Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery.

Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

The ability of this pH-sensitive reporter platform to rapidly screen ligands in nanoparticle format will enable identification and production of targeted NPs with desired lysosome trafficking properties.

Learning from Power Signals: An Automated Approach to Electrical Disturbance Identification within a Power Transmission System.

As power quality becomes a higher priority in the electric utility industry, the amount of disturbance event data continues to grow. Utilities do not have the required personnel to analyze each event by hand. This work presents an automated approach for analyzing power quality events recorded by digital fault recorders and power quality monitors operating within a power transmission system. The automated approach leverages rule-based analytics to examine the time and frequency domain characteristics of the voltage and current signals. Customizable thresholds are set to categorize each disturbance event. The events analyzed within this work include various faults, motor starting, and incipient instrument transformer failure. Analytics for fourteen different event types have been developed. The analytics were tested on 160 signal files and yielded an average accuracy of 99%. Continuous nominal signal data analysis was performed using an approach called the cyclic histogram. The cyclic histogram process is intended to be integrated into the digital fault recorders themselves in order to facilitate the detection of subtle signal variations that are too small to trigger a disturbance event and that can occur over hours or days. In addition to reducing memory requirements by a factor of 320, it is anticipated that cyclic histogram processing will aid in identifying incipient events and identifiers. This project is expected to save engineers time by automating the classification of disturbance events and increasing the reliability of the transmission system by providing near real-time detection and identification of disturbances as well as prevention of problems before they occur.

Optical properties of metasurfaces infiltrated with liquid crystals.

Optical metasurfaces allow the ability to precisely manipulate the wavefront of light, creating many interesting and exotic optical phenomena. However, they generally lack dynamic control over their optical properties and are limited to passive optical elements. In this work, we report the nontrivial infiltration of nanostructured metalenses with three respective nematic liquid crystals of different refractive index and birefringence. The optical properties of the metalens are evaluated after liquid-crystal infiltration to quantify its effect on the intended optical design. We observe a significant modification of the metalens focus after infiltration for each liquid crystal. These optical changes result from modification of local refractive index surrounding the metalens structure after infiltration. We report qualitative agreement of the optical experiments with finite-difference time-domain solver (FDTD) simulation results. By harnessing the tunability inherent in the orientation dependent refractive index of the infiltrated liquid crystal, the metalens system considered here has the potential to enable dynamic reconfigurability in metasurfaces.

Novel mechanisms of action contributing to benralizumab's potent anti-eosinophilic activity.

BACKGROUND: Benralizumab is a humanised, anti-interleukin-5 receptor α monoclonal antibody with anti-eosinophilic activity. Lack of fucose (afucosylation) increases its affinity to CD16a and significantly enhances antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells. Although benralizumab proved clinically efficacious in clinical trials for patients with severe asthma and hypereosinophilic syndrome, in-depth characterisation of its anti-eosinophilic mechanisms of action remains elusive. METHODS: Here, we further investigated the mechanisms involved in benralizumab's anti-eosinophilic activities by employing relevant primary human autologous cell co-cultures and real-time-lapse imaging combined with flow cytometry. RESULTS: In the presence of NK cells, benralizumab induced potent eosinophil apoptosis as demonstrated by the upstream induction of Caspase-3/7 and upregulation of cytochrome c. In addition, we uncovered a previously unrecognised mechanism whereby benralizumab can induce eosinophil phagocytosis/efferocytosis by macrophages, a process called antibody-dependent cellular phagocytosis. Using live cell imaging, we unravelled the stepwise processes leading to eosinophil apoptosis and uptake by activated macrophages. Through careful observations of cellular co-culture assays, we identified a novel role for macrophage-derived tumour necrosis factor (TNF) to further enhance benralizumab-mediated eosinophil apoptosis through activation of TNF receptor 1 on eosinophils. TNF-induced eosinophil apoptosis was associated with cytochrome c upregulation, mitochondrial membrane depolarisation and increased Caspase-3/7 activity. Moreover, activated NK cells were found to amplify this axis through the secretion of interferon-γ, subsequently driving TNF expression by macrophages. CONCLUSIONS: Our data provide deeper insights into the timely appearance of events leading to benralizumab-induced eosinophil apoptosis and suggest that additional mechanisms may contribute to the potent anti-eosinophilic activity of benralizumab in vivo. Importantly, afucosylation of benralizumab strongly enhanced its potency for all mechanisms investigated.

Short-term effects of electronic cigarettes on cerebrovascular function: A time course study.

