Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Deep eutectic solvents as green and sustainable diluents in headspace gas chromatography for the determination of trace level genotoxic impurities in pharmaceuticals.

Genotoxic impurities (GTIs) are potential carcinogens that need to be controlled down to ppm or lower concentration levels in pharmaceuticals under strict regulations. The static headspace gas chromatography (HS-GC) coupled with electron capture detection (ECD) is an effective approach to monitor halogenated and nitroaromatic genotoxins. Deep eutectic solvents (DESs) possess tunable physico-chemical properties and low vapor pressure for HS-GC methods. In this study, zwitterionic and non-ionic DESs have been used for the first time to develop and validate a sensitive analytical method for the analysis of 24 genotoxins at sub-ppm concentrations. Compared to non-ionic diluents, zwitterionic DESs produced exceptional analytical performance and the betaine : 7 (1,4- butane diol) DES outperformed the betaine : 5 (1,4-butane diol) DES. Limits of detection (LOD) down to the 5-ppb concentration level were achieved in DESs. Wide linear ranges spanning over 5 orders of magnitude (0.005-100 µg g-1) were obtained for most analytes with exceptional sensitivities and high precision. The method accuracy and precision were validated using 3 commercially available drug substances and excellent recoveries were obtained. This study broadens the applicability of HS-GC in the determination of less volatile GTIs by establishing DESs as viable diluent substitutes for organic solvents in routine pharmaceutical analysis.

Ionic Liquids in Metal, Photo-, Electro-, and (Bio) Catalysis.

Ionic liquids (ILs) have unique physicochemical properties that make them advantageous for catalysis, such as low vapor pressure, non-flammability, high thermal and chemical stabilities, and the ability to enhance the activity and stability of (bio)catalysts. ILs can improve the efficiency, selectivity, and sustainability of bio(transformations) by acting as activators of enzymes, selectively dissolving substrates and products, and reducing toxicity. They can also be recycled and reused multiple times without losing their effectiveness. ILs based on imidazolium cation are preferred for structural organization aspects, with a semiorganized layer surrounding the catalyst. ILs act as a container, providing a confined space that allows modulation of electronic and geometric effects, miscibility of reactants and products, and residence time of species. ILs can stabilize ionic and radical species and control the catalytic activity of dynamic processes. Supported IL phase (SILP) derivatives and polymeric ILs (PILs) are good options for molecular engineering of greener catalytic processes. The major factors governing metal, photo-, electro-, and biocatalysts in ILs are discussed in detail based on the vast literature available over the past two and a half decades. Catalytic reactions, ranging from hydrogenation and cross-coupling to oxidations, promoted by homogeneous and heterogeneous catalysts in both single and multiphase conditions, are extensively reviewed and discussed considering the knowledge accumulated until now.

Impact of cryopreservation on elastomuscular artery mechanics.

Low temperatures slow or halt undesired biological and chemical processes, protecting cells, tissues, and organs during storage. Cryopreservation techniques, including controlled media exchange and regulated freezing conditions, aim to mitigate the physical consequences of freezing. Dimethyl sulfoxide (DMSO), for example, is a penetrating cryoprotecting agent (CPA) that minimizes ice crystal growth by replacing intracellular water, while polyvinyl alcohol (PVA) is a nonpenetrating CPA that prevents recrystallization during thawing. Since proteins and ground substance dominate the passive properties of soft biological tissues, we studied how different freezing rates, storage temperatures, storage durations, and the presence of cryoprotecting agents (5% [v/v] DMSO + 1 mg/mL PVA) impact the histomechanical properties of the internal thoracic artery (ITA), a clinically relevant blood vessel with both elastic and muscular characteristics. Remarkably, biaxial mechanical analyses failed to reveal significant differences among the ten groups tested, suggesting that mechanical properties are virtually independent of the cryopreservation technique. Scanning electron microscopy revealed minor CPA-independent delamination in rapidly frozen samples, while cryoprotected ITAs had better post-thaw viability than their unprotected counterparts using methyl thiazole-tetrazolium (MTT) metabolic assays, especially when frozen at a controlled rate. These results can be used to inform ongoing and future studies in vascular engineering, physiology, and mechanics.

