Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and iNOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors including head and neck cancers. We show for the first time that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti-PD1, and anti-CTLA4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid-T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid-T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.

Outpatient Patient Controlled Analgesia (PCA) for the Palliative Care Patient.

This study explores the under-researched domain of patient-controlled analgesia (PCA) for cancer pain management in adult outpatients, focusing on the transition from patient-controlled analgesia pumps (PCA pump) to oral medications. While existing literature primarily addresses the use of PCA in inpatient settings, this descriptive study investigates the initiation of outpatient PCA in palliative care patients. The retrospective chart review includes data from all admissions between July 1, 2014, and December 31, 2020. Among the 49 identified patients, 41 were admitted for cancer-related pain, with an indication for PCA such as insufficient pain relief, highly fluctuating pain, or inadequate response to other routes. Of these patients, 13 were successfully transitioned from outpatient PCA to oral opioids. The study underscores the effective use of PCA as a transitional tool following a pain crisis that necessitates inpatient admission. Future research avenues could explore healthcare utilization, length of stay, and required outpatient resources, such as home visits or telehealth, for optimal PCA use in outpatient settings.

Immunologic Predictors of Vaccine Responsiveness in Patients With Lymphoma and Chronic Lymphocytic Leukemia.

Patients with B-cell lymphomas have altered cellular components of vaccine responses due to malignancy and therapy, and the optimal timing of vaccination relative to therapy remains unknown. Severe acute respiratory syndrome coronavirus 2 vaccines created an opportunity for new insights in vaccine timing because patients were challenged with a novel antigen across multiple phases of treatment. We studied serologic messenger RNA vaccine response in retrospective and prospective cohorts with lymphoma and chronic lymphocytic leukemia, paired with clinical and research immune parameters. Reduced serologic response was observed more frequently during active treatment, but nonresponse was also common within observation and posttreatment groups. Total immunoglobulin A and immunoglobulin M correlated with successful vaccine response. In individuals treated with anti-CD19-directed chimeric antigen receptor-modified T cells, nonresponse was associated with reduced B and T follicular helper cells. Predictors of vaccine response varied by disease and therapeutic group, and therefore further studies of immune health during and after cancer therapies are needed to individualize vaccine timing.

Customized passive-dynamic ankle-foot orthoses can improve walking economy and speed for many individuals post-stroke.

BACKGROUND: Passive-dynamic ankle-foot orthoses (PD-AFOs) are often prescribed to address plantar flexor weakness during gait, which is commonly observed after stroke. However, limited evidence is available to inform the prescription guidelines of PD-AFO bending stiffness. This study assessed the extent to which PD-AFOs customized to match an individual's level of plantar flexor weakness influence walking function, as compared to No AFO and their standard of care (SOC) AFO. METHODS: Mechanical cost-of-transport, self-selected walking speed, and key biomechanical variables were measured while individuals greater than six months post-stroke walked with No AFO, with their SOC AFO, and with a stiffness-customized PD-AFO. Outcomes were compared across these conditions using a repeated measures ANOVA or Friedman test (depending on normality) for group-level analysis and simulation modeling analysis for individual-level analysis. RESULTS: Twenty participants completed study activities. Mechanical cost-of-transport and self-selected walking speed improved with the stiffness-customized PD-AFOs compared to No AFO and SOC AFO. However, this did not result in a consistent improvement in other biomechanical variables toward typical values. In line with the heterogeneous nature of the post-stroke population, the response to the PD-AFO was highly variable. CONCLUSIONS: Stiffness-customized PD-AFOs can improve the mechanical cost-of-transport and self-selected walking speed in many individuals post-stroke, as compared to No AFO and participants' standard of care AFO. This work provides initial efficacy data for stiffness-customized PD-AFOs in individuals post-stroke and lays the foundation for future studies to enable consistently effective prescription of PD-AFOs for patients post-stroke in clinical practice. TRIAL REGISTRATION: NCT04619043.

Well-Being and Leadership Within the Emergency Department.

Description This article looks at well-being and the role of leadership from the perspective of emergency medicine. The importance of leadership within the emergency department (ED), emergency medicine writing at large, and the prevention of burnout and compassion fatigue cannot be overstated. This article looks at the need for more research and measured interventions within the ED. It also highlights some measures that could be taken to help improve well-being from a leadership perspective to improve patient safety and outcomes within the ED.

Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway.

