Suppression of Lung Oxidative Stress, Inflammation, and Fibrosis following Nitrogen Mustard Exposure by the Selective Farnesoid X Receptor Agonist Obeticholic Acid.

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause pulmonary injury that can progress to fibrosis. NM toxicity is associated with an influx of inflammatory macrophages in the lung. Farnesoid X receptor (FXR) is a nuclear receptor involved in bile acid and lipid homeostasis that has anti-inflammatory activity. In these studies, we analyzed the effects of FXR activation on lung injury, oxidative stress, and fibrosis induced by NM. Male Wistar rats were exposed to phosphate-buffered saline (vehicle control) or NM (0.125 mg/kg) by intratracheal Penncentury-MicroSprayer aerosolization; this was followed by treatment with the FXR synthetic agonist, obeticholic acid (OCA, 15 mg/kg), or vehicle control (0.13-0.18 g peanut butter) 2 hours later and then once per day, 5 days per week thereafter for 28 days. NM caused histopathological changes in the lung, including epithelial thickening, alveolar circularization, and pulmonary edema. Picrosirius red staining and lung hydroxyproline content were increased, indicative of fibrosis; foamy lipid-laden macrophages were also identified in the lung. This was associated with aberrations in pulmonary function, including increases in resistance and hysteresis. Following NM exposure, lung expression of HO-1 and iNOS, and the ratio of nitrates/nitrites in bronchoalveolar lavage fluid (BAL), markers of oxidative stress increased, along with BAL levels of inflammatory proteins, fibrinogen, and sRAGE. Administration of OCA attenuated NM-induced histopathology, oxidative stress, inflammation, and altered lung function. These findings demonstrate that FXR plays a role in limiting NM-induced lung injury and chronic disease, suggesting that activating FXR may represent an effective approach to limiting NM-induced toxicity. SIGNIFICANCE STATEMENT: In this study, the role of farnesoid-X-receptor (FXR) in mustard vesicant-induced pulmonary toxicity was analyzed using nitrogen mustard (NM) as a model. This study's findings that administration of obeticholic acid, an FXR agonist, to rats reduces NM-induced pulmonary injury, oxidative stress, and fibrosis provide novel mechanistic insights into vesicant toxicity, which may be useful in the development of efficacious therapeutics.

Differential effects of healthcare worker burnout on psychotropic medication use and misuse by occupational level.

PURPOSE: Burnout has been well examined among physicians and other high-wage, high-autonomy healthcare positions. However, lower-wage healthcare workers with less workplace autonomy (e.g., medical assistants, nurses' aides) represent a substantial proportion of the workforce, but remain understudied. We aimed to examine the effects of burnout on psychotropic medication use and misuse and whether these effects differed by occupational level. METHODS: In March 2022, we collected data from a diverse sample of US healthcare workers (N = 200) and examined the cross-sectional relationship between burnout and changes in prescribed psychotropic medication (i.e., starting, stopping, and/or having a change in the dose/frequency) during the COVID-19 pandemic. We also separately examined the relationship between burnout and psychotropic medication misuse (i.e., without a prescription, in greater amounts, more often, longer than prescribed, and/or for a reason other than prescribed). We stratified models by occupational level (prescribers/healthcare administrators vs. other healthcare workers). RESULTS: Greater burnout was associated with higher odds of changes in prescribed psychotropic medication among prescribers/healthcare administrators (aOR = 1.23, 95% CI 1.01, 1.48), but not among other healthcare workers (aOR = 1.04, 95% CI 0.98, 1.10). Greater burnout was not associated with psychotropic medication misuse among prescribers/healthcare administrators (aOR = 0.96, 95% CI 0.82, 1.12) but was associated with increased odds of psychotropic medication misuse among other healthcare workers (aOR = 1.07, 95% CI 1.01, 1.14). CONCLUSIONS: Potential disparities in help-seeking and healthcare access might manifest in non-medical use of prescription drugs among some healthcare workers, which has implications for worker safety and well-being.

Dynamic diagnosis of metamaterials through laser-induced vibrational signatures.