Chronic shedding of a SARS-CoV-2 Alpha variant in wastewater.

BACKGROUND: Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program since 2021. Wastewater samples were collected from on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR and results were reported to the health department. RESULTS: One rural, off-campus site consistently produced higher concentrations of SARS-CoV-2 genome copies. Samples from this site were sequenced and contained predominately a derivative of Alpha variant lineage B.1.1.7, detected from fall 2021 through summer 2023. Mutational analysis of reconstructed genes revealed divergence from the Alpha variant lineage sequence over time, including numerous mutations  in the Spike RBD and NTD. CONCLUSIONS: We discuss the possibility that a chronic SARS-CoV-2 infection accumulated adaptive mutations that promoted long-term infection. This study reveals that small wastewater treatment plants can enhance resolution of rare events and facilitate reconstruction of viral genomes due to the relative lack of contaminating sequences.

Noradrenergic regulation of cue-guided decision making and impulsivity is doubly dissociable across frontal brain regions.

RATIONALE: Win-paired stimuli can promote risk taking in experimental gambling paradigms in both rats and humans. We previously demonstrated that atomoxetine, a noradrenaline reuptake inhibitor, and guanfacine, a selective α2A adrenergic receptor agonist, reduced risk taking on the cued rat gambling task (crGT), a rodent assay of risky choice in which wins are accompanied by salient cues. Both compounds also decreased impulsive premature responding. OBJECTIVE: The key neural loci mediating these effects were unknown. The lateral orbitofrontal cortex (lOFC) and the medial prefrontal cortex (mPFC), which are highly implicated in risk assessment, action selection, and impulse control, receive dense noradrenergic innervation. We therefore infused atomoxetine and guanfacine directly into either the lOFC or prelimbic (PrL) mPFC prior to task performance. RESULTS: When infused into the lOFC, atomoxetine improved decision making score and adaptive lose-shift behaviour in males, but not in females, without altering motor impulsivity. Conversely, intra-PrL atomoxetine improved impulse control in risk preferring animals of both sexes, but did not alter decision making. Guanfacine administered into the PrL, but not lOFC, also altered motor impulsivity in all subjects, though in the opposite direction to atomoxetine. CONCLUSIONS: These data highlight a double dissociation between the behavioural effects of noradrenergic signaling across frontal regions with respect to risky choice and impulsive action. Given that the influence of noradrenergic manipulations on motor impulsivity could depend on baseline risk preference, these data also suggest that the noradrenaline system may function differently in subjects that are susceptible to the risk-promoting lure of win-associated cues.

Predicting Long-Term Stability of an Oral Delivered Antibody Drug Product with Accelerated Stability Assessment Program Modeling.

The oral delivery of protein therapeutics offers numerous advantages for patients but also presents significant challenges in terms of development. Currently, there is limited knowledge available regarding the stability and shelf life of orally delivered protein therapeutics. In this study, a comprehensive assessment of the stability of an orally delivered solid dosage variable domain of heavy-chain antibody (VHH antibody) drug product was conducted. Four stability related quality attributes that undergo change as a result of thermal and humidity stress were identified. Subsequently, these attributes were modeled using an accelerated stability approach facilitated by ASAPprime software. To the best of our knowledge, this is the first time that this approach has been reported for an antibody drug product. We observed overall good model quality and accurate predictions regarding the protein stability during storage. Notably, we discovered that protein aggregation, formed through a degradation pathway, requires additional adjustments to the modeling method. In summary, the ASAP approach demonstrated promising results in predicting the stability of this complex solid-state protein formulation. This study sheds light on the stability and shelf life of orally delivered protein therapeutics, addressing an important knowledge gap in the field.

Monitoring environmental microbiomes: Alignment of microbiology and computational biology competencies within a culturally integrated curriculum and research framework.

