Orphan Nuclear Receptor Family 4A (NR4A) Members NR4A2 and NR4A3 Selectively Modulate Elements of the Monocyte Response to Buffered Hypercapnia.

Hypercapnia occurs when the partial pressure of carbon dioxide (CO2) in the blood exceeds 45 mmHg. Hypercapnia is associated with several lung pathologies and is transcriptionally linked to suppression of immune and inflammatory signalling through poorly understood mechanisms. Here we propose Orphan Nuclear Receptor Family 4A (NR4A) family members NR4A2 and NR4A3 as potential transcriptional regulators of the cellular response to hypercapnia in monocytes. Using a THP-1 monocyte model, we investigated the sensitivity of NR4A family members to CO2 and the impact of depleting NR4A2 and NR4A3 on the monocyte response to buffered hypercapnia (10% CO2) using RNA-sequencing. We observed that NR4A2 and NR4A3 are CO2-sensitive transcription factors and that depletion of NR4A2 and NR4A3 led to reduced CO2-sensitivity of mitochondrial and heat shock protein (Hsp)-related genes, respectively. Several CO2-sensitive genes were, however, refractory to depletion of NR4A2 and NR4A3, indicating that NR4As regulate certain elements of the cellular response to buffered hypercapnia but that other transcription factors also contribute. Bioinformatic analysis of conserved CO2-sensitive genes implicated several novel putative CO2-sensitive transcription factors, of which the ETS Proto-Oncogene 1 Transcription Factor (ETS-1) was validated to show increased nuclear expression in buffered hypercapnia. These data give significant insights into the understanding of immune responses in patients experiencing hypercapnia.

Remdesivir-induced conduction abnormalities: A molecular model-based explanation.

Purpose: Remdesivir use in COVID-19 is associated with cardiac conduction abnormalities from unclear mechanisms. A proposed mechanism is the bioaccumulation of the intermediate metabolite GS-441524 resulting in exogenous activation of cardiac adenosine A1 due to the structural similarity between adenosine and GS-441524. The prolonged half-life of GS-441524 can result in sustained activation of adenosine A1 receptors. In this study, we used molecular modeling of adenosine, GS-441524 and the adenosine A1 receptor to assess the potential mechanistic association of the proposed mechanism. Methods: Adenosine and GS-441524 structures were acquired from the PubChem database. Ligand docking was carried out using UCSF Chimera. Models were chosen based on greatest binding affinity and minimum root mean square deviation. Figures of resulting structural models were prepared using UCSF Chimera or PyMOL 2.3.5. Results: By modeling the interaction between the A1 G protein complex and both adenosine and GS-441524, we found that the proposed mechanism of exogenous A1 receptor activation is feasible based on docking compatibility. Conclusion: The proposed mechanism of exogenous cardiac A1 receptor activation from bioaccumulation of GS-441524 as a cause of observed cardiac conduction abnormalities with the use of remdesivir in COVID-19 is viable. Further studies are needed to assess causality.

Hypercapnia alters mitochondrial gene expression and acylcarnitine production in monocytes.

CO2 is produced during aerobic respiration. Normally, levels of CO2 in the blood are tightly regulated but pCO2 can rise (hypercapnia, pCO2 > 45 mmHg) in patients with lung diseases, for example, chronic obstructive pulmonary disease (COPD). Hypercapnia is a risk factor in COPD but may be of benefit in the context of destructive inflammation. The effects of CO2 per se, on transcription, independent of pH change are poorly understood and warrant further investigation. Here we elucidate the influence of hypercapnia on monocytes and macrophages through integration of state-of-the-art RNA-sequencing, metabolic and metabolomic approaches. THP-1 monocytes and interleukin 4-polarized primary murine macrophages were exposed to 5% CO2 versus 10% CO2 for up to 24 h in pH-buffered conditions. In hypercapnia, we identified around 370 differentially expressed genes (DEGs) under basal and about 1889 DEGs under lipopolysaccharide-stimulated conditions in monocytes. Transcripts relating to both mitochondrial and nuclear-encoded gene expression were enhanced in hypercapnia in basal and lipopolysaccharide-stimulated cells. Mitochondrial DNA content was not enhanced, but acylcarnitine species and genes associated with fatty acid metabolism were increased in hypercapnia. Primary macrophages exposed to hypercapnia also increased activation of genes associated with fatty acid metabolism and reduced activation of genes associated with glycolysis. Thus, hypercapnia elicits metabolic shifts in lipid metabolism in monocytes and macrophages under pH-buffered conditions. These data indicate that CO2 is an important modulator of monocyte transcription that can influence immunometabolic signaling in immune cells in hypercapnia. These immunometabolic insights may be of benefit in the treatment of patients experiencing hypercapnia.

Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Transcriptional Profiling of Monocytes Deficient in Nuclear Orphan Receptors NR4A2 and NR4A3 Reveals Distinct Signalling Roles Related to Antigen Presentation and Viral Response.

The nuclear receptor sub-family 4 group A (NR4A) family are early response genes that encode proteins that are activated in several tissues/cells in response to a variety of stressors. The NR4A family comprises NR4A1, NR4A2 and NR4A3 of which NR4A2 and NR4A3 are under researched and less understood, particularly in the context of immune cells. NR4A expression is associated with multiple diseases e.g. arthritis and atherosclerosis and the development of NR4A-targetting molecules as therapeutics is a current focus in this research field. Here, we use a combination of RNA-sequencing coupled with strategic bioinformatic analysis to investigate the down-stream effects of NR4A2 and NR4A3 in monocytes and dissect their common and distinct signalling roles. Our data reveals that NR4A2 and NR4A3 depletion has a robust and broad-reaching effect on transcription in both the unstimulated state and in the presence of LPS. Interestingly, many of the genes affected were present in both the unstimulated and stimulated states revealing a previously unappreciated role for the NR4As in unstimulated cells. Strategic clustering and bioinformatic analysis identified both distinct and common transcriptional roles for NR4A2 and NR4A3 in monocytes. NR4A2 notably was linked by both bioinformatic clustering analysis and transcription factor interactome analysis to pathways associated with antigen presentation and regulation of MHC genes. NR4A3 in contrast was more closely linked to pathways associated with viral response. Functional studies further support our data analysis pointing towards preferential/selective roles for NR4A2 in the regulation of antigen processing with common roles for NR4A2 and NR4A3 evident with respect to cell migration. Taken together this study provides novel mechanistic insights into the role of the enigmatic nuclear receptors NR4A2 and NR4A3 in monocytes.

From carotenoid intake to carotenoid blood and tissue concentrations - implications for dietary intake recommendations.

There is uncertainty regarding carotenoid intake recommendations, because positive and negative health effects have been found or are correlated with carotenoid intake and tissue levels (including blood, adipose tissue, and the macula), depending on the type of study (epidemiological vs intervention), the dose (physiological vs supraphysiological) and the matrix (foods vs supplements, isolated or used in combination). All these factors, combined with interindividual response variations (eg, depending on age, sex, disease state, genetic makeup), make the relationship between carotenoid intake and their blood/tissue concentrations often unclear and highly variable. Although blood total carotenoid concentrations <1000 nmol/L have been related to increased chronic disease risk, no dietary reference intakes (DRIs) exist. Although high total plasma/serum carotenoid concentrations of up to 7500 nmol/L are achievable after supplementation, a plateauing effect for higher doses and prolonged intake is apparent. In this review and position paper, the current knowledge on carotenoids in serum/plasma and tissues and their relationship to dietary intake and health status is summarized with the aim of proposing suggestions for a "normal," safe, and desirable range of concentrations that presumably are beneficial for health. Existing recommendations are likewise evaluated and practical dietary suggestions are included.

Analysis of Lutein, Zeaxanthin, and Meso-Zeaxanthin in the Organs of Carotenoid-Supplemented Chickens.

The macular carotenoids (i.e., lutein (L), zeaxanthin (Z) and meso-zeaxanthin (MZ)) exhibit anti-inflammatory, antioxidant and optical properties that are believed to support human health and function. Studying the accumulation and distribution of these nutrients in tissues and organs, in addition to the eye, is an important step in understanding how these nutrients might support global human function and health (e.g., heart and brain). Chicken is an appropriate animal model with which to study the accumulation of these carotenoids in organs, as the relevant transport molecules and carotenoid binding proteins for L, Z and MZ are present in both humans and chickens. In this experiment, a sample of 3 chickens that were supplemented with L and MZ diacetate (active group) and a sample of 3 chickens that received a standard diet (control group) were analysed. Both groups were analysed for L, Z and MZ concentrations in the brain, eyes, heart, lung, duodenum/pancreas, jejunum/ileum, kidney and breast tissue. L, Z and MZ were identified in all the organs/tissues analysed from the active group. L and Z were identified in all of the organs/tissues analysed from the control group; while, MZ was identified in the eyes of these animals only. The discovery that MZ is accumulated in the tissues and organs of chickens supplemented with this carotenoid is important, given that it is known that a combination of L, Z and MZ exhibits superior antioxidant capacity when compared to any of these carotenoids in isolation.

Stability of Commercially Available Macular Carotenoid Supplements in Oil and Powder Formulations.

