Modulation of diabetic wound healing using carbon monoxide gas-entrapping materials.

Diabetic wound healing is uniquely challenging to manage due to chronic inflammation and heightened microbial growth from elevated interstitial glucose. Carbon monoxide (CO), widely acknowledged as a toxic gas, is also known to provide unique therapeutic immune modulating effects. To facilitate delivery of CO, we have designed hyaluronic acid-based CO-gas-entrapping materials (CO-GEMs) for topical and prolonged gas delivery to the wound bed. We demonstrate that CO-GEMs promote the healing response in murine diabetic wound models (full-thickness wounds and pressure ulcers) compared to N2-GEMs and untreated controls.

Cerebellar granule cell migration and folia development require Mllt11/Af1q/Tcf7c.

The organization of neurons into distinct layers, known as lamination, is a common feature of the nervous system. This process, which arises from the direct coupling of neurogenesis and neuronal migration, plays a crucial role in the development of the cerebellum, a structure exhibiting a distinct folding cytoarchitecture with cells arranged in discrete layers. Disruptions to neuronal migration can lead to various neurodevelopmental disorders, highlighting the significance of understanding the molecular regulation of lamination. We report a role Mllt11/Af1q/Tcf7c (myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q, also known as Mllt11 transcriptional cofactor 7; henceforth referred to Mllt11) in the migration of cerebellar granule cells (GCs). We now show that Mllt11 plays a role in both the tangential and radial migration of GCs. Loss of Mllt11 led to an accumulation of GC precursors in the rhombic lip region and a reduction in the number of GCs successfully populating developing folia. Consequently, this results in smaller folia and an overall reduction in cerebellar size. Furthermore, analysis of the anchoring centers reveals disruptions in the perinatal folia cytoarchitecture, including alterations in the Bergmann glia fiber orientation and reduced infolding of the Purkinje cell plate. Lastly, we demonstrate that Mllt11 interacts with non-muscle myosin IIB (NMIIB) and Mllt11 loss-reduced NMIIB expression. We propose that the dysregulation of NMIIB underlies altered GC migratory behavior. Taken together, the findings reported herein demonstrate a role for Mllt11 in regulating neuronal migration within the developing cerebellum, which is necessary for its proper neuroanatomical organization.

Early resource scarcity causes cortical astrocyte enlargement and sex-specific changes in the orbitofrontal cortex transcriptome in adult rats.

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN augments maternal behaviors and promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.

Impact of formulation on solid oxygen-entrapping materials to overcome tumor hypoxia.

Tumor hypoxia, resulting from rapid tumor growth and aberrant vascular proliferation, exacerbates tumor aggressiveness and resistance to treatments like radiation and chemotherapy. To increase tumor oxygenation, we developed solid oxygen gas-entrapping materials (O2-GeMs), which were modeled after clinical brachytherapy implants, for direct tumor implantation. The objective of this study was to investigate the impact different formulations of solid O2-GeMs have on the entrapment and delivery of oxygen. Using a Parr reactor, we fabricated solid O2-GeMs using carbohydrate-based formulations used in the confectionary industry. In evaluating solid O2-GeMs manufactured from different sugars, the sucrose-containing formulation exhibited the highest oxygen concentration at 1 mg/g, as well as the fastest dissolution rate. The addition of a surface coating to the solid O2-GeMs, especially polycaprolactone, effectively prolonged the dissolution of the solid O2-GeMs. In vivo evaluation confirmed robust insertion and positioning of O2-GeMs in a malignant peripheral nerve sheath tumor, highlighting potential clinical applications.

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Oral Carbon Monoxide Enhances Autophagy Modulation in Prostate, Pancreatic, and Lung Cancers.

Modulation of autophagy, specifically its inhibition, stands to transform the capacity to effectively treat a broad range of cancers. However, the clinical efficacy of autophagy inhibitors has been inconsistent. To delineate clinical and epidemiological features associated with autophagy inhibition and a positive oncological clinical response, a retrospective analysis of patients is conducted treated with hydroxychloroquine, a known autophagy inhibitor. A direct correlation between smoking status and inhibition of autophagy with hydroxychloroquine is identified. Recognizing that smoking is associated with elevated circulating levels of carbon monoxide (CO), it is hypothesized that supplemental CO can amplify autophagy inhibition. A novel, gas-entrapping material containing CO in a pre-clinical model is applied and demonstrated that CO can dramatically increase the cytotoxicity of autophagy inhibitors and significantly inhibit the growth of tumors when used in combination. These data support the notion that safe, therapeutic levels of CO can markedly enhance the efficacy of autophagy inhibitors, opening a promising new frontier in the quest to improve cancer therapies.