We have developed a flexible undergraduate curriculum that leverages the place-based research of environmental microbiomes to increase the number of Indigenous researchers in microbiology, data science and scientific computing. Monitoring Environmental Microbiomes (MEM) provides a curriculum and research framework designed to integrate an Indigenous approach when conducting authentic scientific research and to build interest and confidence at the undergraduate level. MEM has been successfully implemented as a short summer workshop to introduce computing practices in microbiome analysis. Based on self-assessed student knowledge of topics and skills, increased scientific confidence and interest in genomics careers were observed. We propose MEM be incorporated in a scalable course-based research experience for undergraduate institutions, including tribal colleges and universities, community colleges and other minority serving institutions. This coupled curricular and research framework explicitly considers cultural perspectives, access and equity to train a diverse future workforce that is more informed to engage in microbiome research and to translate microbiome science to benefit community and environmental health.

Label-Free Composition Analysis of Supramolecular Polymer - Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties following applied stresses, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel compositions is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, Bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e. proteins), and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with high resolution mass spectrometry and a Fourier-transform based deconvolution algorithm. To our knowledge, this is the first RPLC-CAD method for characterizing the critical quality attributes of supramolecular hydrogels. We envision this analytical strategy could be generalized to characterize other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

A Novel Technique to Assess Drug Substance Particle Size in a Complex Inhaled Formulation.

Dry powder inhalers, comprising an active pharmaceutical ingredient (API) and carrier excipients, are often used in the delivery of pulmonary drugs. The stability of the API particle size within a formulation blend is a critical attribute for aerodynamic performance but can be challenging to measure. The presence of excipients, typically at concentrations much higher than API, makes measurement by laser diffraction very difficult. This work introduces a novel laser diffraction approach that takes advantage of solubility differences between the API and excipients. The method allows insight into the understanding of drug loading effects on API particle stability of the drug product. Lower drug load formulations show better particle size stability compared with high drug load formulations, likely due to reduced cohesive interactions.

SARS-CoV-2 wastewater monitoring in rural and small metropolitan communities in Central Michigan.

Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program throughout the 2021-2022 academic year. Wastewater samples were collected weekly from ten on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR. Case data reported by Central Michigan District Health Department and CMU were collected and compared with wastewater data. During the delta wave, wastewater detection and on-campus case reports increased rapidly with the start of the academic semester and peaked quickly, compared with a more gradual and prolonged increase in detection and case reports off-campus. During the omicron wave, transmission dynamics were similar on-campus and off-campus. Normalization of on-campus and off-campus wastewater data with pepper mild mottle virus gene expression suggested lower SARS-CoV-2 shedding per person in on-campus compared to off-campus samples during the delta wave, but no difference in virus shedding during the omicron wave. We discuss the possibility that a higher on-campus vaccination rate may have reduced virus shedding per person during the delta wave, but that this effect was lost with the omicron variant. This study suggests that wastewater monitoring is effective in rural and small metropolitan communities when used in conjunction with case reports to understand regional transmission dynamics and the impact of public health policies at a public university on virus shedding in the community.

Biosensors Based on Graphene Oxide Functionalized with Benzothiadiazole-Derived Ligands for the Detection of Cholesterol.

In this work, imidazole- or imidazolium-based benzothiadiazole ligands functionalized on graphene oxide combined with cholesterol oxidase constitute efficient, robust, and easy-to-handle materials with high biosensing activity for the detection of cholesterol by colorimetric methods. The presence of lanthanum(III) supported on graphene oxide as a possible coordinating site for the benzothiadiazole ligands was also evaluated, and its bioactivity was compared to that of the analogous material without the rare-earth metal. Our results demonstrated that graphene oxide functionalized with 4,7-bis-(imidazol-1-yl)-2,1,3-benzothiadiazole exhibited the best performance for the quantification of total cholesterol with a sensitivity of 0.0649 (with lanthanum) and 0.0618 au dL mg-1 (without lanthanum). In addition, these materials presented a better percentage of immobilization (>90%), recovered activity, resistance to storage, and detection range than materials containing 4,7-[1-carboxymethyl-(imidazol-3-ium)]-2,1,3-benzothiadiazole chloride. Therefore, the combination of GO-BTD (Im/Ac)/ChOx (with or without lanthanum) affords efficient biosensors for the colorimetric detection of cholesterol.

Engineered anti-prostate cancer CAR-neutrophils from human pluripotent stem cells.

