Bipolar Clark-Type Oxygen Electrode Arrays for Imaging and Multiplexed Measurements of the Respiratory Activity of Cells.

Various miniature Clark-type oxygen electrodes (COEs), which are typically used to measure dissolved oxygen (DO) concentration in cellular respiration, have been developed since the 1980s. Arrays with individually addressable electrodes that constitute the sensor were used for various applications. However, the large number of leads and contact pads required for connecting the electrodes and the external instrument complicate the electrode layout and make the operation of integrated COE arrays challenging. Here, we fabricated closed bipolar electrochemical systems comprising 6 × 8 and 4 × 4 arrays of COEs for imaging and multiplexed detection. The cathodic compartment was sealed with a hydrophobic oxygen-permeable membrane to separate the internal electrolyte solution from the sample solutions. Using the bipolar Clark-type oxygen electrode (BCOE) arrays and electrochemiluminescence (ECL), we measured the DO concentration at each cathode. The results revealed that the ECL intensity changed linearly with the DO concentration. In addition, we used ECL imaging to investigate the respiratory activity of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in suspensions with different cell densities. The ECL images showed that the ECL intensity changed noticeably with the bacterial density. The bacterial respiratory activity was then qualitatively analyzed based on the ECL images acquired successively over a time duration. Further, we measured the antibiotic efficacy of piperacillin, oxacillin, gentamicin, and cefmetazole against E. coli and P. aeruginosa using the BCOE. We found that the ECL intensity increased with the antibiotic concentration, thus indicating the suppression of the bacterial respiratory activity.

l-Carnitine pretreatment ameliorates heat stress-induced acute kidney injury by restoring mitochondrial function of tubular cells.

A major complication of heat-related illness is the development of acute kidney injury (AKI) and damage to kidney tubular cells. Because kidney tubular cells use fatty acids as a major energy source, impaired fatty acid oxidation (FAO) may be associated with kidney injury due to heat stress. Carnitine is essential in the transportation of fatty acid into mitochondria for FAO. To date, there has been little attention given to the role of carnitine in heat-related illness and AKI. To evaluate the relationship between carnitine inadequacy and heat-related illness severity or AKI, we examined serum carnitine levels in patients with heat-related illness. We also used heat-stressed mice to investigate the effect of l-carnitine pretreatment on various kidney functions such as mitochondrial activity, proinflammatory changes in kidney macrophages, and histological damage. We observed an elevation in serum acylcarnitine levels, indicating carnitine insufficiency in patients with severe heat-related illness and/or AKI. l-Carnitine pretreatment ameliorated ATP production in murine tubular cell mitochondria and prevented a change in the kidney macrophage population dynamics observed in AKI: a decrease in tissue-resident macrophages, influx of bone marrow-derived macrophages, and change toward proinflammatory M1 polarization. In conclusion, carnitine insufficiency may be closely associated with severe heat-related illness and related AKI. Enhancement of the FAO pathway by l-carnitine pretreatment may prevent heat stress-induced AKI by restoring mitochondrial function.NEW & NOTEWORTHY Enhancing fatty acid oxidation (FAO) after acute kidney injury (AKI) improves renal outcomes. This report shows that carnitine insufficiency, which could inhibit FAO, correlates to severe heat-related illness and AKI in a clinical study. We also demonstrate that administering l-carnitine to mice improves mitochondrial respiratory function and prevents deleterious changes in renal macrophage, resulting in improved renal outcomes of heat-induced AKI. l-Carnitine may be an effective preventive treatment for severe heat-related illness and related AKI.

Novel phenotypical and functional sub-classification of liver macrophages highlights changes in population dynamics in experimental mouse models.

