CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance.

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function.

Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication.IMPORTANCEEnterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

Non-Pharmacological Approaches to Pain Management in Residential Aged Care: a Pre-Post-Test Study.

OBJECTIVES: The project aimed to evaluate a pain management program (PMP) using non-pharmacological approaches at five residential aged care facilities (RACFs) in Australia. METHODS: The PMP involved a physiotherapist implementing four sessions per week of treatments (massage therapy, TENS, exercises and stretching, or combinations of these). Ninety-five participants were recruited (average age, 83 years; SD = 7.6; 38% men, 62% women; 56% with dementia). Sessions lasted approximately 10 minutes, and residents' levels of pain were recorded using a 5-point scale before and after each treatment. The intervention period for each participant was the first consecutive 8 weeks in which they received the intervention. RESULTS: Data analyses showed: (1) a small but statistically significant decrease in the number of as required (PRN) medications; and (2) a decrease in average pain ratings from pre-session to post-session from 2.4 (some to moderate pain) to 1.1 (a little pain). Notably, residents with dementia received lower pain ratings than those without. CONCLUSIONS: Non-pharmacological approaches to pain in residential care settings are effective, especially when two or more are combined. Staff working in residential care settings should rely on best practice to recognise pain in residents with dementia. CLINICAL IMPLICATIONS: Non-pharmacological interventions may be effective in reducing pain and reliance on PRN medications in residential care settings, especially when two or more are used. Staff working in residential aged care settings should be provided with training in pain assessment and management, with particular attention to residents with dementia.

Parallel Control over Surface Charge and Wettability Using Polyelectrolyte Architecture: Effect on Protein Adsorption and Cell Adhesion.

Surface charge and wettability, the two prominent physical factors governing protein adsorption and cell adhesion, have been extensively investigated in the literature. However, a comparison between these driving forces in terms of their independent and cooperative effects in affecting adhesion is rarely explored on a systematic and quantitative level. Herein, we formulate a protocol that features two-dimensional control over both surface charge and wettability with limited cross-parameter influence. This strategy is implemented by controlling both the polyion charge density in the layer-by-layer (LbL) assembly process and the polyion side-chain chemical structures. The 2D property matrix spans surface isoelectric points ranging from 5 to 9 and water contact angles from 35 to 70°, with other interferential factors (e.g., roughness) eliminated. The interplay between these two surface variables influences protein (bovine serum albumin, lysozyme) adsorption and 3T3 fibroblast cell adhesion. For proteins, we observe the presence of thresholds for surface wettability and electrostatic driving forces necessary to affect adhesion. Beyond these thresholds, the individual effects of electrostatic forces and wettability are observed. For fibroblast, both surface charge and wettability have an effect on its adhesion. The combined effects of positive charge and hydrophilicity lead to the highest cell adhesion, whereas negative charge and hydrophobicity lead to the lowest cell adhesion. Our design strategy can potentially form the basis for studying the distinct behaviors of electrostatic force or wettability driven interfacial phenomena and serve as a reference in future studies assessing protein adsorption and cell adhesion to surfaces with known charge and wettability within the property range studied here.

Historical analysis of participation in 161 km ultramarathons in North America.

Participation trends in 100 m (161 km) ultramarathon running competitions in North America were examined from race results from 1977 through 2008. A total of 32, 352 finishes accounted for by 9815 unique individuals were identified. The annual number of races and number of finishes increased exponentially over the study period. This growth in number of finishes occurred through a combination of (1) an increase in participation among runners >40 years of age from less than 40% of the finishes prior to the mid-1980s to 65-70% of the finishes since 1996, (2) a growth (p < 0.0001) in participation among women from virtually none in the late 1970s to nearly 20% since 2004, and (3) an increase in the average annual number of races completed by each individual to 1.3. While there has been considerable growth in participation, the 161 km ultramarathon continues to attract a relatively small number of participants compared with running races of shorter distances.