Multiplexed live-cell imaging for drug responses in patient-derived organoid models of cancer.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to simultaneously quantitate cell health and apoptosis. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Early resource scarcity causes cortical astrocyte enlargement and sex-specific changes in the orbitofrontal cortex transcriptome in adult rats.

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. However, Park7, which encodes for the protein DJ-1 that alters astrocyte morphology, was increased by LBN across sex. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.

Health care...associated infections studies project: An American Journal of Infection Control and National Health Care Safety Network data quality collaboration case study..÷Laboratory-identified event reporting validation.

This case study is part of a series centered on the Centers for Disease Control and Prevention/National Healthcare Safety Network (NHSN) health care...associated infection surveillance definitions. This specific case study focuses on the application of the common surveillance concepts included in Laboratory-Identified Event Reporting (Chapter 12 of the NHSN Patient Safety Manual..÷Multidrug-Resistant Organism & Clostridioides difficile Infection Module) used with validation efforts. The intent of the case study series is to foster standardized application of the NHSN surveillance definitions and encourage accurate event determination among infection preventionists.

Low-Cost, High-Pressure-Synthesized Oxygen-Entrapping Materials to Improve Treatment of Solid Tumors.

Tumor hypoxia drives resistance to many cancer therapies, including radiotherapy and chemotherapy. Methods that increase tumor oxygen pressures, such as hyperbaric oxygen therapy and microbubble infusion, are utilized to improve the responses to current standard-of-care therapies. However, key obstacles remain, in particular delivery of oxygen at the appropriate dose and with optimal pharmacokinetics. Toward overcoming these hurdles, gas-entrapping materials (GeMs) that are capable of tunable oxygen release are formulated. It is shown that injection or implantation of these materials into tumors can mitigate tumor hypoxia by delivering oxygen locally and that these GeMs enhance responsiveness to radiation and chemotherapy in multiple tumor types. This paper also demonstrates, by comparing an oxygen (O2 )-GeM to a sham GeM, that the former generates an antitumorigenic and immunogenic tumor microenvironment in malignant peripheral nerve sheath tumors. Collectively the results indicate that the use of O2 -GeMs is promising as an adjunctive strategy for the treatment of solid tumors.

Inpatient Satisfaction With Surgical Resident Care After Elective General and Oncologic Surgery.

OBJECTIVE: To investigate inpatient satisfaction with surgical resident care. BACKGROUND: Surgical trainees are often the primary providers of care to surgical inpatients, yet patient satisfaction with surgical resident care is not well characterized or routinely assessed. METHODS: English-speaking, general surgery inpatients recovering from elective gastrointestinal and oncologic surgery were invited to complete a survey addressing their satisfaction with surgical resident care. Patients positively identified photos of surgical senior residents and interns before completing a modified version of the Consumer Assessment of Healthcare Providers and Systems Surgical Care Survey (S-CAHPS). Adapted S-CAHPS items were scored using the "top-box" method. RESULTS: Ninety percent of recruited patients agreed to participate (324/359, mean age=62.2, 50.3% male). Patients were able to correctly identify their seniors and interns 85% and 83% of the time, respectively ( P =0.14). On a 10-point scale, seniors had a mean rating of 9.23±1.27 and interns had a mean rating of 9.01±1.49 ( P =0.14). Ninety-nine percent of patients agreed it was important to help in the education of future surgeons. CONCLUSIONS: Surgical inpatients were able to recognize their resident physicians with high frequency and rated resident care highly overall, suggesting that they may serve as a willing source of feedback regarding residents' development of core competencies such as interpersonal skills, communication, professionalism, and patient care. Future work should investigate how to best incorporate patient evaluation of surgical resident care routinely into trainee assessment to support resident development.

Associations of cephalad drainage in neonatal veno-venous ECMO - A mixed-effects, propensity score adjusted retrospective analysis of 20 years of ELSO data.

