Protocol to characterize the melanoma tumor immune microenvironment in mice from single cell to flow cytometry analysis.

Here, we present a protocol to study and describe immune cells that surround or infiltrate tumor cells or get through the body of a melanoma syngeneic mice model. We describe steps for creating and establishing the syngeneic mouse model, euthanasia, and tumor or organ harvest. We then detail procedures to rapidly achieve a single-cell suspension from different tissue samples to further quantify and analyze the phenotype of the immune cell population (lymphocytes T and B, tumor-associated macrophages, and myeloid-derived suppressor cells) by flow cytometry.

Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Puerto Rico Health System Resilience After Hurricane Maria: Implications for Disaster Preparedness in the COVID-19 Era.

BACKGROUND: Every year, Puerto Rico faces a hurricane season fraught with potentially catastrophic structural, emotional and health consequences. In 2017, Puerto Rico was hit by Hurricane Maria, the largest natural disaster to ever affect the island. Several studies have estimated the excess morbidity and mortality following Hurricane Maria in Puerto Rico, yet no study has comprehensively examined the underlying health system weaknesses contributing to the deleterious health outcomes. METHODS: A qualitative case study was conducted to assess the ability of the UPR health system to provide patient care in response to Hurricane Maria. An established five key resilience framework and inductive analysis was used to identify factors that affected health system resilience. Thirteen Emergency Medicine Physicians, Family Medicine Physicians, and Hospital Administrators in a University of Puerto Rico (UPR) Community Hospital were interviewed as part of our study. RESULTS: Of the five key resiliency components, three domains were notably weak with respect to UPR resiliency. Prior to the Hurricane, key personnel at the UPR hospital were unaware of the limited capacity of back-up generators at hospitals and were ill-prepared to transfer ICU patients to appropriate hospitals. Post Hurricane, the hospital faced self-regulation challenges when triaging the provision of Hurricane-related emergency services with delivering core health services, in particular for patients with chronic conditions. Finally, during and after the Hurricane, integration of patient care coordination between the UPR hospital ambulances, neighboring hospitals, and national and state government was suboptimal. The two remaining resiliency factors, addressing diverse needs and system adaptiveness in a time of crisis, were seen as strengths. CONCLUSIONS: Hurricane Maria exposed weaknesses in the Puerto Rican health system, notably the lack of awareness about the limited capacity of backup generators, poor patient care coordination, and interruption of medical care for patients with chronic conditions. As in other countries, the current COVID epidemic is taxing the capacity of the Puerto Rico health system, which could increase the likelihood of another health system collapse should another hurricane hit the island. Therefore, a resilience framework is a useful tool to help health systems identify areas of improvement in preparation for possible natural disasters.

The imposition of, but not the propensity for, social subordination impairs exploratory behaviors and general cognitive abilities.

Imposed social subordination, such as that which accompanies physical defeat or alienation, has been associated with impaired cognitive function in both human and non-human animals. Here we examined whether domain-specific and/or domain-general learning abilities (c.f. general intelligence) are differentially influenced by the imposition of social subordination. Furthermore, we assessed whether the impact of subordination on cognitive abilities was the result of imposed subordination per se, or if it reflected deficits intrinsically expressed in subjects that are predisposed to subordination. Subordinate and dominant behaviors were assessed in two groups of CD-1 male mice. In one group (Imposed Stratification), social stratification was imposed (through persistent physical defeat in a colonized setting) prior to the determination of cognitive abilities, while in the second group (Innate Stratification), an assessment of social stratification was made after cognitive abilities had been quantified. Domain-specific learning abilities were measured as performance on individual learning tasks (odor discrimination, fear conditioning, spatial maze learning, passive avoidance, and egocentric navigation) while domain-general learning abilities were determined by subjects' aggregate performance across the battery of learning tasks. We observed that the imposition of subordination prior to cognitive testing decreased exploratory tendencies, moderately impaired performance on individual learning tasks, and severely impaired general cognitive performance. However, similar impairments were not observed in subjects with a predisposition toward a subordinate phenotype (but which had not experienced physical defeat at the time of cognitive testing). Mere colonization, regardless of outcome (i.e., stratification), was associated with an increase in stress-induced serum corticosterone (CORT) levels, and thus CORT elevations were not themselves adequate to explain the effects of imposed stratification on cognitive abilities. These findings indicate that absent the imposition of subordination, individuals with subordinate tendencies do not express learning impairments. This observation could have important ramifications for individuals in environments where social stratification is prevalent (e.g., schools or workplace settings).