OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm are limited. Here, we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa ΔoprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here, we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A ΔoprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective ΔoprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in biofilms.

OprF impacts Pseudomonas aeruginosa biofilm matrix eDNA levels in a nutrient-dependent manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby Δ oprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa Δ oprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of mature P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE: Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A Δ oprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective Δ oprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in mature biofilms.

B'rit shalom: a Jewish ritual alternative to newborn male circumcision.

This paper focuses on the ritual b'rit shalom (also brit shalom), Hebrew for "covenant of peace," which is intentionally positioned as an alternative to b'rit milah, the covenant of circumcision, for infant males. Although b'rit shalom is currently not common in the Jewish community, it might well become more popular. The first section discusses some of the history and practices of b'rit milah, so that the second section can better show the development and potential of b'rit shalom. Since many Americans, including Jews, no longer view circumcision as a cultural necessity, and there is long-running ethical debate about infant male genital cutting, there is reason to explore Jewish infant rituals which do not include circumcision.

Obesity Related Glomerulopathy in Adolescent Women: The Effect of Body Surface Area.

Background: Adolescent obesity, a risk factor for cardiorenal morbidity in adulthood, has reached epidemic proportions. Obesity-related glomerulopathy (ORG) has an early reversible stage of hyperfiltration. Age-appropriate formulae for eGFR, which are standardized to ideal body surface area (BSA) and provide assessment of kidney function in ml/min/1.73 m2, may underestimate prevalence of early ORG. We investigated whether adjusting eGFR to actual BSA more readily identifies early ORG. Methods: We studied a cohort of 22,417 young individuals, aged 12-21 years, from a New York metropolitan multi-institutional electronic health records clinical database. eGFR was calculated in two ways: BSA-standardized eGFR, and absolute eGFR. Hyperfiltration was defined above a threshold of 135 ml/min per 1.73 m2 or 135 ml/min, respectively. The prevalence of hyperfiltration according to each formula was assessed in parallel to creatinine clearance. Results: Serum creatinine values and hyperfiltration prevalence according to BSA-standardized eGFR were similar, 13%-15%, across body mass index (BMI) groups. The prevalence of hyperfiltration determined by absolute eGFR differed across BMI groups: underweight, 2%; normal weight, 6%; overweight, 17%; and obese, 31%. This trend paralleled the rise in creatinine clearance across BMI groups. Conclusions: Absolute eGFR more readily identifies early ORG than the currently used formulae, which are adjusted to a standardized BSA and are not representative of current population BMI measures. Using absolute eGFR in clinical practice and research may improve the ability to identify, intervene, and reverse early ORG, which has great importance with increasing obesity rates.

Death in a Cold Climate: Medical Aid in Dying in Vermont.

What happened when Vermont passed its medical-aid-in-dying bill in 2013? Not what one might hope or expect. In Scripting Death: Stories of Assisted Dying in America (University of California Press, 2021), Mara Buchbinder details, through a host of gripping interviews, the difficulties people experience in actually accessing their legal rights to physician-assisted suicide.

Altered Chromatin States Drive Cryptic Transcription in Aging Mammalian Stem Cells.

A repressive chromatin state featuring trimethylated lysine 36 on histone H3 (H3K36me3) and DNA methylation suppresses cryptic transcription in embryonic stem cells. Cryptic transcription is elevated with age in yeast and nematodes, and reducing it extends yeast lifespan, though whether this occurs in mammals is unknown. We show that cryptic transcription is elevated in aged mammalian stem cells, including murine hematopoietic stem cells (mHSCs) and neural stem cells (NSCs) and human mesenchymal stem cells (hMSCs). Precise mapping allowed quantification of age-associated cryptic transcription in hMSCs aged in vitro. Regions with significant age-associated cryptic transcription have a unique chromatin signature: decreased H3K36me3 and increased H3K4me1, H3K4me3, and H3K27ac with age. Genomic regions undergoing such changes resemble known promoter sequences and are bound by TBP even in young cells. Hence, the more permissive chromatin state at intragenic cryptic promoters likely underlies increased cryptic transcription in aged mammalian stem cells.

Vertebrate cell culture as an experimental approach - limitations and solutions.

Mammalian cell culture has provided the foundation for the incredible expansion of cell biology to uncover the 'inner life of the cell'. The protocols for propagating cells in the laboratory have their origins in the mid-20th century. At that time the focus was on creating cell culture media that kept cells viable and favoured replicative growth. To the extent that oxygen level was considered as an important parameter, it was in the context of ensuring that oxygen was not depleted; the idea that environmental oxygen levels could be toxic was not widely appreciated. We increasingly understand that media composition and oxygen levels have important effects on cellular functions and that maintaining physiologically relevant conditions is necessary to maintain in vivo behaviours. We also understand much about the impact of growing cells that function in a 3D environment in 2D adherent monolayers. In this review, we examine some of the issues affecting standard cell culture approaches and new solutions that address these issues to increase the physiological accuracy of the cellular environment. We have reached a threshold in cell biology in which we know enough about the problems and their solutions to inform useful adjustments to protocols moving forward. This will increase the accuracy and translatability of this reductionist approach to understanding cell behaviours.

