MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism.

BACKGROUND: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AIM: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. METHODS: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. RESULTS: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. CONCLUSIONS: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

Prevalence of lifestyle-related behavioral information in claims data in the U.S.

OBJECTIVE: Given their association with varying health risks, lifestyle-related behaviors are essential to consider in population-level disease prevention. Health insurance claims are a key source of information for population health analytics, but the availability of lifestyle information within claims data is unknown. Our goal was to assess the availability and prevalence of data items that describe lifestyle behaviors across several domains within a large U.S. claims database. METHODS: We conducted a retrospective, descriptive analysis to determine the availability of the following claims-derived lifestyle domains: nutrition, eating habits, physical activity, weight status, emotional wellness, sleep, tobacco use, and substance use. To define these domains, we applied a serial review process with three physicians to identify relevant diagnosis and procedure codes within claims for each domain. We used enrollment files and medical claims from a large national U.S. health plan to identify lifestyle relevant codes filed between 2016 and 2020. We calculated the annual prevalence of each claims-derived lifestyle domain and the proportion of patients by count within each domain. RESULTS: Approximately half of all members within the sample had claims information that identified at least one lifestyle domain (2016 = 41.9%; 2017 = 46.1%; 2018 = 49.6%; 2019 = 52.5%; 2020 = 50.6% of patients). Most commonly identified domains were weight status (19.9-30.7% across years), nutrition (13.3-17.8%), and tobacco use (7.9-9.8%). CONCLUSION: Our study demonstrates the feasibility of using claims data to identify key lifestyle behaviors. Additional research is needed to confirm the accuracy and validity of our approach and determine its use in population-level disease prevention.

Hyperinsulinemia Impairs Clathrin-Mediated Endocytosis of the Insulin Receptor and Activation of Endothelial Nitric Oxide Synthase in Brain Endothelial Cells.

Adequate perfusion of cerebral tissues, which is necessary for the preservation of optimal brain health, depends on insulin signaling within brain endothelial cells. Proper insulin signaling relies on the regulated internalization of insulin bound to the insulin receptor, a process which is disrupted by hyperinsulinemia via an unknown mechanism. Thus, the goal of this study was to characterize the impact of hyperinsulinemia on the regulation of molecular targets involved in cerebral blood flow and insulin receptor internalization into brain endothelial cells. The phosphorylation of molecular targets associated with cerebral blood flow and insulin receptor internalization was assessed in hyperinsulinemic brain endothelial cells. Insulin receptor uptake into cells was also examined in the setting of endocytosis blockade. Our data demonstrate that hyperinsulinemia impairs the activation of endothelial nitric oxide synthase. These data correspond with an impairment in clathrin-mediated endocytosis of the insulin receptor and dysregulated phosphorylation of key internalization effectors. We conclude that hyperinsulinemia alters the phosphorylation of molecular targets involved in clathrin-mediated endocytosis, disrupts signaling through the insulin receptor, and hinders the capacity for blood flow regulation by brain endothelial cells.

Society of General Internal Medicine Position Statement on Social Risk and Equity in Medicare's Mandatory Value-Based Payment Programs.

The Affordable Care Act (2010) and Medicare Access and CHIP Reauthorization Act (2015) ushered in a new era of Medicare value-based payment programs. Five major mandatory pay-for-performance programs have been implemented since 2012 with increasing positive and negative payment adjustments over time. A growing body of evidence indicates that these programs are inequitable and financially penalize safety-net systems and systems that care for a higher proportion of racial and ethnic minority patients. Payments from penalized systems are often redistributed to those with higher performance scores, which are predominantly better-financed, large, urban systems that serve less vulnerable patient populations - a "Reverse Robin Hood" effect. This inequity may be diminished by adjusting for social risk factors in payment policy. In this position statement, we review the literature evaluating equity across Medicare value-based payment programs, major policy reports evaluating the use of social risk data, and provide recommendations on behalf of the Society of General Internal Medicine regarding how to address social risk and unmet health-related social needs in these programs. Immediate recommendations include implementing peer grouping (stratification of healthcare systems by proportion of dual eligible Medicare/Medicaid patients served, and evaluation of performance and subsequent payment adjustments within strata) until optimal methods for accounting for social risk are defined. Short-term recommendations include using census-based, area-level indices to account for neighborhood-level social risk, and developing standardized approaches to collecting individual socioeconomic data in a robust but sensitive way. Long-term recommendations include implementing a research agenda to evaluate best practices for accounting for social risk, developing validated health equity specific measures of care, and creating policies to better integrate healthcare and social services.