NEW FINDINGS: What is the central question of this study? Acute exposure to electronic cigarettes (Ecigs) triggers abnormal vascular responses in systemic arteries; however, effects on cerebral vessels are poorly understood and time for recovery is not known. We hypothesized that exposure to cigarettes or Ecigs would trigger rapid (<4 h) impairment of the middle cerebral artery (MCA) but that this would resolve by 24 h. What is the main finding and its importance? Cigarettes and Ecigs caused similar degree and duration of MCA impairment. We find it takes ~72 hours after exposure for MCA function to return to normal. This suggests that Ecig use is likely to produce similar adverse vascular health outcomes to those seen with cigarette smoke. ABSTRACT: Temporal influences of electronic cigarettes (Ecigs) on blood vessels are poorly understood. In this study, we evaluated a single episode of cigarette versus Ecig exposure on middle cerebral artery (MCA) reactivity and determined how long after the exposure MCA responses took to return to normal. We hypothesized that cigarette and Ecig exposure would induce rapid (<4 h) reduction in MCA endothelial function and would resolve within 24 h. Sprague-Dawley rats (4 months old) were exposed to either air (n = 5), traditional cigarettes (20 puffs, n = 16) or Ecigs (20-puff group, n = 16; or 60-puff group, n = 12). Thereafter, the cigarette and Ecig groups were randomly assigned for postexposure vessel myography testing on day 0 (D0, 1-4 h postexposure), day 1 (D1, 24-28 h postexposure), day 2 (D2, 48-52 h postexposure) and day 3 (72-76 h postexposure). The greatest effect on endothelium-dependent dilatation was observed within 24 h of exposure (∼50% decline between D0 and D1) for both cigarette and Ecig groups, and impairment persisted with all groups for up to 3 days. Changes in endothelium-independent dilatation responses were less severe (∼27%) and shorter lived (recovering by D2) compared with endothelium-dependent dilatation responses. Vasoconstriction in response to serotonin (5-HT) was similar to endothelium-independent dilatation, with greatest impairment (∼45% for all exposure groups) at D0-D1, returning to normal by D2. These data show that exposure to cigarettes and Ecigs triggers a similar level/duration of cerebrovascular dysfunction after a single exposure. The finding that Ecig (without nicotine) and cigarette (with nicotine) exposure produce the same effects suggesting that nicotine is not likely to be triggering MCA dysfunction, and that vaping (with/without nicotine) has potential to produce the same vascular harm and/or disease as smoking.

Antibacterial Monoclonal Antibodies Do Not Disrupt the Intestinal Microbiome or Its Function.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Localized cytokine responses to total knee arthroplasty and total knee revision complications.

BACKGROUND: The study of localized immune-related factors has proven beneficial for a variety of conditions, and one area of interest in the field of orthopaedics is the impact of implants and localized infections on immune response. Several cytokines have shown increased systemic concentrations (in serum/plasma) in response to implants and infection, but tissue-level cytokines have not been investigated as thoroughly. METHODS: This exploratory study investigated tissue-level cytokines in a cohort of patients (N = 17) in response to total knee arthroplasty and total knee revision to better understand the immune response to implants and localized infection (e.g., prosthetic joint infection). The overall goal of this study was to provide insight into the localized cytokine response of tissues and identify tissue-level markers specific to inflammation caused by implants vs. inflammation caused by infection. Tissues were collected across several anatomical locations and assayed with a panel of 20 human inflammatory cytokines to understand spatial differences in cytokine levels. RESULTS: In this study, six cytokines were elevated in implanted joints, as compared to native joints: IL-10, IL-12p70, IL-13, IL-17A, IL-4, and TNF-α (p < 0.05). Seven cytokines showed infection-dependent increases in localized tissues: IL-1α, IL-1ß, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1ß (p < 0.05). CONCLUSIONS: This study demonstrated that differences exist in tissue-level cytokines in response to presence of implant, and some cytokines were specifically elevated for infection; these responses may be informative of overall tissue health. These results highlight the utility of investigating localized cytokine concentrations to offer novel insights for total knee arthroplasty and total knee revision procedures, as well as their complications. Ultimately, this information could provide additional, quantitative measurements of tissue to aid clinical decision making and patient treatment options.

Metabolite-Based Modification of Poly(l-lysine) for Improved Gene Delivery.

