Comparison of Intubating Conditions with Succinylcholine Versus Rocuronium in the Prehospital Setting.

OBJECTIVE: Rapid sequence intubation (RSI) is frequently performed by emergency medical services (EMS). We investigated the relationship between succinylcholine and rocuronium use and time until first laryngoscopy attempt, first-pass success, and Cormack-Lehane (CL) grades. METHODS: We included adult patients for whom prehospital RSI was attempted from July 2015 through June 2022 in a retrospective, observational study with pre-post analysis. Timing was verified using recorded defibrillator audio in addition to review of continuous ECG, pulse oximetry, and end-tidal carbon dioxide waveforms. Our primary exposure was neuromuscular blocking agent (NMBA) used, either rocuronium or succinylcholine. Our prespecified primary outcome was the first attempt Cormack-Lehane view. Key secondary outcomes were first laryngoscopy attempt success rate, timing from NMBA administration to first attempt, number of attempts, and hypoxemic events. RESULTS: Of 5,179 patients in the EMS airway registry, 1,475 adults received an NMBA while not in cardiac arrest. Cormack-Lehane grades for succinylcholine and rocuronium were similar: grade I (64%, 59% [95% CI 0.64-1.09]), grade II (16%, 21%), grade III (18%, 16%), grade IV (3%, 3%). The median interval from NMBA administration to start of the first attempt was 57 s for succinylcholine and 83 s for rocuronium (mean difference 28 [95% CI 20-36] seconds). First attempt success was 84% for succinylcholine and 83% for rocuronium. Hypoxemic events were present in 25% of succinylcholine cases and 23% of rocuronium cases. CONCLUSIONS: Prehospital use of either rocuronium or succinylcholine is associated with similar Cormack-Lehane grades, first-pass success rates, and rates of peri-intubation hypoxemia.

Improved Neutron Lifetime Measurement with UCNτ.

We report an improved measurement of the free neutron lifetime τ_{n} using the UCNτ apparatus at the Los Alamos Neutron Science Center. We count a total of approximately 38×10^{6} surviving ultracold neutrons (UCNs) after storing in UCNτ's magnetogravitational trap over two data acquisition campaigns in 2017 and 2018. We extract τ_{n} from three blinded, independent analyses by both pairing long and short storage time runs to find a set of replicate τ_{n} measurements and by performing a global likelihood fit to all data while self-consistently incorporating the ß-decay lifetime. Both techniques achieve consistent results and find a value τ_{n}=877.75±0.28_{stat}+0.22/-0.16_{syst} s. With this sensitivity, neutron lifetime experiments now directly address the impact of recent refinements in our understanding of the standard model for neutron decay.

DNA methylation and transcriptome aberrations mediated by ERα in mouse seminal vesicles following developmental DES exposure.

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health.

Essential Oils and Health.

Essential oils (EOs) have risen in popularity over the past decade. These oils function in society as holistic integrative modalities to traditional medicinal treatments, where many Americans substitute EOs in place of other prescribed medications. EOs are found in a multitude of products including food flavoring, soaps, lotions, shampoos, hair styling products, cologne, laundry detergents, and even insect repellents. EOs are complex substances comprised of hundreds of components that can vary greatly in their composition depending upon the extraction process by the producer or the origin of the plant. Thus, making it difficult to determine which pathways in the body are affected. Here, we review the published research that shows the health benefits of EOs as well as some of their adverse effects. In doing so, we show that EOs, as well as some of their individual components, possess antimicrobial, antiviral, antibiotic, anti-inflammatory, and antioxidant properties as well as purported psychogenic effects such as relieving stress, treating depression, and aiding with insomnia. Not only do we show the health benefits of using EOs, but we also indicate risks associated with their use such as their endocrine disrupting properties leading to the induction of premature breast growth in young adolescents. Taken together, there are many positive and potentially negative risks to human health associated with EOs, which make it important to bring awareness to all their known effects on the human body.

Inferring distributional shifts of epidemiologically important North and Central American sandflies from Pleistocene to future scenarios.

Nine sandfly species (Diptera: Psychodidae) are suspected or proven vectors of Leishmania spp. in the North and Central America region. The ecological niches for these nine species were modelled in three time periods and the overlaps for all time periods of the geographic predictions (G space), and of ecological dimensions using pairwise comparisons of equivalent niches (E space), were calculated. Two Nearctic, six Neotropical and one species in both bioregions occupied a reduced number of distribution areas. The ecological niche projections for most sandfly species other than Lutzomyia shannoni and Lutzomyia ovallesi have not expanded significantly since the Pleistocene. Only three species increase significantly to 2050, whereas all others remain stable. Lutzomyia longipalpis shared a similar ecological niche with more species than any other, although both L. longipalpis and Lutzomyia olmeca olmeca had conserved distributions over time. Climate change, at both regional and local levels, will play a significant role in the temporal and spatial distributions of sandfly species.