We previously identified that the concentration of zeaxanthin in some commercially available carotenoid supplements did not agree with the product's label claim. The conclusion of this previous work was that more quality assurance was needed to guarantee concordance between actual and declared concentrations of these nutrients i.e., lutein (L) zeaxanthin (Z) and meso-zeaxanthin (MZ) in commercially available supplements. Since this publication, we performed further analyses using different commercially available macular carotenoid supplements. Three capsules from one batch of eight products were analysed at two different time points. The results have been alarming. All of the powder filled products (n = 3) analysed failed to comply with their label claim (L: 19-74%; Z: 57-73%; MZ: 83-97%); however, the oil filled soft gel products (n = 5) met or were above their label claim (L: 98-122%; Z: 117-162%; MZ: 97-319%). We also identified that the carotenoid content of the oil filled capsules were stable over time (e.g., L average percentage change: -1.7%), but the powder filled supplements degraded over time (e.g., L average percentage change: -17.2%). These data are consistent with our previous work, and emphasize the importance of using carotenoid interventions in oil based formulas rather than powder filled formulas.

Mineral-Biochar Composites: Molecular Structure and Porosity.

Dramatic changes in molecular structure, degradation pathway, and porosity of biochar are observed at pyrolysis temperatures ranging from 250 to 550 °C when bamboo biomass is pretreated by iron-sulfate-clay slurries (iron-clay biochar), as compared to untreated bamboo biochar. Electron microscopy analysis of the biochar reveals the infusion of mineral species into the pores of the biochar and the formation of mineral nanostructures. Quantitative (13)C nuclear magnetic resonance (NMR) spectroscopy shows that the presence of the iron clay prevents degradation of the cellulosic fraction at pyrolysis temperatures of 250 °C, whereas at higher temperatures (350-550 °C), the clay promotes biomass degradation, resulting in an increase in both the concentrations of condensed aromatic, acidic, and phenolic carbon species. The porosity of the biochar, as measured by NMR cryoporosimetry, is altered by the iron-clay pretreatment. In the presence of the clay, at lower pyrolysis temperatures, the biochar develops a higher pore volume, while at higher temperature, the presence of clay causes a reduction in the biochar pore volume. The most dramatic reduction in pore volume is observed in the kaolinite-infiltrated biochar at 550 °C, which is attributed to the blocking of the mesopores (2-50 nm pore) by the nonporous metakaolinite formed from kaolinite.

Anterior capsulotomy integrity after femtosecond laser-assisted cataract surgery.

OBJECTIVE: To compare the incidence of anterior capsular tears after femtosecond laser-assisted cataract surgery (FLACS) versus phacoemulsification cataract surgery (PCS) and to assess the ultrastructural features of anterior capsulotomy specimens (FLACS and PCS) using electron microscopy. DESIGN: Prospective, multicenter, comparative cohort case series. PARTICIPANTS: Consecutive patients undergoing FLACS or PCS. METHODS: A prospective cohort study of all patients (n = 1626) undergoing FLACS or PCS by 2 surgeons from centers A and B was undertaken to compare the incidence of anterior capsule tears. Anterior lens capsules were collected by 4 surgeons from centers A, B, C, and D using 3 different commercially available femtosecond platforms, each with latest version upgrades. Lens capsule tissue was prepared for scanning electron microscopy (SEM) using a total of 10 samples for patients undergoing PCS, and 40 samples for patients undergoing FLACS. MAIN OUTCOME MEASURES: Incidence of anterior capsule tear and comparative ultrastructural features of capsular samples from both PCS and FLACS cases. RESULTS: There was a significantly increased rate of anterior capsule tears in the FLACS group (15/804 [1.87%]) when compared with the PCS group (1/822 [0.12%]; P = 0.0002, Fisher exact test). In 7 cases, the anterior capsule tear extended to the posterior capsule. Because all cases had occurred in complete capsulotomy, the integrity of the anterior capsule was questioned in the FLACS group. Subsequent SEM sampling showed irregularity at the capsule margin, as well as multiple apparently misplaced laser pits in normal parts of the tissue. Aberrant pits were approximately 2 to 4 µm apart and occurred at a range of 10 to 100 µm radially from the capsule edge. CONCLUSIONS: Laser anterior capsulotomy integrity seems to be compromised by postage-stamp perforations and additional aberrant pulses, possibly because of fixational eye movements. This can lead to an increased rate of anterior capsule tears, and extra care should be taken during surgery after femtosecond laser pretreatment has been performed. A learning curve may account for some of the increased complication rate with FLACS. However, the SEM features raise safety concerns for capsular integrity after FLACS and warrant further investigation.

HLA-DR specific monoclonal antibodies block lymphoproliferative response to measles vaccine in vitro: a pilot study.