Synthetic gene delivery systems employ multiple functions to enable safe and effective transport of DNA to target cells. Here, we describe metabolite-based poly(l-lysine) (PLL) modifiers that improve transfection by imparting both pH buffering and nanoparticle stabilization functions within a single molecular unit. PLL modifiers were based on morpholine (M), morpholine and niacin (MN), or thiomorpholine (TM). PLL modification with (MN) or (TM) imparted buffering function over the pH range of 5-7 both in solution and live cells and enhanced the stability of PLL DNA nanoparticles, which exhibited higher resistance to polyanion exchange and prolonged blood circulation. These properties translated into increased transfection efficiency in vitro coupled with reduced toxicity compared to unmodified PLL and PLL(M). Furthermore, PEG-PLL(MN) DNA nanoparticles transfected muscle tissue in vivo for >45 days following intramuscular injection. These polymer modifiers demonstrate the successful design of multifunctional units that improve transfection of synthetic gene delivery systems while maintaining biocompatibility.

The impact of cytokine responses in the intra- and extracellular signaling network of a traumatic injury.

Investigations of cellular responses involved in injury and repair processes have generated valuable information contributing to the advancement of wound healing and treatments. Intra- and extracellular regulators of healing mechanisms, such as cytokines, signaling proteins, and growth factors, have been described to possess significant roles in facilitating optimal recovery. This study explored a collection of 30 spatiotemporal responses comprised of cytokines (IL-1α, IL-1ß, IL-2, IL-6, TNF-α, MIP-1α), intracellular proteins (Akt, c-Jun, CREB, ERK1/2, JNK, MEK1, p38, p53, p90RSK), phosphorylated proteins (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-GSK-3α/ß, p-HSP27, p-IκBα, p-JNK, p-MEK1, p-p38, p-p70S6K, p-p90RSK, p-STAT2, p-STAT3), and a protease (Caspase-3), measured in skeletal muscle tissue following a traumatic injury (rodent Gustilo IIIB fracture). To optimize the analysis of context-specific data sets, a network centrality parameter approach was used to assess the impact of each response in relation to all other measured responses. This approach identified proteins that were substantially amplified and potentially central in the wound healing network by evaluation of their corresponding centrality parameter, radiality. Network analysis allowed us to distinguish the progression of healing that occurred at certain time points and regions of injury. Notably, new tissue formation was proposed to occur by 168 h post-injury in severely injured tissue, while tissue 1-cm away from the site of injury that experienced relatively minor injury appeared to exhibit signs of new tissue formation as early as 24 h post-injury. In particular, hallmarks of inflammation, cytokines IL-1ß, IL-6, and IL-2, appear to have a pronounced impact at earlier time points (0-24 h post-injury), while intracellular proteins involved in cell proliferation, differentiation, or proteolysis (c-Jun, CREB, JNK, p38, p-c-Jun; p-MEK1, p-p38, p-STAT3) are more significant at later times (24-168 h). Overall, this study demonstrates the feasibility of a network analysis approach to extract significant information and also offers a spatiotemporal visualization of the intra- and extracellular signaling responses that regulate healing mechanisms.

In vitro cytotoxicity assessment of a West Virginia chemical spill mixture involving 4-methylcyclohexanemethanol and propylene glycol phenyl ether.

Thousands of gallons of industrial chemicals, crude 4-methylcyclohexanemethanol (MCHM) and propylene glycol phenyl ether (PPh), leaked from industrial tanks into the Elk River in Charleston, West Virginia, USA, on January 9, 2014. A considerable number of people were reported to exhibit symptoms of chemical exposure and an estimated 300,000 residents were advised not to use or drink tap water. At the time of the spill, the existing toxicological data of the chemicals were limited for a full evaluation of the health risks, resulting in concern among those in the impacted regions. In this preliminary study, we assessed cell viability and plasma membrane degradation following a 24-h exposure to varying concentrations (0-1000 µM) of the two compounds, alone and in combination. Evaluation of different cell lines, HEK-293 (kidney), HepG2 (liver), H9c2 (heart), and GT1-7 (brain), provided insight regarding altered cellular responses in varying organ systems. Single exposure to MCHM or PPh did not affect cell viability, except at doses much higher than the estimated exposure levels. Certain co-exposures significantly reduced metabolic activity and increased plasma membrane degradation in GT1-7, HepG2, and H9c2 cells. These findings highlight the importance of examining co-exposures to fully understand the potential toxic effects.

Spatiotemporal phosphoprotein distribution and associated cytokine response of a traumatic injury.

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/ß, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.

Deguelin and Its Role in Chronic Diseases.