On the use of stereodynamical effects to control cold chemical reactions: The H + D2 ⟷ D + HD case study.

Quantum calculations are reported for the stereodynamic control of the H + D2 ↔ D + HD chemical reaction in the energy range of 1-50 K. Stereodynamic control is achieved by a formalism similar to that reported by Perreault et al. [Nat. Chem. 10, 561 (2018)] in recent experimental works in which the alignment of the molecular bond axis relative to the incident relative velocity is controlled by selective preparations of the molecule in a specific or superposition of magnetic projection quantum numbers of the initial molecular rotational level. The approach presented here generalizes the experimental scheme of Perreault et al. and offers additional degree of control through various experimental preparations of the molecular alignment angle. Illustrative results presented for the H + D2 and D + HD reactions show significant control with the possibility of turning the reaction completely on or off with the appropriate stereodynamic preparation of the molecular state. Various scenarios for maximizing and minimizing the reaction outcomes are identified with the selective preparation of molecular rotational states.

Optimization of Heavy Metal Sensors Based on Transcription Factors and Cell-Free Expression Systems.

Many bacterial mechanisms for highly specific and sensitive detection of heavy metals and other hazards have been reengineered to serve as sensors. In some cases, these sensors have been implemented in cell-free expression systems, enabling easier design optimization and deployment in low-resource settings through lyophilization. Here, we apply the advantages of cell-free expression systems to optimize sensors based on three separate bacterial response mechanisms for arsenic, cadmium, and mercury. We achieved detection limits below the World Health Organization-recommended levels for arsenic and mercury and below the short-term US Military Exposure Guideline levels for all three. The optimization of each sensor was approached differently, leading to observations useful for the development of future sensors: (1) there can be a strong dependence of specificity on the particular cell-free expression system used, (2) tuning of relative concentrations of the sensing and reporter elements improves sensitivity, and (3) sensor performance can vary significantly with linear vs plasmid DNA. In addition, we show that simply combining DNA for the three sensors into a single reaction enables detection of each target heavy metal without any further optimization. This combined approach could lead to sensors that detect a range of hazards at once, such as a panel of water contaminants or all known variants of a target virus. For low-resource settings, such "all-hazard" sensors in a cheap, easy-to-use format could have high utility.

Five-year results of a randomised controlled trial comparing cemented and cementless Oxford unicompartmental knee replacement using radiostereometric analysis.

BACKGROUND: Cementless fixation is an alternative to cemented unicompartmental knee replacement (UKR). The aim of this study was to determine if cementless UKR fixation is as good as cemented by comparing the five-year migration measured radiostereometric analysis (RSA) in a randomised controlled trial. METHODS: Thirty-nine patients were randomised to receive either a cemented or a cementless Oxford UKR and were studied at intervals up to five years to assess migration with RSA and radiolucencies with radiographs. RESULTS: During the first year there was a small and significant amount of migration, predominantly in an anterior direction, of both the cemented (0.24 mm, SD 0.32, p = 0.01) and cementless (0.26 mm, SD 0.31, p = 0.00) femoral components. Thereafter there was no significant migration in any direction. At no stage was there any significant difference between the migrations of the cemented or cementless femoral components. During the first year, particularly the first three months, the cementless tibial components subsided 0.28 mm (SD 0.19, p = 0.00). This was significantly (p = 0.00) greater than the subsidence of the cemented tibial component (0.09, SD 0.19, p = 0.28). Between the second and fifth years there was no significant migration of either cemented or cementless tibial components. At five years radiolucent lines occurred significantly less with cementless (one partial) compared to cemented (six partial and one complete) tibial components. CONCLUSIONS: As, between two and five years, there was no significant migration of cemented or cementless components, and no significant difference between them, we conclude that cementless fixation is as reliable as cemented. It may be better as there are fewer radiolucent lines.

Prediction of a Feshbach Resonance in the Below-the-Barrier Reactive Scattering of Vibrationally Excited HD with H.