Immunotherapy is a powerful technique where immune cells are modified to improve cytotoxicity against cancerous cells to treat cancers that do not respond to surgery, chemotherapy, or radiotherapy. Expressing chimeric antigen receptor (CAR) in immune cells, typically T lymphocytes, is a practical modification that drives an immune response against cancerous tissue. CAR-T efficacy is suboptimal in solid tumors due to the tumor microenvironment (TME) that limits T lymphocyte cytotoxicity. In this study, we demonstrate that neutrophils differentiated from human pluripotent stem cells modified with AAVS1-inserted CAR constructs showed a robust cytotoxic effect against prostate-specific membrane antigen (PSMA) expressing LNCaP cells as a model for prostate cancer in vitro. Our results suggest that engineered CAR can significantly enhance the neutrophil anti-tumor effect, providing a new avenue in treating prostate cancers.

Temporal Dynamics Underlying Prelimbic Prefrontal Cortical Regulation of Action Selection and Outcome Evaluation during Risk/Reward Decision-Making.

Risk/reward decision-making is a dynamic process that includes periods of deliberation before action selection and evaluation of the action outcomes that bias subsequent choices. Inactivation of the prelimbic (PL) cortex has revealed its integral role in updating decision biases in the face of changes in probabilistic reward contingencies, yet how phasic PL signals during different phases of the decision process influence choice remains unclear. We used temporally specific optogenetic inhibition to selectively disrupt PL activity coinciding with action selection and outcome phases to examine how these signals influence choice. Male rats expressing the inhibitory opsin eArchT within PL excitatory neurons were well trained on a probabilistic discounting task, entailing choice between small/certain versus large/risky rewards, the probability of which varied over a session (50-12.5%). During testing, brief light pulses suppressed PL activity before choice or after different outcomes. Prechoice suppression reduced bias toward more preferred/higher utility options and disrupted how recent outcomes influenced subsequent choice. Inhibition during risky losses induced a similar profile, but here, the impact of reward omissions were either amplified or diminished, relative to the context of the estimated profitability of the risky option. Inhibition during large or small reward receipt reduced risky choice when this option was more profitable, suggesting these signals can both reinforce rewarded risky choices and also act as a relative value comparator signal that augments incentive for larger rewards. These findings reveal multifaceted contributions by the PL in implementing decisions and integrating action-outcome feedback to assign context to the decision space.SIGNIFICANCE STATEMENT The PL prefrontal cortex plays an integral role in guiding risk/reward decisions, but how activity in this region during different phases of the decision process influences choice is unclear. By using temporally specific optogenetic manipulations of this activity, the present study unveiled previously uncharacterized and differential contributions by PL in implementing decision policies and how evaluation of decision outcomes shape subsequent choice. These findings provide novel insight into the dynamic processes engaged by the PL that underlie action selection in situations involving reward uncertainty that may aid in understanding the mechanism underlying normal and aberrant decision-making processes.

Impact of interprofessional geriatric teamwork on students' perceptions of older persons and collaborative practice.

Health professions programs lack sufficient exposure to geriatric education in curricula. The Seniors Assisting in Geriatric Education (SAGE) Program exposes interprofessional (IP) teams of health professions students to older adults. To determine the impact of an interprofessional geriatric educational experience on student perceptions of team collaboration and older adults. IP teams of three or four students (n = 662) representing eight disciplines from two institutions were paired with an older adult to promote person-centered care over three semesters. Students completed two online questionnaires (pre- and post-SAGE Program, ~10 min). 136 students completed both questionnaires. Three IP collaborative practice sub-competencies under the Roles & Responsibilities and Interprofessional Communication Core Competencies increased significantly from pre- to post-SAGE Program (p ≤ 0.002). Comparison of the means for attitudes toward geriatric patients revealed statistically significant improvement in one item, Compassion (p < .002). The SAGE Program had a positive impact on IP collaborative practice and attitudes toward older people in some, but not all, areas.

3D biocomposite culture enhances differentiation of dopamine-like neurons from SH-SY5Y cells: A model for studying Parkinson's disease phenotypes.