Mechanical metamaterials at the microscale exhibit exotic static properties owing to their engineered building blocks1-4, but their dynamic properties have remained substantially less explored. Their design principles can target frequency-dependent properties5-7 and resilience under high-strain-rate deformation8,9, making them versatile materials for applications in lightweight impact resistance10-12, acoustic waveguiding7,13 or vibration damping14,15. However, accessing dynamic properties at small scales has remained a challenge owing to low-throughput and destructive characterization8,16,17 or lack of existing testing protocols. Here we demonstrate a high-throughput, non-contact framework that uses MHz-wave-propagation signatures within a metamaterial to non-destructively extract dynamic linear properties, omnidirectional elastic information, damping properties and defect quantification. Using rod-like tessellations of microscopic metamaterials, we report up to 94% direction-dependent and rate-dependent dynamic stiffening at strain rates approaching 102 s-1, as well as damping properties three times higher than their constituent materials. We also show that frequency shifts in the vibrational response allow for characterization of invisible defects within the metamaterials and that selective probing allows for the construction of experimental elastic surfaces, which were previously only possible computationally. Our work provides a route for accelerated data-driven discovery of materials and microdevices for dynamic applications such as protective structures, medical ultrasound or vibration isolation.

Tunable Mechanical Response of Self-Assembled Nanoparticle Superlattices.

Self-assembled nanoparticle superlattices (NPSLs) are an emergent class of self-architected nanocomposite materials that possess promising properties arising from precise nanoparticle ordering. Their multiple coupled properties make them desirable as functional components in devices where mechanical robustness is critical. However, questions remain about NPSL mechanical properties and how shaping them affects their mechanical response. Here, we perform in situ nanomechanical experiments that evidence up to an 11-fold increase in stiffness (∼1.49 to 16.9 GPa) and a 5-fold increase in strength (∼88 to 426 MPa) because of surface stiffening/strengthening from shaping these nanomaterials via focused-ion-beam milling. To predict the mechanical properties of shaped NPSLs, we present discrete element method (DEM) simulations and an analytical core-shell model that capture the FIB-induced stiffening response. This work presents a route for tunable mechanical responses of self-architected NPSLs and provides two frameworks to predict their mechanical response and guide the design of future NPSL-containing devices.

Growth rules for irregular architected materials with programmable properties.

Biomaterials display microstructures that are geometrically irregular and functionally efficient. Understanding the role of irregularity in determining material properties offers a new path to engineer materials with superior functionalities, such as imperfection insensitivity, enhanced impact absorption, and stress redirection. We uncover fundamental, probabilistic structure-property relationships using a growth-inspired program that evokes the formation of stochastic architectures in natural systems. This virtual growth program imposes a set of local rules on a limited number of basic elements. It generates materials that exhibit a large variation in functional properties starting from very limited initial resources, which echoes the diversity of biological systems. We identify basic rules to control mechanical properties by independently varying the microstructure's topology and geometry in a general, graph-based representation of irregular materials.

Promoting simulation-based training in radiology: a homemade phantom for the practice of ultrasound-guided procedures.

OBJECTIVE: Ultrasound-guided intervention is an essential skill for many radiologists and critical for accurate diagnosis and treatment in many radiology subspecialties. Simulation using phantoms have demonstrated statistically significant benefits for trainees within the literature. We propose a novel phantom model which the authors feel is ideal for training clinical radiology trainees in the performance of ultrasound-guided procedures. METHODS: The recipe to prepare a homemade phantom is described. Results of a local survey from trainees preparing and using the phantom are also presented. RESULTS: This realistic training simulation model can be adapted to suit a variety of biopsy devices and procedures including soft tissue biopsy and cyst aspiration. The phantom mimics the sonographic appearances of soft tissue and biopsy targets can be concealed within. The phantom was easily prepared by 22 trainees (Likert score 4.5) and it functioned well (Likert score of 4.7). CONCLUSION: In summary, our phantom model is ideal for training clinical radiology trainees in the performance of ultrasound-guided core biopsy. The availability and low cost of the model, combined with the ease of preparation and reproducibility, make this an efficient and effective addition to the training process. ADVANCES IN KNOWLEDGE: A low cost easily handmade phantom recipe is described that could be easily implemented in training schemes.