The Joint Utilization Management Program-Implementation of a Bundle Payment Model and Comparison Between Year 1 and 2 Results.

BACKGROUND: Health care spending is projected to increase throughout the next decade alongside the number of total joint arthroplasties (TJAs) performed. Such growth places significant financial burden on the economic system. To address these concerns, Bundled Payments for Care Improvement (BPCI) is becoming a favorable reimbursement model. The aim of this study is to present the outcomes with BPCI model focused on the post-acute care (PAC) phase and compare the outcomes between years 1 and 2 of implementation. METHODS: The Joint Utilization Management Program (JUMP) was implemented in January 2014. Inclusion criteria were Medicare patients undergoing primary unilateral in-patient TJA procedures, outpatient procedures that resulted in an in-hospital admission, and trauma episodes that required TJA. Scorecards monitoring surgeons' performance and tracking length of stay (LOS) in the PAC setting were established. The data generated from these scorecards guided percentage sum-allocation from the total gain-shared sum among the participating providers. RESULTS: A total of 683 JUMP patients were assessed over two years. PAC utilization decreased between 2014 and 2015. The average LOS was longer in year 1 than year 2 (4.50 vs 3.19 days). In-patient rehabilitation (IPR) decreased from 6.45% to 3.22%, with a decrease in IPR average LOS of 1.47 days. The rate of 30-day readmission was lower for JUMP patients in 2015 than 2014 (8.77% vs 10.56%), with day of readmission being earlier (11.91 days vs 13.71 days) in 2014. CONCLUSION: Under the BPCI program, our experience with the JUMP model demonstrates higher efficiency of care in the PAC setting through reduced LOS, IPR admission rates, and 30-day readmission rate.

Development of a Microchip CE-HPMS Platform for Cell Growth Monitoring.

Improvements were made to a previously developed platform coupling microchip capillary electrophoresis (CE) with high pressure mass spectrometry (HPMS). The RF drive frequency was increased to over 30 MHz from less than 10 MHz, and the ion trap was scaled down to 100 µm critical dimensions. A stretched length ion trap was used to improve sensitivity, and a tube lens was used to improve ion transmission. Detection of the 20 common amino acids was demonstrated, resulting in an average improvement of signal-to-noise of 28 times and an average improvement in peak width of 2.6 times over those obtained in previous work. Consumption of amino acids by cells in growth media was monitored over time using the improved CE-HPMS platform, and several amino acids were shown to be consumed at different rates, demonstrating the potential for real-time bioreactor monitoring.

Impact of climate change on vector transmission of Trypanosoma cruzi (Chagas, 1909) in North America.

Climate change can influence the geographical range of the ecological niche of pathogens by altering biotic interactions with vectors and reservoirs. The distributions of 20 epidemiologically important triatomine species in North America were modelled, comparing the genetic algorithm for rule-set prediction (GARP) and maximum entropy (MaxEnt), with or without topographical variables. Potential shifts in transmission niche for Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) (Chagas, 1909) were analysed for 2050 and 2070 in Representative Concentration Pathway (RCP) 4.5 and RCP 8.5. There were no significant quantitative range differences between the GARP and MaxEnt models, but GARP models best represented known distributions for most species [partial-receiver operating characteristic (ROC) > 1]; elevation was an important variable contributing to the ecological niche model (ENM). There was little difference between niche breadth projections for RCP 4.5 and RCP 8.5; the majority of species shifted significantly in both periods. Those species with the greatest current distribution range are expected to have the greatest shifts. Positional changes in the centroid, although reduced for most species, were associated with latitude. A significant increase or decrease in mean niche elevation is expected principally for Neotropical 1 species. The impact of climate change will be specific to each species, its biogeographical region and its latitude. North American triatomines with the greatest current distribution ranges (Nearctic 2 and Nearctic/Neotropical) will have the greatest future distribution shifts. Significant shifts (increases or decreases) in mean elevation over time are projected principally for the Neotropical species with the broadest current distributions. Changes in the vector exposure threat to the human population were significant for both future periods, with a 1.48% increase for urban populations and a 1.76% increase for rural populations in 2050.