Studies of underlying neurodegenerative processes in Parkinson's Disease (PD) have traditionally utilized cell cultures grown on two-dimensional (2D) surfaces. Biomimetic three-dimensional (3D) cell culture platforms have been developed to better emulate features of the brain's natural microenvironment. We here use our bioengineered brain-like tissue model, composed of a silk-hydrogel composite, to study the 3D microenvironment's contributions on the development and performance of dopaminergic-like neurons (DLNs). Compared with 2D culture, SH-SY5Y cells differentiated in 3D microenvironments were enriched for DLNs concomitant with a reduction in proliferative capacity during the neurodevelopmental process. Additionally, the 3D DLN cultures were more sensitive to oxidative stresses elicited by the PD-related neurotoxin 1-methyl-4-phenylpyridinium (MPP). MPP induced transcriptomic profile changes specific to 3D-differentiated DLN cultures, replicating the dysfunction of neuronal signaling pathways and mitochondrial dynamics implicated in PD. Overall, this physiologically-relevant 3D platform resembles a useful tool for studying dopamine neuron biology and interrogating molecular mechanisms underlying neurodegeneration in PD.

Gadolinium Deposition Disease: A Case Report and the Prevalence of Enhanced MRI Procedures Within the Veterans Health Administration.

Background: Gadolinium (Gd) usage in the Veterans Health Administration is increasing and patients with renal disease are frequently exposed. Gd is not entirely eliminated within 24 hours after administration, which may pose long-term adverse effects. Case Presentation: A Vietnam-era veteran aged > 70 years presented for evaluation of Gd-based contrast agent-induced chronic multisymptom illness. In the course of his routine clinical care, he was exposed to repeated Gd-enhanced magnetic resonance imaging studies. After his second Gd-based contrast agent exposure, he noted rash, pain, headaches, and hoarseness. Years after the exposure to the contrast agents, he continued to have detectable Gd in urine and serum. Conclusions: Practitioners should be aware of long-term intracellular Gd retention (including the brain) as patients increasingly turn to consultants with concerns about Gd deposition disease. Data from patient advocates demonstrate that Gd is eliminated in intermediate and long phases, which may represent a multicompartment model. The commercialization of Gd use in imaging studies is outpacing the science addressing the long-term consequences of harboring this alien, toxic, nonphysiologic rare earth metal.

A synthetic tactic to substitute axial ligands in sterically demanding Ru(II)porphyrinates.

We report a synthetic strategy that allows for the preparation of sterically encumbered heteroleptic Ru(II)porphyrinates with the desired configuration of stable/inert and weak/labile axial ligands to direct reactions between substrates to exclusively occur at the sterically encumbered face. To demonstrate the method, we describe the synthesis of a strapped-Ru(II)porphyrinate bearing a stable/inert triphenylphosphine (PPh3) bulky axial ligand coordinated exo to the central cavity and a weak/labile methanol molecule coordinated at the internal axial position. With this axial ligand configuration, the reported Ru(II)porphyrinate exclusively promotes carbene transfer reactions to olefins through the central cavity, which has been verified by the selective formation of cycloprane-linked [2]rotaxanes.

Emergency Medical Services Demand: An Analysis of County-Level Social Determinants.

OBJECTIVES: Variations in the demand for Emergency Medical Services (EMS) exist when observed at a local level. This unspecified heterogeneity leads to an investigation of social factors contributing to EMS demand. METHODS: Data for this study were collected from publicly available EMS reports from Florida and Oklahoma for 2009 - 2015. Health and social data were gathered from County health rankings and roadmap reports. Data were combined into a single dataset, and pooled ordinary-least-squares models with time-fixed effects were utilized for tests of inference. EMS call volume was log-transformed to derive a semi-elasticity function. RESULTS: A total of 874 county-year observations were analyzed. Increases in poor/fair health (95% CI: 0.6% - 3.9%), binge drinking (95% CI: 1.6% - 3.5%), teen birth rate (95% CI: 1.1% - 5.2%), unemployment rate (95% CI: 0.5% - 3.9%), and violent crime rate (95% CI: 1.0% - 3.0%) were associated with an increase in the EMS demand rate. CONCLUSION: The data supports the notion that some community measures have an effect on EMS demand as counties with higher levels of poor health, binge drinking, teen births, unemployment, and violent crime saw higher EMS demand. These factors may have been treated as spurious, or overlooked by policy makers and EMS leadership.