Effectiveness of percutaneous vacuum-assisted excision (VAE) of breast lesions of uncertain malignant potential (B3 lesions) as an alternative to open surgical biopsy.

OBJECTIVES: Traditionally B3 breast lesions are treated surgically, but overtreatment is a concern, as the majority have a final benign diagnosis. A national screening program introduced vacuum-assisted excision (VAE) for managing B3 lesions in late 2016. This retrospective study aimed to assess the outcomes associated with this approach. METHODS: All B3 lesions diagnosed between 01/2017 and 12/2019 were identified at two centres. Information was obtained on the initial biopsy and final histology, and method of VAE image guidance, needle size and number of cores. Lesions were excluded if there was cancer elsewhere in the breast at the time of diagnosis; the lesion was not suitable for VAE due to position in the breast or had B3 pathology for which open biopsy was still required. The final decision to offer VAE was always made at a multidisciplinary meeting (MDM). Risk difference was used to test the significance at p ≤ .05. RESULTS: In total, 258 B3 lesions were diagnosed, 105 (40.7%) met the inclusion criteria and underwent VAE. VAE was performed under X-ray (89/105) or ultrasound guidance (16/105), taking an average of 18.5 cores with the 10-G needle or 10.8 cores with the 7-G needle. Nine cases (8.6%) were upgraded to a malignant diagnosis following VAE. Malignancy was found in 15.5% (9/58) of B3 lesions with epithelial atypia, but in none without atypia (0/47) (p = .004). No new lesions or malignancy has occurred at the site of the VAE with an average mammographic follow-up of 2.2 years. CONCLUSION: Upgrade to malignancy following VAE was uncommon (8.6%) and associated with atypia in the initial biopsy. VAE is an alternative approach to the management of B3 lesions, reducing open surgical procedures. KEY POINTS: • Upgrade to malignancy after a vacuum-assisted excision of a B3 breast lesion is uncommon with an 8.6% upgrade rate. • The risk of a malignant diagnosis after a vacuum-assisted excision was significantly higher for B3 lesions with atypia compared to those without (+15.5% difference, p = .004).

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

Combined Intranasal Nanoemulsion and RIG-I Activating RNA Adjuvants Enhance Mucosal, Humoral, and Cellular Immunity to Influenza Virus.

Current influenza virus vaccines are focused on humoral immunity and are limited by the short duration of protection, narrow cross-strain efficacy, and suboptimal immunogenicity. Here, we combined two chemically and biologically distinct adjuvants, an oil-in-water nanoemulsion (NE) and RNA-based agonists of RIG-I, to determine whether the diverse mechanisms of these adjuvants could lead to improved immunogenicity and breadth of protection against the influenza virus. NE activates TLRs, stimulates immunogenic apoptosis, and enhances cellular antigen uptake, leading to a balanced TH1/TH2/TH17 response when administered intranasally. RIG-I agonists included RNAs derived from Sendai and influenza viral defective interfering RNAs (IVT DI, 3php, respectively) and RIG-I/TLR3 agonist, poly(I:C) (pIC), which induce IFN-Is and TH1-polarized responses. NE/RNA combined adjuvants potentially allow for costimulation of multiple innate immune receptor pathways, more closely mimicking patterns of activation occurring during natural viral infection. Mice intranasally immunized with inactivated A/Puerto Rico/8/1934 (H1N1) (PR/8) adjuvanted with NE/IVT DI or NE/3php (but not NE/pIC) showed synergistic enhancement of systemic PR/8-specific IgG with significantly greater avidity and virus neutralization activity than the individual adjuvants. Notably, NE/IVT DI induced protective neutralizing titers after a single immunization. Hemagglutinin stem-specific antibodies were also improved, allowing recognition of heterologous and heterosubtypic hemagglutinins. All NE/RNAs elicited substantial PR/8-specific sIgA. Finally, a unique cellular response with enhanced TH1/TH17 immunity was induced with the NE/RNAs. These results demonstrate that the enhanced immunogenicity of the adjuvant combinations was synergistic and not simply additive, highlighting the potential value of a combined adjuvant approach for improving the efficacy of vaccination against the influenza virus.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer.