Deguelin is one of four major naturally occurring rotenoids isolated from root extracts and is best recognized as a NADH: ubiquinone oxidoreductase (complex I) inhibitor, resulting in significant alterations in mitochondrial function. Deguelin has also been implicated as a regulator of apoptosis through signaling pathways, such as the (PI3K)/Akt pathway, as well as an initiator of cell cycle arrest. Consequently, this compound has accrued great interest as a potential chemopreventive and chemotherapeutic. Additionally, deguelin exposure has been linked to Parkinson's disease (PD). PD is a neurodegenerative disorder, characterized by a substantial loss of dopaminergic neurons in the substantia nigra, as well the manifestation of symptoms such as bradykinesia, rigidity, and rest tremor. While exploring the genetic impact of PD is imperative, environmental factors, such as exposure to pesticides, herbicides, and insecticides, have also been connected to the development of PD. The etiology and pathogenesis of PD are yet to be fully understood and elucidated, but mitochondrial dysfunction is gaining recognition as a molecular hallmark of PD. In fact, deguelin has been reported to elicit PD-like symptoms (degeneration of the dopaminergic pathway) in rats administered with deguelin (6 mg/kg/day for 6 days), possibly through the inhibition of mitochondrial complex I. Further research investigating the mechanisms by which deguelin inhibits central cellular processes is essential in order to advance any prospective research addressing potential applications and risks of deguelin.

Spatial cytokine distribution following traumatic injury.

Temporal changes in cytokine concentrations following traumatic injuries have been extensively studied. Less is known regarding spatial differences in cytokine concentrations following traumatic injury. The primary aim of this study was to determine the spatial relationship between cytokines and the zone of injury (ZOI). Muscle and vessel tissues obtained from rats subjected to an open femoral fracture were analyzed to determine if spatial cytokine gradients exist that could potentially be used as biomarkers of the ZOI. Samples were collected at 4 time points following fracture from 3 distinct locations: at the fracture site, 1-cm away from the fracture, and from the opposite leg. The concentrations of IL-6, IL-1α, IL-1ß, IL-2, GM-CSF, TNF-α, and MIP-1α were quantified in each sample. Temporally and spatially regulated variations in cytokine concentrations were found. IL-6 showed the most promise as a ZOI biomarker with statistically different spatial concentrations that were inversely proportional to the distance from the fracture in both tissues. IL-1ß and IL-2 also showed spatial differences in concentration in both tissues, while GM-CSF, MIP-1α, and TNF-α showed spatial differences in vessel samples. These results demonstrate that spatial cytokine gradients exist following traumatic injury, representing potential biomarkers that may be used to define the ZOI.

An approach to investigate intracellular protein network responses.

Modern toxicological evaluations have evolved to consider toxicity as a perturbation of biological pathways or networks. As such, toxicity testing approaches are shifting from common end point evaluations to pathway based approaches, where the degree of perturbation of select biological pathways is monitored. These new approaches are greatly increasing the data available to toxicologists, but methods of analyses to determine the inter-relationships between potentially affected pathways are needed to fully understand the consequences of exposure. An approach to construct dose-response curves that use graph theory to describe network perturbations among three disparate mitogen-activated protein kinase (MAPK) pathways is presented. Mitochondrial stress was induced in human hepatocytes (HepG2) by exposing the cells to increasing doses of the complex I inhibitor, deguelin. The relative phosphorylation responses of proteins involved in the regulation of the stress response were measured. Graph theory was applied to the phosphorylation data to obtain parameters describing the network perturbations at each individual dose tested. The graph theory results depicted the dynamic nature of the relationship between p38, JNK, and ERK1/2 under conditions of mitochondrial stress and revealed shifts in the relationships between these MAPK pathways at low doses. The inter-relationship, or crosstalk, among these 3 traditionally linear MAPK cascades was further probed by coexposing cells to deguelin plus SB202190 (JNK and p38 inhibitor) or deguelin plus SB202474 (JNK inhibitor). The cells exposed to deguelin plus SB202474 resulted in significantly decreased viability, which could be visualized and attributed to the decrease of ERK1/2 network centrality. The approach presented here allows for the construction and visualization of dose-response curves that describe network perturbations induced by chemical stress, which provides an informative and sensitive means of assessing toxicological effects on biological systems.

Fentanyl overdose: Temporal effects and prognostic factors in SKH1 mice.