Quantum reactive scattering calculations on the vibrational quenching of HD due to collisions with H were carried out employing an accurate potential energy surface. The state-to-state cross sections for the chemical reaction HD(v = 1, j = 0) + H → D + H2(v' = 0, j') at collision energies between 1 and 10 000 cm-1 are presented, and a Feshbach resonance in the low-energy regime, below the reaction barrier, is observed for the first time. The resonance is attributed to coupling with the vibrationally adiabatic potential correlating to the v = 1, j = 1 level of the HD molecule, and it is dominated by the contribution from a single partial wave. The properties of the resonance, such as its dynamic behavior, phase behavior, and lifetime, are discussed.

Peri-operative administration of tranexamic acid in lower limb arthroplasty: a multicentre, prospective cohort study.

In the UK, tranexamic acid is recommended for all surgical procedures where expected blood loss exceeds 500 ml. However, the optimal dose, route and timing of administration are not known. This study aimed to evaluate current practice of peri-operative tranexamic acid administration. Patients undergoing primary total hip arthroplasty, total knee arthroplasty or unicompartmental knee arthroplasty during a 2-week period were eligible for inclusion in this prospective study. The primary outcome was the proportion of patients receiving tranexamic acid in the peri-operative period. Secondary outcomes included: dose, route and timing of tranexamic acid administration; prevalence of pre- and postoperative anaemia; estimated blood loss; and red blood cell transfusion rates. In total, we recruited 1701 patients from 56 NHS hospitals. Out of these, 1523 (89.5%) patients received tranexamic acid and of those, 1052 (69.1%) received a single dose of 1000 mg intravenously either pre- or intra-operatively. Out of the 1701 patients, 571 (33.6%) and 1386 (81.5%) patients were anaemic (haemoglobin < 130 g.l-1 ) in the pre- and postoperative period, respectively. Mean (SD) estimated blood loss for all included patients was 792 (453) ml and 54 patients (3.1%) received a red blood cell transfusion postoperatively. The transfusion rate for patients with pre-operative anaemia was 6.5%, compared with 1.5% in patients without anaemia. Current standard of care in the UK is to administer 1000 mg of tranexamic intravenously either pre- or intra-operatively. Approximately one-third of patients present for surgery with anaemia, although the overall red blood cell transfusion rate is low. These data provide useful comparators when assessing the efficacy of tranexamic acid and other patient blood management interventions in future studies.

Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery.

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.

The role of intra-operative cell salvage in patient blood management for revision hip arthroplasty: a prospective cohort study.

Cell salvage is an important component of blood management in patients undergoing revision hip arthroplasty surgery. However concerns regarding efficacy and patient selection remain. The aims of this study were to describe intra-operative blood loss, cell salvage re-infusion volumes and red blood cell transfusion rates for revision hip procedures and to identify factors associated with the ability to salvage sufficient blood intra-operatively to permit processing and re-infusion. Data were collected from a prospective cohort of 664 consecutive patients undergoing revision hip surgery at a single tertiary centre from 31 March 2015 to 1 April 2018. Indications for revision surgery were aseptic (n = 393 (59%)) fracture (n = 160 (24%)) and infection (n = 111 (17%)). Salvaged blood was processed and re-infused when blood loss exceeded 500 ml. Mean (SD) intra-operative blood loss was 1038 (778) ml across all procedures. Salvaged blood was re-infused in 505 of 664 (76%) patients. Mean (SD) re-infusion volume was 253 (169) ml. In total, 246 of 664 (37%) patients received an allogeneic red blood cell transfusion within 72 h of surgery. Patients undergoing femoral component revision only (OR (95%CI) 0.41 (0.23-0.73)) or acetabular component revision only (0.53 (0.32-0.87)) were less likely to generate sufficient blood salvage volume for re-infusion compared with revision of both components. Compared with aseptic indications, patients undergoing revision surgery for infection (1.87 (1.04-3.36)) or fracture (4.43 (2.30-8.55)) were more likely to generate sufficient blood salvage volume for re-infusion. Our data suggest that cell salvage is efficacious in this population. Cases where the indication is infection or fracture and where both femoral and acetabular components are to be revised should be prioritised.

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle.

Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway's kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.

Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles.