BACKGROUND: Neurologic complications can occur during neonatal Veno-Venous (VV) ECMO. The addition of a cephalad drainage cannula (i.e., VVDL+V) to dual lumen cannulation (i.e., VVDL) has been advocated to reduce such complications, but previous studies have presented mixed results. METHODS: Data from the ECMO Registry of the Extracorporeal Life Support Organization was used to extract all neonates (≤28 days old) who underwent VV ECMO for respiratory support between 2000 and 2019. Primary outcomes were mortality, conversion to Veno-Arterial (VA) ECMO, pump flows, and complications. A mixed-effects, propensity score adjusted analysis was performed. RESULTS: 4,275 neonates underwent VV ECMO, 581 (13.6%) via VVDL+V cannulation, and 3,694 (86.4%) via VVDL. On unadjusted analyses, VVDL+V patients had higher rates of mortality (25.5% vs 19.0%, p<0.001), conversion to VA ECMO (14.5% vs 4.1%, p<0.001), and higher pump flows at 4 h from ECMO initiation (112.7 vs 105.5 mL/Kg/min, p<0.001), but lower at 24 h (100.3 vs 104.0 mL/Kg/min, p = 0.004), and a higher proportion of them experienced hemorrhagic (29.3% vs 18.3%, p<0.001), cardiovascular (60.8% vs 45.8%, p<0.001), and mechanical (42.5% vs 32.6%, p<0.001) complications compared to VVDL patients. After adjusting for propensity scores and the multi-level nature of ELSO data, there were no differences in neurologic outcomes, pump flows, or mortality. Rather, VVDL+V cannulation was associated with higher rates of conversion to VA ECMO (adjusted odds ratio [AOR] 43.3, 95% CI 24.3 - 77.4, p<0.001), and increased mechanical (AOR 2.2, 95% CI 1.6 - 3.0, p<0.001) and hemorrhagic (AOR 2.0, 95% CI 1.4 - 3.0, p<0.001) complications. CONCLUSIONS: In this analysis, VVDL+V cannulation was not associated with any improvement in neurologic outcomes, pump flows, or mortality, but was rather associated with higher rates of conversion to Veno-Arterial ECMO, mechanical, and hemorrhagic complications.

Peripheral T-Cells, B-Cells, and Monocytes from Multiple Sclerosis Patients Supplemented with High-Dose Vitamin D Show Distinct Changes in Gene Expression Profiles.

Vitamin D is a steroid hormone that has been widely studied as a potential therapy for multiple sclerosis and other inflammatory disorders. Pre-clinical studies have implicated vitamin D in the transcription of thousands of genes, but its influence may vary by cell type. A handful of clinical studies have failed to identify an in vivo gene expression signature when using bulk analysis of all peripheral immune cells. We hypothesized that vitamin D's gene signature would vary by immune cell type, requiring the analysis of distinct cell types. Multiple sclerosis patients (n = 18) were given high-dose vitamin D (10,400 IU/day) for six months as part of a prospective clinical trial (NCT01024777). We collected peripheral blood mononuclear cells from participants at baseline and again after six months of treatment. We used flow cytometry to isolate three immune cell types (CD4+ T-cells, CD19+ B-cells, CD14+ monocytes) for RNA microarray analysis and compared the expression profiles between baseline and six months. We identified distinct sets of differentially expressed genes and enriched pathways between baseline and six months for each cell type. Vitamin D's in vivo gene expression profile in the immune system likely differs by cell type. Future clinical studies should consider techniques that allow for a similar cell-type resolution.

Abstinence-Dependent Effects of Long-Access Cocaine Self-Administration on Nucleus Accumbens Astrocytes Are Observed in Male, But Not Female, Rats.

Accumulating evidence indicates significant consequences for astrocytes associated with drug abuse. For example, reductions in structural features and synaptic colocalization of male rat nucleus accumbens (NAc) astrocytes are observed following short-access (ShA; 2 h/d) self-administration and extinction from cocaine, methamphetamine, and heroin. However, it is unknown whether these observations extend to other rodent models of drug abuse, how enduring these effects may be, and whether similar effects are observed in female rats. Here, we assess the effects of long-access (LgA; 6 h/d) cocaine self-administration and abstinence on NAc astrocytes separately in male and female rats, employing a commonly used behavioral approach to investigate the incubation of cocaine craving. NAc astrocytes from male rats exhibit extensive (∼40%) reductions in surface area, volume, and postsynaptic colocalization 45 d but not 24 h after the last self-administration session. In contrast, no effect of self-administration and abstinence was observed in astrocytes from female rats. Moreover, no effect of LgA self-administration and abstinence was observed on NAc GLT-1 expression in female rats, an effect that has been well described in males. These results indicate striking and sexually dimorphic effects of abstinence subsequent to LgA self-administration on astrocytes. Taken together, these results indicate a pivotal role of prolonged abstinence in the effects of cocaine self-administration on NAc astrocytes, and extend a growing body of evidence regarding sex differences in the cellular consequences of drug self-administration in the brain.