Fentanyl exposure and overdose are growing concerns in public health and occupational safety. This study aimed to establish parameters of fentanyl lethality in SKH1 mice for future overdose research. Lethality was determined using the up-down procedure, with subjects monitored post-administration using pulse oximetry (5 min) and then whole-body plethysmography (40 min). Following the determination of subcutaneous dose-response, [18F]Fluorodeoxyglucose positron emission tomography (18 F-FDG PET) was performed after LD10 fentanyl at 40 min, 6 h, 24 h or 7 days post-dose. LD10 and LD50 were observed to be 110 and 135 mg/kg, respectively, and consistent with four-parameter logistic fit values of 111.2 and 134.6 mg/kg (r2  = 0.9996). Overdose (LD10 or greater) yielded three distinct cardiovascular groups: survival, non-survival with blood oxygen saturation (SpO2) minimum ≥37% and non-survival with SpO2 <37%. Breaths per minute, minute volume and inspiratory quotient were significantly different between surviving and non-surviving animals for up to 40 min post-injection. 18 F-FDG PET revealed decreased glucose uptake in the heart, lungs and brain for up to 24 h. These findings provide critical insights into fentanyl lethality in SKH1 mice, including non-invasive respiratory effects and organ-specific impacts that are invaluable for future translational studies investigating the temporal effects of fentanyl overdose.

140 H/D isotopomers identified by long-range NMR hyperfine shifts in ruthenium(III) ammine complexes. Hyperconjugation in Ru-NH3 bonding.

(1)H NMR spectra of the paramagnetic cyanide-bridged mixed-valence compound [(η(5)-C5H5)Fe(CO)2(µ-CN)Ru(NH3)5](CF3SO3)3 (I) have been obtained in several solvents. When traces of partially deuterated water are present, instead of a single cyclopentadienyl (Cp) resonance shifted by the hyperfine interaction, numerous well-resolved resonances are observed. The spectra were simulated satisfactorily by giving the appropriate statistical weight to 140 possible H/D isotopomers formed by deuteration in the five ruthenium(III) ammine ligands. The proliferation of distinct resonances occurs because (a) the hyperfine shifts (HSs) due to each sequential deuteration in a single ammine are different and (b) while deuteration in an ammine cis to the cyanide bridge causes a downfield shift, in the trans ammine it causes an upfield shift that is nearly twice as large. All of these shifts exhibit a 1/T dependence, but temperature-independent components, due to large second-order Zeeman effects at the Ru(III) center, are also present. Combining the results of density functional theory calculations with data from metal-metal charge-transfer optical transitions and with the effect of solvent-induced NMR HSs, it is argued that Fermi contact shifts at the Cp protons are insignificant compared to those due to the dipolar (pseudocontact) mechanism. Analytical expressions are presented for the dependence of the HS on the tetragonal component of the ligand field at the Ru(III) ion. The tetragonal field parameter, defined as the energy by which the 4d(xy) orbital exceeds the mean t(2g) orbital energy, was found to be 147, 52, and 76 cm(-1), in dimethylformamide, acetone, and nitromethane, respectively. The effects of deuteration show that there is a significant component of hyperconjugation in the Ru-ammine interaction and that ND3 is a weaker π donor than NH3. A single deuteration in an axial ammine increases the tetragonal field parameter (ν) by +2.8 cm(-1), resulting in a HS of -37 ppb in the Cp proton resonance, whereas a single deuteration in an equatorial ammine decreases the field by -1.5 cm(-1) with a HS of +20 ppb, despite a nominal separation of seven chemical bonds. We analyze the origin of this remarkable sensitivity, which relies on the favorable characteristics of the Ru(III) low-spin t(2g)(5) configuration, having a spin-orbit coupling constant ζ ≈ 950 cm(-1).

Opioids and Vitamin C: Known Interactions and Potential for Redox-Signaling Crosstalk.

Opioids are among the most widely used classes of pharmacologically active compounds both clinically and recreationally. Beyond their analgesic efficacy via µ opioid receptor (MOR) agonism, a prominent side effect is central respiratory depression, leading to systemic hypoxia and free radical generation. Vitamin C (ascorbic acid; AA) is an essential antioxidant vitamin and is involved in the recycling of redox cofactors associated with inflammation. While AA has been shown to reduce some of the negative side effects of opioids, the underlying mechanisms have not been explored. The present review seeks to provide a signaling framework under which MOR activation and AA may interact. AA can directly quench reactive oxygen and nitrogen species induced by opioids, yet this activity alone does not sufficiently describe observations. Downstream of MOR activation, confounding effects from AA with STAT3, HIF1α, and NF-κB have the potential to block production of antioxidant proteins such as nitric oxide synthase and superoxide dismutase. Further mechanistic research is necessary to understand the underlying signaling crosstalk of MOR activation and AA in the amelioration of the negative, potentially fatal side effects of opioids.