BACKGROUND: The US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients. METHODS: We used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy. RESULTS: Monotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC). CONCLUSIONS: These results support combining α-PD-1 therapy with induction of IFN-α/ß signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Steric Inhibition of 5' UTR Regulatory Elements Results in Upregulation of Human CFTR.

Cystic fibrosis (CF) is an autosomal recessive monogenic disease caused by mutations in the CFTR gene. Therapeutic approaches that are focused on correcting CFTR protein face the challenge of the heterogeneity in CFTR mutations and resulting defects. Thus, while several small molecules directed at CFTR show benefit in the clinic for subsets of CF patients, these drugs cannot treat all CF patients. Additionally, the clinical benefit from treatment with these modulators could be enhanced with novel therapies. To address this unmet need, we utilized an approach to increase CFTR protein levels through antisense oligonucleotide (ASO)-mediated steric inhibition of 5' UTR regulatory elements. We identified ASOs to upregulate CFTR protein expression and confirmed the regulatory role of the sites amenable to ASO-mediated upregulation. Two ASOs were investigated further, and both increased CFTR protein expression and function in cell lines and primary human bronchial epithelial cells with distinct CF genotypes. ASO treatment further increased CFTR function in almost all CF genotypes tested on top of treatment with the FDA approved drug Symdeko (ivacaftor and tezacaftor). Thus, we present a novel approach to CFTR therapeutic intervention, through ASO-mediated modulation of translation.

Photocontrolled Release of Doxorubicin Conjugated through a Thioacetal Photocage in Folate-Targeted Nanodelivery Systems.

Despite their proven ability for precise and targeted release, nanoplatform systems for photocontrolled delivery often face formidable synthetic challenges, in part due to the paucity of advanced linker strategies. Here, we report on a novel linker strategy using a thioacetal ortho-nitrobenzaldehyde (TNB) cage, demonstrating its application for delivery of doxorubicin (Dox) in two nanoscale systems. This photocleavable linker, TNB(OH), which presents two identical arms, each terminated with a hydroxyl functionality, was prepared in a single step from 6-nitroveratraldehyde. TNB(OH) was used to cross-link Dox to a folate receptor (FAR)-targeting poly(amidoamine) dendrimer conjugate G5(FA)n=5.4(Dox)m=5.1, and also used to prepare an upconversion nanocrystal (UCN) conjugate, UCN-PPIX@(Dox)(G5FA), a larger core/shell nanostructure. In this core/shell nanostructure, the UCN core emits UV and visible light luminescence upon near-infrared (NIR) excitation, allowing for the photocleavage of the TNB linker as well as the photostimulation of protoporphyrin IX (PPIX) coupled as a cytotoxic photosensitizer. Drug-release experiments performed in aqueous solutions with long-wavelength ultraviolet A (UVA) light showed that Dox release occurred rapidly from its TNB linked form or from its dendrimer conjugated form with comparable decay kinetics. Cellular toxicity studies in FAR-overexpressing KB carcinoma cells demonstrated that each nanoconjugate lacked intrinsic cytotoxicity until exposed to UVA or NIR (980 nm) (for the UCN nanoconjugate), which resulted in induction of potent cytotoxicity. In summary, this new TNB strategy offers synthetic convenience in drug conjugation chemistry with the ability for the temporal control of drug activation at the delivery site.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV) (Part 1 - small molecules, peptides and small molecule biomarkers by LCMS).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This white paper is published in 3 parts due to length. This part (Part 1) discusses the recommendations for small molecules, peptides and small molecule biomarkers by LCMS. Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in the Bioanalysis journal, issue 23.

Light-controlled active release of photocaged ciprofloxacin for lipopolysaccharide-targeted drug delivery using dendrimer conjugates.

We report an active delivery mechanism targeted specifically to Gram(-) bacteria based on the photochemical release of photocaged ciprofloxacin carried by a cell wall-targeted dendrimer nanoconjugate.

Positive youth development programs for adolescents with greater psychosocial needs: subjective outcome evaluation over 3 years.