Delivery of therapeutic carbon monoxide by gas-entrapping materials.

Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

Medical Students and the Drive for a Single Right Answer: Teaching Complexity and Uncertainty.

The arrival of Generation Z, the next generation of medical learners, has been accompanied by efforts to adapt teaching practices for this new group of students. Many have identified challenges associated with addressing the needs of modern medical learners. One particular trend we have observed is that medical students are increasingly requesting an "answer key" for all aspects of their medical education. Students often expect to have the correct answers readily available to them to ensure they have reached the correct conclusion and to determine the precise knowledge they need to master. Yet, for much of medicine, and particularly in the care of critically ill patients with multisystem disease in intensive care units, answers are uncertain, and the body of knowledge is ever-growing. Students' regular requests for solutions to be provided to them threaten to undermine their development into critically thinking, self-sufficient physicians. We outline three potential contributors to this multifactorial problem and offer corresponding pedagogical solutions. Specifically, we address how prioritizing outcomes over process, discomfort with uncertainty, and fear of faltering can cause students to seek excessive levels of support that may ultimately do more harm than good. Addressing students' concerns in these three key areas will not only serve students well during their undergraduate medical training but will also equip them with the skills needed to succeed in the clinical realm. To produce physicians capable of navigating an increasingly uncertain world, medical educators will need to help students appreciate that finding the answer is more complex than being provided an answer key.

A multisite study of medical student perspectives on the core surgical clerkship.

BACKGROUND: The surgical clerkship is the primary surgical learning experience for medical students. This study aims to understand student perspectives on the surgery clerkship both before and after the core surgical rotation. METHODS: Medical students at 4 academic hospitals completed pre and postclerkship surveys that included open-ended questions regarding (1) student learning goals and concerns and (2) how surgical clerkship learning could be enhanced. Thematic analysis was performed, and interrater reliability was calculated. RESULTS: Ninety-one percent of students completed both a pre and postclerkship survey (n =162 of 179), generating 320 preclerkship and 270 postclerkship responses. Mean kappa coefficients were 0.83 and 0.82 for pre and postclerkship primary themes, respectively. Thematic analysis identified 5 broad themes: (1) core learning expectations, (2) understanding surgical careers, culture, and work, (3) inhabiting the role of a surgeon, (4) inclusion in the surgical team, and (5) the unique role of the medical student on clinical clerkships. Based on these themes, we propose a learner-centered model of a successful surgical clerkship that satisfies discrete student learning and goals and career objectives while ameliorating the challenges of high-stakes clinical surgical environments such as the operating room. CONCLUSION: Understanding student perspectives on the surgery clerkship, including preclerkship motivations and concerns and postclerkship reflections on surgical learning, revealed potential targets of intervention to improve the surgery clerkship. Future investigation may elucidate whether the proposed model of the elements of a successful surgery clerkship learning facilitates improvement of the surgical learning environment and enhanced surgical learning.

Non-English Primary Language is Associated with Emergency Surgery for Diverticulitis.

BACKGROUND: Language barriers can limit access to care for patients with a non-English primary language (NEPL). The objective of this study was to define the association between primary language and emergency versus elective surgery among diverticulitis patients. MATERIALS AND METHODS: Retrospective cohort study of adult patients from the 2009-2014 New Jersey State Inpatient Database. Patients were included if they had primary language data and underwent a partial colon resection for diverticulitis. Primary language was dichotomized into NEPL versus English primary language (EPL). The primary outcome was surgical admission type - urgent/emergent (referred to as "emergency") versus elective. Descriptive and multivariable analyses were performed. RESULTS: A total of 9,453 patients underwent surgery for diverticulitis, of which 592 (6.3%) had NEPL. Among NEPL patients, 300 (51%) had Spanish as primary language and 292 (49%) had another non-Spanish primary language. Patients with NEPL and EPL were similar in age (median age 58 versus 59 years; P = 0.54) and sex (52% versus 53% female; P = 0.45). Patients with NEPL were less likely to have commercial insurance (45% versus 59%; P <0.001). On multivariable analysis, compared to patients with EPL, NEPL was associated with increased odds of emergency surgery for diverticulitis (OR 1.35; 95% Confidence Interval 1.13-1.62; P = 0.001) CONCLUSION: Patients with NEPL have higher odds of emergency versus elective surgery for diverticulitis compared to patients with EPL. Further research is needed to examine differences in referral pathways, patient-provider communication, and health literacy that may hinder access to elective surgery in patients with diverticulitis.