manage. Consequently, patient‐directed delivery of anti‐inflammatories is an essential component of future therapeutic strategies for the management of this disorder. The design and application of a light‐responsive red blood cell (RBC)‐conveyed dexamethasone (Dex) construct that enables targeted drug delivery upon illumination of the inflamed site is described. The red wavelength (650 nm) responsive nature of the phototherapeutic is validated using tissue phantoms mimicking the light absorbing properties of various skin types. Furthermore, photoreleased Dex has the same impact on cellular responses as conventional Dex. Murine RBCs containing the photoactivatable therapeutic display comparable circulation properties as fluorescently labeled RBCs. In addition, a single dose of light‐targeted Dex delivery is fivefold more effective in suppressing inflammation than the parent drug, delivered serially over multiple days. These results are consistent with the notion that the circulatory system be used as an on‐command drug depot, providing the means to therapeutically target diseased sites both efficiently and effectively.

Cobalamin has shown promise as a light-sensitive drug delivery platform owing to its ease of modification and the high quantum yields for drug photorelease. However, studies to date on the general photochemistry of alkyl cobalamins have primarily focused on methyl and adenosyl-substituted derivatives, the natural cofactors present in various enzymatic species. We describe the synthesis and photolytic behavior of cobalamin conjugates comprised of different combinations of fluorophores and β-axial ligands. In general, cobalamin conjugates containing β-axial alkyl substituents undergo efficient photolysis under aqueous conditions, with quantum yields up to >40%. However, substituents that are large and hydrophobic, or unable to readily support the presumed radical intermediate, suffer less efficient photolysis (<15%) than smaller, water-soluble, analogs. By contrast, quantum yields improve by 2-fold in DMF for cobalamins containing large hydrophobic β-axial substituents. This suggests that drug release from carriers comprised of membranous compartments, such as liposomes, may be significantly more efficient than the corresponding photorelease in an aqueous environment. Finally, we explored the impact of fluorophores on the photolysis of alkyl cobalamins under tissue-mimetic conditions. Cobalamins substituted with efficient photon-capturing fluorophores display up to 4-fold enhancements in photolysis relative to unsubstituted derivatives. In summary, we have shown that the photosensitivity of alkyl cobalamin conjugates can be tuned by altering the Co-appended alkyl moiety, modulating the polarity of the environment (solvent), and installing photon-capturing fluorophores onto the cobalamin framework.

Peptide bioreporters were developed to perform multiplexed measurements of the activation of epidermal growth factor receptor kinase (EGFR), Akt kinase (Akt/protein kinase B), and proteases/peptidases in single cells. The performance characteristics of the three reporters were assessed by measuring the reporter's proteolytic stability, kinetic constants for EGFR and Akt, and dephosphorylation rate. The reporter displaying optimal performance was composed of 6-carboxyfluorescein (6-FAM) on the peptide N-terminus, an Akt substrate sequence employing a threonine phosphorylation site for Akt, followed by a tri-D arginine linker, and finally an EGFR substrate sequence bearing a phosphatase-resistant 7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-htc) residue as the EGFR phosphorylation site. Importantly, use of a single electrophoretic condition separated the mono- and diphosphorylated products as well as proteolytic forms permitting the quantitation of multiple enzyme activities simultaneously using a single reporter. Because the Akt and EGFR substrates were linked, a known ratio (EGFR/Akt) of the reporter was loaded into cells. A photoactivatable version of the reporter was synthesized by adding two 4,5-dimethoxy-2-nitrobenzyl (DMNB) moieties to mask the EGFR and Akt phosphorylation sites. The DMNB moieties were readily photocleaved following exposure to 360 nm light, unmasking the phosphorylation sites on the reporter. The new photoactivatable reporter permitted multiplexed measurements of kinase signaling and proteolytic degradation in single cells in a temporally controlled manner. This work will facilitate the development of a new generation of multiplexed activity-based reporters capable of light-initiated measurement of enzymatic activity in single cells.