Behavioural and electrophysiological responses of Triatoma dimidiata nymphs to conspecific faecal volatiles.

The behavioural and electrophysiological (electroantennography) responses of the first two instars of Triatoma dimidiata (Hemiptera: Reduviidae) Latreille to fresh and dry faecal headspace volatile extracts from fifth instar conspecific nymphs and synthetic compounds were analysed in this study. Recently emerged nymphs (3-5 days) aggregated around filter paper impregnated with dry faeces and around filter paper impregnated with extracts from both fresh and dry faeces. Older first instars (10-15 days) and second instars aggregated around filter paper impregnated with fresh and dry faeces, and their respective headspace extracts. Dry faecal volatile extracts elicited the strongest antennal responses, followed by fresh faecal extracts. Gas chromatography-mass spectrometry analysis of dried faecal headspace volatiles demonstrated the presence of 12 compounds: 2-ethyl-1-hexanol, 1,2,4-trimethylbenzene, n-octadecane, n-nonadecane, n-eicosane, n-heneicosane, n-tricosane, n-pentaeicosane, n-hexaeicosane, n-octaeicosane, nonanal, and 4-methyl quinazoline. In fresh faecal headspace extracts, only nonanal was clearly detected, although there were other trace compounds, including several unidentified sesquiterpenes. Four of the 11 compounds tested individually elicited aggregation behaviour at concentrations of 100 ng/µL and 1 µg/µL. A blend containing these four components also mediated the aggregation of nymphs. These volatiles may be valuable for developing monitoring methods and designing sensitive strategies to detect and measure T. dimidiata infestation.

Self-injurious behaviours in rhesus macaques: Potential glial mechanisms.

BACKGROUND: Self-injurious behaviour (SIB) can be classified as intentional, direct injuring of body tissue usually without suicidal intent. In its non-suicidal form it is commonly seen as a clinical sign of borderline personality disorder, autism, PTSD, depression, and anxiety affecting a wide range of ages and conditions. In rhesus macaques SIB is most commonly manifested through hair plucking, self-biting, self-hitting, and head banging. SIB in the form of self-biting is observed in approximately 5-15% of individually housed monkeys. Recently, glial cells are becoming recognised as key players in regulating behaviours. METHOD: The goal of this study was to determine the role of glial activation, including astrocytes, in macaques that had displayed SIB. To this end, we performed immunohistochemistry and next generation sequence of brain tissues from rhesus macaques with SIB. RESULTS: Our studies showed increased vimentin, but not nestin, expression on astrocytes of macaques displaying SIB. Initial RNA Seq analyses indicate activation of pathways involved in tissue remodelling, neuroinflammation and cAMP signalling. CONCLUSIONS: Glia are most probably activated in primates with self-injury, and are therefore potential novel targets for therapeutics.

Liver mitochondrial oxygen consumption and efficiency of milk production in lactating Holstein cows supplemented with copper, manganese and zinc.

The objective of this study was to examine the relationship between mitochondrial proton leak and feed efficiency with supplementation of different levels of Cu, Mn and Zn (Bioplex, Alltech) at levels above Nutrient Requirements of Dairy Cattle (NRC, 2001). Milk yield and composition, mineral excretion in milk and faeces, feed efficiency and liver mitochondrial proton leak were measured in 60 Holstein dairy cows at approximately 70 days in milk on a commercial dairy. Treatments reflect total Cu, Mn and Zn intake/day and are as follows: (i) High: 444 mg/day Cu, 3492 mg/day Mn, 2234 mg/day Zn; (ii) Med: 436 mg/day Cu, 3002 mg/day Mn, 2047 mg/day Zn; (iii) Low: 420 mg/day Cu, 2764 mg/day Mn, 2186 mg/day Zn; (iv) LowMn: 391 mg/day Cu, 2617 mg/day Mn, 1849 mg/day Zn; and (v) Control: 264 mg/day Cu, 2850 mg/day Mn, 1593 mg/day Zn. Proton leak-dependent respiration was lowest in Control (p < .10). However, measures of efficiency were greatest in Med and least in High (p < .10). Therefore, measures of efficiency did not reflect efficiency due to low proton leak and there appears to be an upper limit to beneficial supplementation of Cu, Mn and Zn.

Coupling Microchip Electrospray Ionization Devices with High Pressure Mass Spectrometry.