The sequence fitness of a llama single-domain antibody with an unusually high thermal stability is explored by a combined computational and experimental study. Starting with the X-ray crystallographic structure, RosettaBackrub simulations were applied to model sequence-structure tolerance profiles and identify key substitution sites. From the model calculations, an experimental site-directed mutagenesis was used to produce a panel of mutants, and their melting temperatures were determined by thermal denaturation. The results reveal a sequence fitness of an excess stability of approximately 12 °C, a value taken from a decrease in the melting temperature of an electrostatic charge-reversal substitution in the CRD3 without a deleterious effect on the binding affinity to the antigen. The tolerance for the disruption of antigen recognition without loss in the thermal stability was demonstrated by the introduction of a proline in place of a tyrosine in the CDR2, producing a mutant that eliminated binding. To further assist the sequence design and the selection of engineered single-domain antibodies, an assessment of different computational strategies is provided of their accuracy in the detection of substitution "hot spots" in the sequence tolerance landscape.

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability.

Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.

Bacterial bioreactors: Outer membrane vesicles for enzyme encapsulation.

Bacterial membrane vesicles, whether naturally occurring or engineered for enhanced functionality, have significant potential as tools for bioremediation, enzyme catalysis, and the development of therapeutics such as vaccines and adjuvants. In many instances, the vesicles themselves and the naturally occurring proteins are sufficient to lend functionality. Alternatively, additional function can be conveyed to these biological nanoparticles through the directed packaging of peptides and proteins, specifically recombinant enzymes chosen to mediate a specific reaction or facilitate a controlled response. Here we will detail mechanisms for directing the packaging of recombinant proteins and peptides into the nascent membrane vesicles (MVs) of Gram-negative bacteria with a focus on both active and passive packaging using both cellular machinery and engineered molecular systems. Additionally, we detail some of the more common methods for bacterial MVs purification, quantitation, and characterization as these methods are requisite for any subsequent experimentation or processing of MV reagents.

Primary stability of a short bone-conserving femoral stem: a two-year randomized controlled trial using radiostereometric analysis.

Aims: The aim of this study was to determine the stability of a new short femoral stem compared with a conventional femoral stem in patients undergoing cementless total hip arthroplasty (THA), in a prospective randomized controlled trial using radiostereometric analysis (RSA). Patients and Methods: A total of 53 patients were randomized to receive cementless THA with either a short femoral stem (MiniHip, 26 patients, mean age: 52 years, nine male) or a conventional length femoral stem (MetaFix, 23 patients, mean age: 53 years, 11 male). All patients received the same cementless acetabular component. Two-year follow-up was available on 38 patients. Stability was assessed through migration and dynamically inducible micromotion. Radiographs for RSA were taken postoperatively and at three, six, 12, 18, and 24 months. Results: At two years, there was significantly less subsidence (inferior migration) of the short femoral stem (head, 0.26 mm, 95% confidence interval (CI) 0.08 to 0.43, sd 0.38; tip, 0.11 mm, 95% CI -0.08 to 0.31, sd 0.42) compared with the conventional stem (head, 0.62 mm, 95% CI 0.34 to 0.90, sd 0.56, p = 0.02; tip, 0.43 mm, 95% CI 0.21 to 0.65, sd 0.44, p = 0.03). There was no significant difference in dynamically inducible micromotion, rate of complications or functional outcome. Conclusion: This study demonstrates that the short femoral stem has a stable and predictable migration. However, longer-term survival analysis still needs to be determined. Cite this article: Bone Joint J 2018;100-B:1148-56.

Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase.

While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.

Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.

Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.

Symmetry and the geometric phase in ultracold hydrogen-exchange reactions.

Quantum reactive scattering calculations are reported for the ultracold hydrogen-exchange reaction and its non-reactive atom-exchange isotopic counterparts, proceeding from excited rotational states. It is shown that while the geometric phase (GP) does not necessarily control the reaction to all final states, one can always find final states where it does. For the isotopic counterpart reactions, these states can be used to make a measurement of the GP effect by separately measuring the even and odd symmetry contributions, which experimentally requires nuclear-spin final-state resolution. This follows from symmetry considerations that make the even and odd identical-particle exchange symmetry wavefunctions which include the GP locally equivalent to the opposite symmetry wavefunctions which do not. It is shown how this equivalence can be used to define a constant which quantifies the GP effect and can be obtained solely from experimentally observable rates. This equivalence reflects the important role that discrete symmetries play in ultracold chemistry and highlights the key role that ultracold reactions can play in understanding fundamental aspects of chemical reactivity more generally.