FOXO3 regulates a common genomic program in aging and glioblastoma stem cells.

Background: Glioblastoma (GBM) is an aggressive, age-associated malignant glioma that contains populations of cancer stem cells. These glioma stem cells (GSCs) evade therapeutic interventions and repopulate tumors due to their existence in a slowly cycling quiescent state. Although aging is well known to increase cancer initiation, the extent to which the mechanisms supporting GSC tumorigenicity are related to physiological aging remains unknown. Aims: Here, we investigate the transcriptional mechanisms by which Forkhead Box O3 (FOXO3), a transcriptional regulator that promotes healthy aging, affects GSC function and the extent to which FOXO3 transcriptional networks are dysregulated in aging and GBM. Methods and results: We performed transcriptome analysis of clinical GBM tumors and observed that high FOXO3 activity is associated with gene expression signatures of stem cell quiescence, reduced oxidative metabolism, and improved patient outcomes. Consistent with these findings, we show that elevated FOXO3 activity significantly reduces the proliferation of GBM-derived GSCs. Using RNA-seq, we find that functional ablation of FOXO3 in GSCs rewires the transcriptional circuitry associated with metabolism, epigenetic stability, quiescence, and differentiation. Since FOXO3 has been implicated in healthy aging, we then investigated the extent to which it regulates common transcriptional programs in aging neural stem cells (NSCs) and GSCs. We uncover a shared transcriptional program and, most strikingly, find that FOXO3-regulated pathways are associated with altered mitochondrial functions in both aging and GBM. Conclusions: This work identifies a FOXO-associated transcriptional program that correlates between GSCs and aging NSCs and is enriched for metabolic and stemness pathways connected with GBM and aging.

Trends in the use of dual mobility bearings in hip arthroplasty.

AIMS: Dual mobility (DM) bearings are an attractive treatment option to obtain hip stability during challenging primary and revision total hip arthroplasty (THA) cases. The purpose of this study was to analyze data submitted to the American Joint Replacement Registry (AJRR) to characterize utilization trends of DM bearings in the USA. METHODS: All primary and revision THA procedures reported to AJRR from 2012 to 2018 were analyzed. Patients of all ages were included and subdivided into DM and traditional bearing surface cohorts. Patient demographics, geographical region, hospital size, and teaching affiliation were assessed. Associations were determined by chi-squared analysis and logistic regression was performed to assess outcome variables. RESULTS: A total of 406,900 primary and 34,745 revision THAs were identified, of which 35,455 (8.7%) and 8,031 (23.1%) received DM implants respectively. For primary THA, DM usage increased from 6.7% in 2012 to 12.0% in 2018. Among revision THA, DM use increased from 19.5% in 2012 to 30.6% in 2018. Patients < 50 years of age had the highest rates of DM implantation in every year examined. For each year of increase in age, there was a 0.4% decrease in the rate of DM utilization (odds ratio (OR) 0.996 (95% confidence interval (CI) 0.995 to 0.997); p < 0.001). Females were more likely to receive a DM implant compared to males (OR 1.077 (95% CI 1.054 to 1.100); p < 0.001). Major teaching institutions and smaller hospitals were associated with higher rates of utilization. DM articulations were used more commonly for dysplasia compared with osteoarthritis (OR 2.448 (95% CI 2.032 to 2.949); p < 0.001) during primary THA and for instability (OR 3.130 (95% CI 2.751 to 3.562) vs poly-wear; p < 0.001) in the revision setting. CONCLUSION: DM articulations showed a marked increase in utilization during the period examined. Younger patient age, female sex, and hospital characteristics such as teaching status, smaller size, and geographical location were associated with increased utilization. DM articulations were used more frequently for primary THA in patients with dysplasia and for revision THA in patients being treated for instability. Cite this article: Bone Joint J 2020;102-B(7 Supple B):27-32.

Manipulation Under Anesthesia After Total Knee: Who Still Requires a Revision Arthroplasty?