During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues. We identified the putative Rim pathway proteins Rim101 and Rra1 in the human skin colonizing fungus Malassezia sympodialis. Gene deletion by transconjugation and homologous recombination revealed that Rim101 and Rra1 are required for M. sympodialis growth at higher pH. Additionally, comparative transcriptional analysis of the mutant strains compared to wild-type suggested mechanisms for fungal adaptation to alkaline conditions. These pH-sensing signaling proteins are required for optimal growth in a murine model of atopic dermatitis, a pathological condition associated with increased skin pH. Together these data elucidate both conserved and phylum-specific features of microbial adaptation to extracellular stresses.

Student experiences with a molecular biotechnology course containing an interactive 3D immersive simulation and its impact on motivational beliefs.

The development and use of virtual laboratories to augment traditional in-person skills training continues to grow. Virtual labs have been implemented in a number of diverse educational settings, which have many purported benefits including their adaptability, accessibility, and repeatability. However, few studies have evaluated the impact of virtual laboratories outside of academic achievement and skills competencies, especially in biotechnology. In this study, an interdisciplinary team of content experts, video game researchers, instructional designers, and assessment experts developed a 3D immersive simulation designed to teach novice scientists the technical skills necessary to perform sterile mammalian cell culture technique. Unique to the simulation development process is the recreation of an immersive experience through the capture of details in the real-world lab where participants have the freedom of choice in their actions, while receiving immediate feedback on their technical skills as well as procedural execution. However, unlike an in-person laboratory course, students are able to iterate and practice their skills outside of class time and learn from their mistakes. Over the course of two semesters, we used a mixed-methods study design to evaluate student attitudes towards the simulation and their science motivational beliefs. Students' self-efficacy and science identity were assessed after engaging with the simulation prior to the physical laboratory. Our results show that students' science identity remained unchanged while their science self-efficacy increased. Furthermore, students had positive perceptions of the benefits of the virtual simulation. These data suggest that the virtual cell culture simulation can be a useful pedagogical training tool to support students' motivational beliefs that is both accessible and easy to implement.

Mapping protein binding sites by photoreactive fragment pharmacophores.

Fragment screening is a popular strategy of generating viable chemical starting points especially for challenging targets. Although fragments provide a better coverage of chemical space and they have typically higher chance of binding, their weak affinity necessitates highly sensitive biophysical assays. Here, we introduce a screening concept that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity handle that enables high hit rates by LC-MS-based detection. The sensitivity of our screening protocol was further improved by a target-conjugated photocatalyst. We have designed, synthesized, and screened 100 diazirine-tagged fragments against three benchmark and three therapeutically relevant protein targets of different tractability. Our therapeutic targets included a conventional enzyme, the first bromodomain of BRD4, a protein-protein interaction represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several fragment hits against all three targets and identified their binding sites via enzymatic digestion, structural studies and modeling. Our results revealed that this protocol outperforms screening traditional fully functionalized and photoaffinity fragments in better exploration of the available binding sites and higher hit rates observed for even difficult targets.

Racial and economic segregation and diabetes mortality in the USA, 2016-2020.

BACKGROUND: The purpose of this study was to examine the association between racial and economic segregation and diabetes mortality among US counties from 2016 to 2020. METHODS: We conducted a cross-sectional ecological study that combined county-level diabetes mortality data from the National Vital Statistics System and sociodemographic information drawn from the 2016-2020 American Community Survey (n=2380 counties in the USA). Racialized economic segregation was measured using the Index Concentration at the Extremes (ICE) for income (ICEincome), race (ICErace) and combined income and race (ICEcombined). ICE measures were categorised into quintiles, Q1 representing the highest concentration and Q5 the lowest concentration of low-income, non-Hispanic (NH) black and low-income NH black households, respectively. Diabetes was ascertained as the underlying cause of death. County-level covariates included the percentage of people aged ≥65 years, metropolitan designation and population size. Multilevel Poisson regression was used to estimate the adjusted mean mortality rate and adjusted risk ratios (aRR) comparing Q1 and Q5. RESULTS: Adjusted mean diabetes mortality rate was consistently greater in counties with higher concentrations of low-income (ICEincome) and low-income NH black households (ICEcombined). Compared with counties with the lowest concentration (Q1), counties with the highest concentration (Q5) of low-income (aRR 1.96; 95% CI 1.81 to 2.11 for ICEincome), NH black (aRR 1.32; 95% CI 1.18 to 1.47 for ICErace) and low-income NH black households (aRR 1.70; 95% CI 1.56 to 1.84 for ICEcombined) had greater diabetes mortality. CONCLUSION: Racial and economic segregation is associated with diabetes mortality across US counties.