STUDY OBJECTIVES: This study examined the views of 153,761 students participating in a positive youth development program designed for participants with greater psychosocial needs (the Tier 2 Program) in the context of the Project P.A.T.H.S. in Hong Kong. The program was implemented in the extension phase of the project from 2009/10 to 2011/12 school years. DESIGN: A validated subjective outcome evaluation scale was used to assess the views of the program participants toward the program qualities, implementer qualities, and program effectiveness after completion of the program. Nine datasets were used which were derived from the aggregated reports submitted by social service providers designing the Tier 2 Program. RESULTS: Participants generally held favorable views of program qualities, implementer qualities, as well as program effectiveness of the Tier 2 Program. Some small grade and program differences on subjective outcome evaluation were also found. Both program qualities and implementer qualities were significant predictors of program effectiveness in different grades. CONCLUSION: Consistent with the findings of the initial phase of the Project P.A.T.H.S., the present study suggests that the Tier 2 Program of the Project P.A.T.H.S. in Hong Kong is perceived favorably by program participants and its perceived effectiveness was high. Significant but small grade and program approach differences on subjective outcome evaluation were found. Both program and implementer qualities were predictive of perceived program effectiveness in different grades.

Development of a credit-bearing service leadership subject for university students in Hong Kong.

Given that service industries contribute to 93% of Hong Kong's GDP, an important question that should be asked is whether the education system can promote the development of service leadership among young people in Hong Kong. Unfortunately, service leadership programs specifically designed for university students are lacking in Hong Kong. In this paper, the basic tenets of the Service Leadership and Management framework are presented. Based on the fundamental postulation that effective service leadership is a function of moral character, competence and care (E=MC(2)), a subject called "Service Leadership" has recently been developed at The Hong Kong Polytechnic University. This paper outlines the objectives, intended learning outcomes, syllabus, assessment, and references of the subject. The proposed evaluation strategies are also described.

Evaluation of the Project P.A.T.H.S. (extension phase) based on the perspective of the program participants.

Subjective outcome evaluation data were collected from 236 schools (n=87,943 students) after students had participated in the Tier 1 Program in the second year of the Extension Phase of the Project P.A.T.H.S. in Hong Kong. Using schools as the units of analysis, results showed that the program and implementers were perceived in a positive manner and approximately four-fifths of the participants regarded the program as helpful to them. There were some significant grade differences in the subjective outcome evaluation findings with small effect size. Multiple regression analyses showed that whereas perceived qualities of the program positively predicted perceived effectiveness of the program, perceived qualities of implementers negatively predicted program effectiveness. The present study suggests that irrespective of cohorts at different times, junior secondary school students perceived the program to be beneficial to them. The theoretical and practical implications of the present findings are discussed.

Helping adolescents with greater psychosocial needs: the extension phase of the Project P.A.T.H.S. in Hong Kong.

Subjective outcome evaluation data were collected from 24,198 students of 236 schools after completion of the Tier 2 Program in the second year of the Extension Phase of the Project P.A.T.H.S. in Hong Kong. Using schools as units of analysis, results showed that the program, implementers, and effectiveness were perceived in a positive manner. Although subjective outcome evaluation findings differed across participant types, grades, and program types did not show significant differences on the outcome measures. Consistent with previous reports, perceived effectiveness of the program was significantly predicted by student perceptions about the program and program instructor. These findings provide support for the claim that the Tier 2 Program is effective in promoting positive development among adolescents with greater psychosocial needs in the extension phase of the Project P.A.T.H.S. in Hong Kong.

Evaluation of Project P.A.T.H.S. by the program implementers: findings based on the extension phase.

A total of 236 schools participated in the Project P.A.T.H.S. in the 2010/2011 school year. After completion of the Tier 1 Program, subjective outcome evaluation data were collected from 3275 program implementers. Based on the consolidated findings based on schools as units, results showed that participants had positive perceptions of the program, implementers, and benefits of the program. More than four-fifths of the implementers regarded the program as helpful to the program participants. Multiple regression analysis revealed that perceived qualities of the program and the program implementers predicted perceived effectiveness of the program. Grade differences were not significant. The present study provides additional support for the effectiveness of the Tier 1 Program of the Project P.A.T.H.S. in Hong Kong.