BACKGROUND: Stiffness after total knee arthroplasty (TKA) is a multifactorial complication involving patient, implant, surgical technique, and rehabilitation, occasionally necessitating manipulation under anesthesia (MUA) or revision. Few modern databases contain sufficient longitudinal information of all factors. We characterized MUA after primary TKA and identified independent risk factors for revision TKA after MUA from the American Joint Replacement Registry. METHODS: We retrospectively reviewed primary TKAs for American Joint Replacement Registry patients ≥65 years from January 1, 2012 to 31 March, 2019. We linked these to the Centers for Medicare and Medicaid Services database to identify MUA and revision TKA procedure codes. We compared groups with chi-squared testing, identifying independent risk factors for subsequent revision with binary logistic regression presented as odds ratios with 95% confidence intervals. RESULTS: Of 664,604 primary TKAs, 3918 (0.6%) underwent MUA after a median of 2.0 ± 1.0 months. Revision surgery occurred in 131 (3.4%) MUA patients after a median of 9.0 months. Timing of MUA was not different between revision and no revision patients (P = .09). Patients undergoing MUA compared to no MUA were older (age 71.5 vs 70.7, P < .01), predominantly female (63.9% vs 61.2%, P < .01), current/former tobacco users (24.2% vs 13.3%, P < .01), with osteoarthritis diagnoses (98.0% vs 84.3%, P < .01). Independent risk factors for revision after MUA were male gender (1.56, 1.09-2.22). CONCLUSION: The incidence of MUA after primary TKA is low (0.6%) in Medicare patients ≥65 years of age; 3.4% progress to revision after a median of 9 months. Being male was significantly associated with revision TKA after MUA.

Efference Copies: Hair Cells Are the Link.

Animals must distinguish external stimuli from self-generated sensory input to guide appropriate behaviors. A recent study elucidates a cellular mechanism by which zebrafish perform this distinction while maintaining sensitivity to external environmental signals.

Living with Alzheimer Disease and Other Types of Dementia: Stories from Caregivers.

This symposium includes twelve personal narratives from people who have provided care to a spouse, parent, another relative, or friend with Alzheimer disease or related dementias (ADRD). People with ADRDs often face years of cognitive decline with memory and thinking that eventually require help from others to assist with their daily activities. Most people caring for older adults in the US are unpaid family members, friends, or other informal caregivers. People providing care often experience emotional and physical stress, or financial burdens. This symposium also includes three commentaries by experts in the fields of bioethics and philosophy, justice in healthcare, family caregiving, and end of life choices. These narratives provide a forum for exploring caregiver needs, suffering, benefits, and joys, as well as opportunities to improve the way we support caregivers and people with dementia and Alzheimer disease.

Development, Validation and Pilot Field Deployment of a Custom Mouthpiece for Head Impact Measurement.

The objective of this study was to develop a mouthpiece sensor with improved head kinematic measurement for use in non-helmeted and helmeted sports through laboratory validation and pilot field deployment in female youth soccer. For laboratory validation, data from the mouthpiece sensor was compared to standard sensors mounted in a headform at the center of gravity as the headform was struck with a swinging pendulum. Linear regression between peak kinematics measured from the mouthpiece and headform showed strong correlation, with r2 values of 0.95 (slope = 1.02) for linear acceleration, 1.00 (slope = 1.00) for angular velocity, and 0.97 (slope = 0.96) for angular acceleration. In field deployment, mouthpiece data were collected from four female youth soccer players and time-synchronized with film. Film-verified events (n = 915) were observed over 9 practices and 5 games, and 632 were matched to a corresponding mouthpiece event. This resulted in an overall sensitivity of 69.2% and a positive predictive value of 80.3%. This validation and pilot field deployment data demonstrates that the mouthpiece provides highly accurate measurement of on-field head impact data that can be used to further study the effects of impact exposure in both helmeted and non-helmeted sports.

Single-cell analysis reveals T cell infiltration in old neurogenic niches.

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.

Hispanic Hospice Utilization: Integrative Review and Meta-analysis.

Hospice is patient-centered end-of-life care. Hispanics are underrepresented among hospice patients (7.1%) relative to the U.S. population (17%). We conducted a systematic review of the literature and meta-analysis to understand this underrepresentation further. In palliative care, Hispanic hospice enrollment is comparable to that of non-Hispanic Whites (NHWs) (RR 1.02, 95% CI: 0.93-1.12; z=0.49; p = .627). However, in cases of heart failure (OR 0.49, 95% CI 0.37-0.66) and stroke (OR 0.77, 95% CI 0.63-0.94), Hispanics are much less likely to use hospice than NHWs. Cancer studies are mixed, but in meta-analysis were significant for lower relative hospice use in Hispanics (RR 0.96, 95% CI: 0.94-0.99; z=3.01; p=.003). It remains unclear whether using census and insurance data in statistical analysis provides valid results since the Hispanic population is younger, healthier, and less likely to be insured. Health equity in hospice may be better represented by hospice quality rather than hospice enrollment rates.

Could We Be Marsupials? Very Premature Babies and Artificial Wombs.

It is often pointed out that one cannot be "a little bit pregnant," but pregnancy's borders are no longer so crisp. At Children's Hospital of Philadelphia, scientists have created an artificial womb in which "extremely premature" lambs were nurtured for four weeks, enough to make them ready to meet the world. The goal is to advance this technology until it is available for very premature human infants. At present, we put preemies into neonatal intensive care units, which are extremely stressful for the babies and their families, and the things we do to try to support them often cause serious damage. If successful, the artificial womb could be a terrific technology. The implications are remarkable. Would humans become somewhat like marsupials? Now, one is either born or not. Would this technology create a new, intermediate stage? What would this mean ethically and legally?