Intrinsic Gata4 expression sensitizes the aortic root to dilation in a Loeys-Dietz syndrome mouse model.

Loeys-Dietz syndrome (LDS) is an aneurysm disorder caused by mutations that decrease transforming growth factor-ß (TGF-ß) signaling. Although aneurysms develop throughout the arterial tree, the aortic root is a site of heightened risk. To identify molecular determinants of this vulnerability, we investigated the heterogeneity of vascular smooth muscle cells (VSMCs) in the aorta of Tgfbr1 M318R/+ LDS mice by single cell and spatial transcriptomics. Reduced expression of components of the extracellular matrix-receptor apparatus and upregulation of stress and inflammatory pathways were observed in all LDS VSMCs. However, regardless of genotype, a subset of Gata4-expressing VSMCs predominantly located in the aortic root intrinsically displayed a less differentiated, proinflammatory profile. A similar population was also identified among aortic VSMCs in a human scRNAseq dataset. Postnatal VSMC-specific Gata4 deletion reduced aortic root dilation in LDS mice, suggesting that this factor sensitizes the aortic root to the effects of impaired TGF-ß signaling.

Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

In Vitro Cell Surface Marker Expression on Mesenchymal Stem Cell Cultures does not Reflect Their Ex Vivo Phenotype.

Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most 'mesenchymal' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture.

Comparative Pathogenesis of Two Lineages of Powassan Virus Reveals Distinct Clinical Outcome, Neuropathology, and Inflammation.

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus.

Early intestinal ultrasound in severe ulcerative colitis identifies patients at increased risk of 1-year treatment failure and colectomy.

BACKGROUND AND AIMS: Reliable and easily accessible objective markers of disease activity to predict long-term treatment outcomes in severe ulcerative colitis (UC) are missing. We aimed to investigate if intestinal ultrasound (IUS) might predict long-term outcomes in hospitalized patients with severe UC treated with intravenous corticosteroids. METHODS: Hospitalized patients with severe UC and IUS inflammation (bowel wall thickness (BWT)>3.0mm) starting IV corticosteroids were recruited at three university hospitals in Denmark. IUS was performed before treatment, 48±24 hours (h), 6±1 days, and 3 months after treatment initiation. Time until colectomy or need for new interventions was registered together with Mayo score at 3 months and partial Mayo score (pMayo) at 12-months. Follow-up time was 12 months. RESULTS: Fifty-six patients were included in the final analysis. Forty-five (80%) patients needed intervention, including 9 colectomies, during the 12-month follow-up. After 48±24h: No patient with a BWT<3mm needed a colectomy, p=0.04. BWT≥4mm showed an increased risk of colectomy (odds ratio 9.5 (95%CI 1.5-186), p=0.03), while a BWT≥3mm showed an increased risk of intervention (3.6 (1.1-12.5), p=0.03). A BWT≥4mm resulted in a significantly shorter time until both colectomy, p=0.03, and treatment intensification (mean days 75 (95%CI24-127) vs. 176 (119-233), p=0.005. However, neither IUS parameters nor pMayo score, CRP, hemoglobin, or p-albumin could predict remission at 3- and 12-months. CONCLUSION: BWT assessed at 48h post intravenous corticosteroid initiation in patients hospitalized with severe UC may identify patients with an increased risk of short- and long-term colectomy and predict a more aggressive short-term disease course.

Discovery of an Azabicyclo[2.1.1]hexane Piperazinium Salt and Its Application in Medicinal Chemistry via a Rearrangement.

Herein we report the discovery of an azabicyclo[2.1.1]hexane piperazinium methanesulfonate salt from an unexpected rearrangement reaction in the preparation of ligand-directed degraders (LDDs). This bench-stable compound was found to be a versatile electrophile in a ring-opening reaction with various types of nucleophiles. Its utility as a versatile medicinal chemistry building block is further demonstrated in the synthesis of an LDD compound targeting degradation of the androgen receptor.

Sequenced treatment effectiveness for posttraumatic stress (STEPS) trial: A protocol for a pragmatic comparative effectiveness trial with baseline results.