A microchip electrospray ionization source was coupled with high pressure mass spectrometry (HPMS). A continuous atmospheric inlet consisting of a stainless steel capillary and DC ion optics was designed to conduct ions into the mass spectrometer. Infusions of amino acids and peptides were performed and detected with a miniature cylindrical ion trap (mini-CIT)-based mass spectrometer operated at ≥1 Torr with air as the buffer gas. Detection of glycine and thymopentin (separately) demonstrated the mass range of the mini-CIT detector could span from m/z 75 to 681. A microchip capillary electrophoresis (CE) separation with mini-CIT detection was performed, and the results were compared with detection using a commercial instrument (Waters Synapt G2). Comparable separation efficiencies were observed with both mass spectrometers as detectors, with about 6 times better signal-to-noise observed on the Synapt G2. Comparison of mass spectra in the two systems reveals similar features observed, but with wider peak widths in the mini-CIT than on the Synapt G2 as expected due to high-pressure operation.

Use of Ice-Nucleating Proteins To Improve the Performance of Freeze-Thaw Valves in Microfluidic Devices.

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.

High pressure mass spectrometry of volatile organic compounds with ambient air buffer gas.

RATIONALE: There are many chemical measurement scenarios that would benefit from hand portable mass spectrometry tools including forensics, environmental monitoring, and safety and security. High pressure mass spectrometry (HPMS) facilitates miniaturization by significantly reducing vacuum system requirements. Previous work demonstrated HPMS using helium buffer gas, but HPMS conducted using ambient air would further reduce the size and weight of a portable instrument while also reducing logistical demands by eliminating the need for a helium supply. METHODS: Mass spectrometry was performed at pressures exceeding 1 Torr with ambient air as the buffer gas. A glow discharge electron ionization source and a miniature cylindrical ion trap mass analyzer with a radius of 0.5 mm were used. Mass analysis was possible at these pressures with increased radiofrequency (RF) drive frequencies (10 MHz) compared with commercial ion traps (~1 MHz). A differentially pumped chamber was used so that mass spectrometry could be performed at high pressures and detection performed at low pressures with an electron multiplier. RESULTS: HPMS with air buffer gas was demonstrated using a suite of volatile organic compounds (VOCs). The glow discharge ionization source was optimized for operation using air. Mass spectral peak widths increased a factor of 8 compared with helium, as expected, but useful chemical information was still acquired. A mixture of VOCs was detected with ambient air as the buffer gas, showing that valuable mass information can be gained using HPMS without the requirement of an onboard buffer gas source. CONCLUSIONS: HPMS significantly reduces the pumping requirements required for miniature mass spectrometers and the use of ambient air buffer gas further reduces size, weight, and logistics requirements. Mass analysis at high pressures of ambient air is another important step for the development of hand portable mass spectrometers. Copyright © 2016 John Wiley & Sons, Ltd.

The influence of Shc proteins and high-fat diet on energy metabolism of mice.

The purpose of this study was to determine if Shc proteins influence the metabolic response to acute (7 days) feeding of a high-fat diet (HFD). To this end, whole animal energy expenditure (EE) and substrate oxidation were measured in the Shc knockout (ShcKO) and wild-type (WT) mice fed a control or HFD. The activities of enzymes of glycolysis, the citric acid cycle, electron transport chain (ETC), and ß-oxidation were also investigated in liver and skeletal muscle of ShcKO and WT animals. The study showed that ShcKO increases (P < .05) EE adjusted for either total body weight or lean mass. This change in EE could contribute to decreases in weight gain in ShcKO versus WT mice fed an HFD. Thus, our results indicate that Shc proteins should be considered as potential targets for developing interventions to mitigate weight gain on HFD by stimulating EE. Although decreased levels of Shc proteins influenced the activity of some enzymes in response to high-fat feeding (eg, increasing the activity of acyl-CoA dehydrogenase), it did not produce concerted changes in enzymes of glycolysis, citric acid cycle, or the ETC. The physiological significance of observed changes in select enzyme activities remains to be determined. SIGNIFICANCE OF THE STUDY: We report higher EE in ShcKO versus WT mice when consuming the HFD. Although decreased levels of Shc proteins influenced the activity of a central enzyme of ß-oxidation in response to high-fat feeding, it did not produce concerted changes in enzymes of glycolysis, citric acid cycle, or the ETC. Thus, an increase in EE in response to consumption of an HFD may be a mechanism that leads to decreased weight gain previously reported in ShcKO mice with long-term consumption of an HFD.

Lack of glucuronidation products of trans-resveratrol in plasma and urine of cats.