Liver macrophages are critical components of systemic immune system defense mechanisms. F4/80high Kupffer cells (KCs) are the predominant liver-resident macrophages and the first immune cells to contact pathogens entering the liver. F4/80low monocyte-derived macrophages (MoMφs) are essential macrophages that modulate liver immune functions. Here we report a novel method of identifying subpopulations of these two populations using traditional flow cytometry and examine each subpopulation for its putative roles in the pathogenesis of an experimental non-alcoholic steatohepatitis model. Using male C57BL/6 mice, we isolated and analyzed liver non-parenchymal cells by flow cytometry. We identified F4/80high and F4/80low macrophage populations and characterized subpopulations using uniform manifold approximation and projection. We identified three subpopulations in F4/80high macrophages: CD163(+) KCs, CD163(-) KCs, and liver capsular macrophages. CD163(+) KCs had higher phagocytic and bactericidal activities and more complex cellular structures than CD163(-) KCs. We also identified four subpopulations of F4/80low MoMφs based on Ly6C and MHC class II expression: infiltrating monocytes, pro-inflammatory MoMφs, Ly6C(-) monocytes, and conventional dendritic cells. CCR2 knock-out mice expressed lower levels of these monocyte-derived cells, and the count varied by subpopulation. In high-fat- and cholesterol-diet-fed mice, only one subpopulation, pro-inflammatory MoMφs, significantly increased in count. This indicates that changes to this subpopulation is the first step in the progression to non-alcoholic steatohepatitis. The community can use our novel subpopulation and gating strategy to better understand complex immunological mechanisms in various liver disorders through detailed analysis of these subpopulations.

In vitro study on the effect of fibrinogen γ-chain peptide-coated ADP-encapsulated liposomes on postcardiopulmonary bypass coagulopathy using patient blood.

BACKGROUND: Fibrinogen γ-chain peptide-coated, adenosine 5'-diphosphate (ADP)-encapsulated liposomes (H12-ADP-liposomes) are potent hemostatic adjuvants that promote platelet thrombi formation at bleeding sites. Although we have reported the efficacy of these liposomes in a rabbit model of cardiopulmonary bypass coagulopathy, we are yet to address the possibility of their hypercoagulative potential, especially in human beings. OBJECTIVES: Considering its future clinical applications, we herein investigated the safety of using H12-ADP-liposomes in vitro using blood samples from patients who had received platelet transfusion after cardiopulmonary bypass surgeries. METHODS: Ten patients receiving platelet transfusions after cardiopulmonary bypass surgery were enrolled. Blood samples were collected at the following 3 points: at the time of incision, at the end of the cardiopulmonary bypass, and immediately after platelet transfusion. After incubating the samples with H12-ADP-liposomes or phosphate-buffered saline (PBS, as a control), blood coagulation, platelet activation, and platelet-leukocyte aggregate formation were evaluated. RESULTS: Patients' blood incubated with H12-ADP-liposomes did not differ from that incubated with PBS in coagulation ability, degree of platelet activation, and platelet-leukocyte aggregation at any of the time points. CONCLUSION: H12-ADP-liposomes did not cause abnormal coagulation, platelet activation, or platelet-leukocyte aggregation in the blood of patients who received platelet transfusion after a cardiopulmonary bypass. These results suggest that H12-ADP-liposomes could likely be safely used in these patients, providing hemostasis at the bleeding sites without causing considerable adverse reactions. Future studies are needed to ensure robust safety in human beings.

Early treatment with C-reactive protein-derived peptide reduces septic acute kidney injury in mice via controlled activation of kidney macrophages.

The mortality rate for acute kidney injury (AKI) due to sepsis remains high, and effective therapies based on its pathogenesis remain elusive. Macrophages are crucial for clearing bacteria from vital organs, including the kidney, under septic conditions. Excessive macrophage activation results in organ injury. C-reactive protein (CRP) peptide (174-185), a functional product of proteolyzed CRP in vivo, effectively activates macrophages. We investigated the therapeutic efficacy of synthetic CRP peptide on septic AKI, focusing on effects on kidney macrophages. Mice underwent cecal ligation and puncture (CLP) to induce septic AKI and were intraperitoneally administered 20 mg/kg of synthetic CRP peptide 1 h post-CLP. Early CRP peptide treatment improved AKI while still clearing infection. Ly6C-negative kidney tissue-resident macrophages did not significantly increase at 3 h after CLP, while Ly6C-positive monocyte-derived macrophages significantly accumulated in the kidney 3 h post-CLP. CRP peptide augmented the phagocytic ROS production in both subtypes of kidney macrophage at 3 h. Interestingly, both subtypes of macrophage increased ROS production 24 h post-CLP compared to the control group, while CRP peptide treatment maintained ROS production at the same level seen 3 h post-CLP. Although bacterium-phagocytic kidney macrophages produced TNF-α, CRP peptide reduced bacterial propagation and tissue TNF-α levels in the septic kidney at 24 h. Although both subsets of kidney macrophages showed populations of M1 at 24 h post-CLP, CRP peptide therapy skewed the macrophages population toward M2 at 24 h. CRP peptide alleviated murine septic AKI via the controlled activation of kidney macrophages and is an excellent candidate for future human therapeutic studies.