Objectives: SARS-CoV-2 has challenged health service provision worldwide. This work evaluates safe surgical pathways and standard operating procedures implemented in the high volume, global city of London during the first wave of SARS-CoV-2 infection. We also assess the safety of minimally invasive surgery(MIS) for anatomical lung resection. Methods: This multicentre cohort study was conducted across all London thoracic surgical units, covering a catchment area of approximately 14.8 Million. A Pan-London Collaborative was created for data sharing and dissemination of protocols. All patients undergoing anatomical lung resection 1st March-1st June 2020 were included. Primary outcomes were SARS-CoV-2 infection, access to minimally invasive surgery, post-operative complication, length of intensive care and hospital stay (LOS), and death during follow up. Results: 352 patients underwent anatomical lung resection with an median age of 69 (IQR: 35-86) years. Self-isolation and pre-operative screening were implemented following the UK national lockdown. Pre-operative SARS-CoV-2 swabs were performed in 63.1% and CT imaging in 54.8%. 61.7% of cases were performed minimally invasively. Median LOS was 6 days. Significant complications developed in 7.3% of patients (Clavien-Dindo Grade 3-4). There were 6 deaths(1.7%) and 12 re-admissions(3.4%). Seven patients(2.0%) were diagnosed with SARS-CoV-2 infection, two of whom died (28.5%). Conclusions: Major elective surgery can be safely undertaken via open and MIS approaches at the peak of a viral pandemic if precautionary measures are implemented. High volume surgery should continue during further viral peaks to minimise health service burden and potential harm to cancer patients.Funding Statement: Nil.Declaration of Interests: None of the authors have conflict of interests to declare.Ethics Approval Statement: Institutional approval was obtained from all Trusts contributing to the collaborative. Approval was granted by each hospital trust for data sharing, collaborative work and retrospective review. Formal individual informed consent was not required due to the retrospective nature of the study.

More than four decades have passed since the first example of a light-activated (caged) compound was described. In the intervening years, a large number of light-responsive derivatives have been reported, several of which have found utility under a variety of in vitro conditions using cells and tissues. Light-triggered bioactivity furnishes spatial and temporal control, and offers the possibility of precision dosing and orthogonal communication with different biomolecules. These inherent attributes of light have been advocated as advantageous for the delivery and/or activation of drugs at diseased sites for a variety of indications. However, the tissue penetrance of light is profoundly wavelength-dependent. Only recently have phototherapeutics that are photoresponsive in the optical window of tissue (600-900 nm) been described. This Review highlights these recent discoveries, along with their limitations and clinical opportunities. In addition, we describe preliminary in vivo studies of prospective phototherapeutics, with an emphasis on the path that remains to be navigated in order to translate light-activated drugs into clinically useful therapeutics. Finally, the unique attributes of phototherapeutics is highlighted by discussing several potential disease applications.

We have developed a technology that facilitates the targeted delivery of glucocorticoids to arthritic joints thereby side-stepping systemic immune suppression while potentially improving efficacy. Our technology loads phototherapeutics inside of red blood cell (RBC) carriers. This ensures that the inactive, RBC-contained phototherapeutic drug is transported throughout the body and released from the RBC carrier only after triggered release by long-wavelength light. We have demonstrated that medications bound to vitamin B12 can be photo-released from RBCs using tissue penetrating, long-wavelength red, far-red, and near infrared light in the collagen antibody-induced arthritis model.

Trauma represents the leading cause of death in patients younger than 45 years old. The introduction of multidisciplinary trauma teams has resulted in an improvement in patients` outcome. The thoracic surgeon plays an invaluable role as a member of the multidisciplinary team that includes an emergency physician, surgical figure, an anaesthetist, a radiology technician plus a number of nurses. Thorough knowledge of the physiological mechanism of injury and an intensive training have contributed massively in improving the outcomes. Different surgical approaches have to be considered in order to provide the best outcome to the acute patient with chest injury.

Protein therapeutics are a powerful class of drugs known for their selectivity and potency. However, the potential efficacy of these therapeutics is commonly offset by short circulatory half-lives and undesired action at otherwise healthy tissue. We describe herein a targeted protein delivery system that employs engineered red blood cells (RBCs) as carriers and light as the external trigger that promotes hemolysis and drug release. RBCs internally loaded with therapeutic proteins are readily surface modified with a dormant hemolytic peptide. The latter is activated via easily assigned wavelengths that extend into the optical window of tissue. We have demonstrated that photorelease transpires with spatiotemporal control and that the liberated proteins display the anticipated biological effects in vitro. Furthermore, we have confirmed targeted delivery of a clot-inducing enzyme in a mouse model. Finally, we anticipate that this strategy is not limited to RBC carriers but also should be applicable to nano- and microtransporters comprised of bilayer lipid membranes.