Humoral and cell-mediated immune responses are important in protection against measles. Non-response to vaccination has been associated with specific HLA-DR and HLA-DQ alleles; however, little is known about the relative importance of these alleles in the cellular immune response induced by measles virus vaccine. To investigate the role of HLA-DR/DQ class II restriction, a small pilot study was conducted. Lymphoproliferation assays using class II DR and DQ-specific monoclonal antibodies (MoAb) were performed at one week and two weeks post immunization with MMRII vaccine. The mean stimulation index (SI) was 4.4 and 5.3 at one and two weeks with reductions in SI of 47.6% and 70.2%, respectively, following the addition of DR-specific MoAb (p<0.001). These results clearly show that a significant proportion of the cell-mediated immune response to measles virus vaccine, as measured by SI, is HLA-DR restricted.

Clinical effectiveness and cost benefit of universal versus targeted methicillin-resistant Staphylococcus aureus screening upon admission in hospitals.

OBJECTIVE: To conduct an exploratory study to evaluate the clinical effectiveness and cost benefit of universal versus targeted screening for methicillin-resistant Staphylococcus aureus (MRSA) to prevent hospital-acquired MRSA infections. DESIGN: Prospective, interventional study, using a case-control design, difference-in-differences, and cost-benefit analyses. SETTING: Two community hospitals in Wisconsin. PATIENTS: Consecutive sample of 15,049 adult admissions from April 2009 to July 2010. INTERVENTIONS: MRSA surveillance performed by polymerase chain reaction (PCR) on samples collected from all adult patients (aged over 18 years) within 30 days before or upon an admission to the hospital. During a 9-month baseline period, targeted screening was conducted at both hospitals. During the 5-month intervention period, all patients admitted to the intervention hospital were screened for MRSA. Infection control measures were consistent at both hospitals. RESULTS: Universal screening was associated with an increase in admission screening of 43.58 percentage points (P< .01), an increase in MRSA detection of 2.95 percentage points (P< .01), and a small, nonsignificant decline in hospital-acquired MRSA infections of 0.12 percentage points (P< .01). The benefit-to-cost ratio was 0.50, indicating that for every dollar spent on universal versus targeted screening, only $0.50 is recovered in avoided costs of hospital-acquired MRSA infection. CONCLUSION: Compared with targeted screening, universal screening increased the rate of detection of MRSA upon hospital admission but did not significantly reduce the rate of hospital-acquired MRSA infection. Universal screening was associated with higher costs of care and was not cost beneficial.

Chitosan tissue scaffolds by emulsion templating.

A novel method for producing porous chitosan gels with controllable pore size and volume fraction has been developed. Complex-shaped 3D objects can be fabricated. Chitosan gels produced with the method are believed to be suitable candidates for tissue scaffolds. The method involves creating an oil-in-water emulsion in which the water phase contains chitosan and a temperature-activated cross-linking agent. The emulsion can then be poured or injected into a mould with the shape of the desired object. The chitosan is cross-linked by heating the emulsion to about 75 degrees C for about 15 min. The gelled object is then washed to remove the oil phase and surfactant. The gels were then dried in air, and further washed in ethanol. Scanning electron microscopy was then used to observe the pore size and fraction. The amount of porosity is directly proportional to the amount of oil phase. The pore size is controlled by the size of the oil droplets which is controlled primarily by the amount of surfactant added.

Delayed reimplantation arthroplasty for candidal prosthetic joint infection: a report of 4 cases and review of the literature.

Fungal prosthetic joint infection (PJI) is rare, with Candida species being the most frequently reported pathogen in the medical literature. The risk of relapse following delayed reimplantation arthroplasty for candidal PJI is unknown. We describe 4 new cases and summarize 6 previously reported cases of candidal PJI treated with delayed reimplantation arthroplasty. Ninety percent of the patients received antifungal therapy. Eight patients received amphotericin B either alone or in combination with other antifungals. One patient received fluconazole alone. The median duration of time from resection arthroplasty to reimplantation for total hip and total knee arthroplasties was 8.6 and 2.3 months, respectively. Eight patients did not have relapse of candidal PJI following delayed reimplantation arthroplasty after a median duration of follow-up of 50.7 months (range, 2--73 months). Candidal PJI can be successfully treated with delayed reimplantation arthroplasty after receipt appropriate antifungal therapy.

Lost bits: particle shedding with polyvinyl chloride intravenous administration sets.

Silicone particles have been demonstrated in the effluent from silicone intravenous (IV) tubing. It has been widely suspected that polyvinyl chloride (PVC) particles are also lost. We sought to clarify the situation in a carefully controlled laboratory setting using the apparatus and flow rates common in a paediatric setting, using scanning electron microscope techniques (SEM), we found that particles were indeed shed from IV tubing during use, but they were not PVC.