Implementing Pool-Based Surveillance Testing for SARS-CoV-2 at the Army Public Health Center Laboratory and across the Army Public Health Laboratory Enterprise.

With limited clinical resources, burgeoning testing requests from Army and other Service units to clinical laboratories, and the continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the military population, the Army Public Health Laboratory (APHL) Enterprise was tasked to establish surveillance testing capabilities for active duty military populations in an expedient manner. Following a proof-of-concept study conducted by Public Health Command-Pacific, Public Health Command-Europe was the first public health laboratory to offer the capability to assess for SARS-CoV-2 in pooled samples, followed closely by the Army Public Health Center (APHC) at Aberdeen Proving Grounds, MD, paralleling the spread of the SARS-CoV-2 virus from China to Europe to the continental US. The APHLs have selected pool sizes of up to 10 samples per pool based on the best evidence available at the time of method development and validation. Real-Time quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) assays using RNA extracts from pooled nasopharyngeal swabs preserved in viral transport media were selected to assess the presence of SARS-CoV-2. The rapid development of initial surveillance testing capabilities depended on existing equipment in each laboratory, with a plan to implement full operational capability using additional staff and common high-throughput platforms. APHL Enterprise has successfully used existing resources to begin to address the changing and complex needs for COVID-19 testing within the Army population. Successful implementation of pooled surveillance testing at the APHC Laboratory has enabled more than 8,600 Soldiers to avoid clinical testing to date. The APHC Laboratory alone has tested over 10,000 samples and prevented approximately 8,600 soldiers from seeking testing with clinical diagnostic assays.

DRoP: Automated detection of conserved solvent-binding sites on proteins.

Water and ligand binding play critical roles in the structure and function of proteins, yet their binding sites and significance are difficult to predict a priori. Multiple solvent crystal structures (MSCS) is a method where several X-ray crystal structures are solved, each in a unique solvent environment, with organic molecules that serve as probes of the protein surface for sites evolved to bind ligands, while the first hydration shell is essentially maintained. When superimposed, these structures contain a vast amount of information regarding hot spots of protein-protein or protein-ligand interactions, as well as conserved water-binding sites retained with the change in solvent properties. Optimized mining of this information requires reliable structural data and a consistent, objective analysis tool. Detection of related solvent positions (DRoP) was developed to automatically organize and rank the water or small organic molecule binding sites within a given set of structures. It is a flexible tool that can also be used in conserved water analysis given multiple structures of any protein independent of the MSCS method. The DRoP output is an HTML format list of the solvent sites ordered by conservation rank in its population within the set of structures, along with renumbered and recolored PDB files for visualization and facile analysis. Here, we present a previously unpublished set of MSCS structures of bovine pancreatic ribonuclease A (RNase A) and use it together with published structures to illustrate the capabilities of DRoP.

Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD.

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.

Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV.

A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.

Assembly and maturation of a T = 4 quasi-equivalent virus is guided by electrostatic and mechanical forces.

Nudaurelia capensis w virus (NωV) is a eukaryotic RNA virus that is well suited for the study of virus maturation. The virus initially assembles at pH 7.6 into a marginally stable 480-Å procapsid formed by 240 copies of a single type of protein subunit. During maturation, which occurs during apoptosis at pH 5.0, electrostatic forces guide subunit trajectories into a robust 410-Å virion that is buttressed by subunit associated molecular switches. We discuss the competing factors in the virus capsid of requiring near-reversible interactions during initial assembly to avoid kinetic traps, while requiring robust stability to survive in the extra-cellular environment. In addition, viruses have a variety of mechanisms to deliver the genome, which must remain off while still inside the infected cell, yet turn on under the proper conditions of infection. We conclude that maturation is the process that provides a solution to these conflicting requirements through a program that is encoded in the procapsid and that leads to stability and infectivity.

Dynamic and geometric analyses of Nudaurelia capensis ω virus maturation reveal the energy landscape of particle transitions.

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T = 4, eukaryotic, single-stranded ribonucleic acid virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diameter = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed maximum likelihood variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e., uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly two to four times the variance of the first two particles. Without maturation cleavage, the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3 Å while the mature particle had an RMSD of 11 Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity.

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras.

Analysis of binding site hot spots on the surface of Ras GTPase.

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the "off" and "on" allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.