Introduction50 million patients are diagnosed with arthritis yearly, with a total of 20% of the nation suffering from this ailment. No curative treatment for arthritis currently exists, only therapeutics that mitigate its symptoms while inducing severe side effects from chronic systemic exposure. The current inadequacy of treatment highlights the need for innovative drug delivery methods. We have developed a photosensitive method of drug delivery that can be spatiotemporally controlled to treat arthritis and reduce systemic exposure to therapeutics, such as dexamethasone, that cause severe side effects. We hypothesize that by conveying a photo‐responsive vitamin B12‐dexamethasone (B12‐dex) conjugate within red blood cells (RBCs) we will be able to localize delivery of dex, treat arthritis with an overall lower amount of dex, and diminish systemic exposure.Methods and ResultsAlkyl‐cobalamin derivatives of vitamin B12 are known to contain an intrinsically photosensitive axial cobalt‐carbon bond. We employed this property to create a light responsive drug platform that is conveyed throughout the circulatory system by RBCs. First, we have demonstrated that the B12‐drug phototherapeutic agents can be loaded via a hypotonic swelling protocol and trapped within RBCs due to the membrane impermeability of B12. Second, we installed a Cy5 “antenna” on B12, enabling the phototherapeutic to respond to long wavelength (650 nm) tissue‐penetrating light. Third, we employed intravital imaging to demonstrate that RBC‐conveyed phototherapeutics are retained in circulation for up to 2 hours, whereas free B12‐drug conjugates rapidly diffuse into surrounding tissue.Building off of these results, we synthesized and loaded a B12‐dex conjugate into RBCs and investigated the therapeutic efficacy of this agent using a collagen antibody induced mouse model of arthritis. Arthritic mice were treated with intraperitoneal (IP) dex at a standard dose, B12‐dex RBCs, and B12‐water RBCs. Dex or water from B12‐drug RBCs was released locally to the arthritic paw via a 650 nm 3 mW laser. As expected, standard of care dex caused arthritis to go into remission while B12‐water RBCs did not treat arthritis. However, B12‐dex RBCs also successfully induced remission of arthritis with a 3‐fold lower dose of dex relative to IP dex. Thus, we successfully treated arthritic mice using B12‐dex RBCs while systemically delivering a much lower dose to achieve comparable remission to IP dex treatment.ConclusionsWe have demonstrated that B12‐dex, conveyed by RBCs, offers a novel method for the treatment of arthritis. This technology potentially addresses three key limitations of current arthritis therapy: (1) the inability to selectively deliver high quantities of a drug to the inflamed joint, (2) moderate to severe systemic side effects from long‐term exposure, and (3) the inability to direct therapeutics in a patient‐directed fashion. Future work will further probe the issue of selectivity and side effects by investigating the therapeutic index of B12‐dex as compared to IP dex.Support or Funding InformationSupported by the Cancer Cell Biology Training Program (NIH T32 CA071341) and the Rheumatology Research Foundation Innovative Research AwardModel of Light‐Mediated Delivery of Dexamethasone from B12‐Dexamethasone RBCsFigure 1Structure of B12‐Dexamethasone: Cy5 antenna is highlighted in red, Dex in blue, and B12 scaffold in blackFigure 2

Herein, the use of red blood cells (RBCs) as carriers of cytoplasmically interned phototherapeutic agents is described. Photolysis promotes drug release from the RBC carrier thereby providing the means to target specific diseased sites. This strategy is realized with a vitamin B12-taxane conjugate (B12-TAX), in which the drug is linked to the vitamin via a photolabile CoC bond. The conjugate is introduced into mouse RBCs (mRBCs) via a pore-forming/pore-resealing procedure and is cytoplasmically retained due to the membrane impermeability of B12. Photolysis separates the taxane from the B12 cytoplasmic anchor, enabling the drug to exit the RBC carrier. A covalently appended Cy5 antenna sensitizes the conjugate (Cy5-B12-TAX) to far red light, thereby circumventing the intense light absorbing properties of hemoglobin (350-600 nm). Microscopy and imaging flow cytometry reveal that Cy5-B12-TAX-loaded mRBCs act as drug carriers. Furthermore, intravital imaging of mice furnish a real time assessment of circulating phototherapeutic-loaded mRBCs as well as evidence of the targeted photorelease of the taxane upon photolysis. Histopathology confirms that drug release occurs in a well resolved spatiotemporal fashion. Finally, acoustic angiography is employed to assess the consequences of taxane release at the tumor site in Nu/Nu-tumor-bearing mice.

Schwannoma is a benign encapsulated tumor of the nerve sheath. Amongst other sites, it develops in the posterior mediastinum in the costovertebral sulcus. We herein present a case of a 68-year-old woman with an incidental finding of a subcarinal mass. Radiological and histopathological studies were suggestive of schwannoma. Therefore, the mass was completely resected through a right thoracotomy, and a definite histopathological diagnosis was established. Although the subcarinal area is a rare site for this tumor to appear, the schwannoma should be considered as part of the differential diagnosis of lesions in the subcarinal region. Treatment of choice is the nerve-sparing surgical excision of the mass with excellent prognosis. A review of the literature on this topic was performed.