BACKGROUND: There have only been two efficacy trials reporting a head-to-head comparison of medications and psychotherapy for PTSD, and neither was conducted in primary care. Therefore, this protocol paper describes a pragmatic trial that compares outcomes of primary care patients randomized to initially receive a brief trauma-focused psychotherapy or a choice of three antidepressants. In addition, because there are few trials examining the effectiveness of subsequent treatments for patients not responding to the initial treatment, this pragmatic trial also compares the outcomes of those switching or augmenting treatments. METHOD: Patients screening positive for PTSD (n = 700) were recruited from the primary care clinics of 7 Federally Qualified Health Centers (FQHC) and 8 Department of Veterans Affairs (VA) Medical Centers and randomized in the ratio 1:1:2 to one of three treatment sequences: 1) selective serotonin reuptake inhibitor (SSRI) followed by augmentation with Written Exposure Therapy (WET), 2) SSRI followed by a switch to serotonin-norepinephrine reuptake inhibitor (SNRI), or 3) WET followed by a switch to SSRI. Participants complete surveys at baseline, 4 months, and 8 months. The primary outcome is PTSD symptom severity as measured by the PTSD Checklist (PCL-5). RESULTS: Average PCL-5 scores (M = 52.8, SD = 11.1) indicated considerable severity. The most common bothersome traumatic event for VA enrollees was combat (47.8%), and for FQHC enrollees was other (28.2%), followed by sexual assault (23.4%), and child abuse (19.8%). Only 22.4% were taking an antidepressant at baseline. CONCLUSION: Results will help healthcare systems and clinicians make decisions about which treatments to offer to patients.

mRNA Display in Cell Lysates Enables Identification of Cyclic Peptides Targeting the BRD3 Extraterminal Domain.

mRNA display is a powerful technology to screen libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands. These cyclic peptides are particularly well suited to challenging protein targets that have been difficult to drug with small molecules. However, target choice can still be limited as screens are typically performed against purified proteins which often demands the use of isolated domains and precludes the use of aggregation-prone targets. Here, we report a method to perform mRNA display selections in mammalian cell lysates without the need for prior target purification, vastly expanding the potential target scope of mRNA display. We have applied the methodology to identify low to sub-nanomolar peptide binders for two targets: a NanoLuc subunit (LgBiT) and full-length bromodomain-containing protein 3 (BRD3). Our cyclic peptides for BRD3 were found to bind to the extraterminal (ET) domain of BRD3 and the closely related BRD proteins, BRD2 and BRD4. While many chemical probes exist for the bromodomains of BRD proteins, the ET domain is relatively underexplored, making these peptides valuable additions to the BRD toolbox.

All-Soft-Tissue Meniscus Allograft Transplantation With Circumferential Suture Tape Augmentation to Mitigate Hoop Stress and Promote Centralization.

Meniscus allograft transplantation (MAT) is a technically challenging procedure. Bone plugs, slot techniques, and all-soft-tissue fixation techniques have been described in the past. Each technique comes with advantages and disadvantages. Native menisci have circumferential collagen fibers to help resist hoop stress during loading cycles. Although hoop stress resistance is a known function of the menisci, its recreation in MAT has only been targeted indirectly through anatomic root placement. The authors describe the use of a high-tensile suture tape (i.e. InternalBrace) to promote centralization by directly mitigating hoop stresses through recreation of peripheral meniscus tensioning in MAT.

The Effect of Concurrent Use of Opioids and Gabapentin on Fall Risk in Older Adults.

Falls pose a significant threat to older adults, resulting in injuries and mortality. Concurrently prescribed opioids and gabapentin for pain management may increase fall risks in older patients. This study aimed to estimate fall risks associated with the concurrent use of gabapentin and opioids, comparing them to opioid monotherapy in older adults. A retrospective case-control study of 1,813 patients aged 65-89 on chronic opioid therapy (2017-2020), excluding those with a fall history, analysis focused on the first fall occurrence. Logistic regression assessed the association between concurrent gabapentin and opioid use and fall events. Out of eligible patients, 122 (6.73%) experienced falls during opioid therapy, with 232 (12.80%) having concurrent gabapentin use. Concurrent use significantly increased fall risk (AOR = 1.73; 95% CI: 1.08-2.78). Being female, aged ≥81, and having more chronic conditions also increased risk. Mitigating fall risk in older adults requires education on prevention, exploring alternative pain management, and careful consideration of prescribing. Further research is crucial to understand adverse events linked to combined opioid and gabapentin use in the geriatric population.