Resveratrol has generated interest in cats due to reported health benefits. Cats have low activity of ß-glucuronidase, and we hypothesized they could not form two common resveratrol metabolites, resveratrol-3-O-glucuronide and resveratrol-4'-O-glucuronide. Resveratrol, 3 mg/cat/day, was given orally to intact male (n = 5) and female cats (n = 5) for 4 weeks. A control group (8 intact males) was used for comparison. Plasma and urine were collected weekly and analysed using high-pressure liquid chromatography coupled with tandem mass spectrometry. Resveratrol and resveratrol-3-O-sulphate, but no glucuronide metabolites, were detected in plasma and urine. Median (range 10-90th percentile) plasma resveratrol for control and treatment groups was 0.46 ng/ml (0.02-1.74 ng/ml) and 0.96 ng/ml (0.65-3.21 ng/ml). Median (range) plasma resveratrol-3-O-sulphate for control and treatment groups was 6.32 ng/ml (2.55-10.29 ng/ml) and 11.45 ng/ml (1.47-53.29 ng/ml). Plasma resveratrol differed from control in week 4, while plasma resveratrol-3-O-sulphate was different in all weeks (p < 0.05). Median (range) urine resveratrol for control and treatment groups was 0.28 ng/ml (0.05-1.59 ng/ml) and 19.98 ng/ml (8.44-87.54 ng/ml). Median (range) urine resveratrol-3-O-sulphate for control and treatment groups was 26.71 ng/ml (10.50-75.58 ng/ml) and 108.69 ng/ml (11.83-231.05 ng/ml). All time points for urine resveratrol and resveratrol-3-O-sulphate were significantly different from control (p < 0.05), except for weeks 1, 3 and 4 for resveratrol. The results support our hypothesis that cats are unlikely able to glucuronidate resveratrol, most likely due to a reduction in the activity of ß-glucuronidase.

Liver mitochondrial oxygen consumption and proton leak kinetics in broilers supplemented with dietary copper or zinc following coccidiosis challenge.

Mitochondrial respiration was assessed in sixteen 7-day-old broilers as a subset of a larger study assessing the effects of Cu and Zn supplementation above requirements with a coccidiosis challenge on gain/feed ratio. The birds were selected from four treatments (four birds/treatment): a control diet (Cu 15 mg/kg and Zn 60 mg/kg) + coccidiosis challenge (CC), a Cu diet with 245 mg/kg Cu from tribasic copper chloride (TBCC) + CC, a negative control diet (Cu 15 mg/kg and Zn 60 mg/kg) - CC and a Zn diet with 2000 mg/kg Zn from ZnO. The diets were composed of 49% corn, 40% soybean meal, 6.2% vegetable oil (diet dry matter = 90.62%, crude protein = 21.37%, fat = 7.7%, metabolizable energy = 12.1 MJ/day) and were fed for 14 days. Birds were dissected, and approximately one gram of liver tissue was used for mitochondrial oxygen consumption and proton leak kinetics assays. Respiratory control ratio and mitochondrial proton leak assessed by calculating rates of oxygen consumption at 175mV membrane potential were greater for the negative control group, but there were no differences in average gain/feed among treatments. In summary, broilers that did not undergo coccidiosis challenge had lower proton leak and higher respiratory control ratio. However, the impact of supplementation of Cu and Zn above requirements did not appear to prevent changes in respiratory control ratio and proton leak kinetics with coccidiosis challenge.

ZpdN, a Plasmid-Encoded Sigma Factor Homolog, Induces pBS32-Dependent Cell Death in Bacillus subtilis.

The ancestral Bacillus subtilis strain 3610 contains an 84-kb plasmid called pBS32 that was lost during domestication of commonly used laboratory derivatives. Here we demonstrate that pBS32, normally present at 1 or 2 copies per cell, increases in copy number nearly 100-fold when cells are treated with the DNA-damaging agent mitomycin C. Mitomycin C treatment also caused cell lysis dependent on pBS32-borne prophage genes. ZpdN, a sigma factor homolog encoded by pBS32, was required for the plasmid response to DNA damage, and artificial expression of ZpdN was sufficient to induce pBS32 hyperreplication and cell death. Plasmid DNA released by cell death was protected by the capsid protein ZpbH, suggesting that the plasmid was packaged into a phagelike particle. The putative particles were further indicated by CsCl sedimentation but were not observed by electron microscopy and were incapable of killing B. subtilis cells extracellularly. We hypothesize that pBS32-mediated cell death releases a phagelike particle that is defective and unstable. IMPORTANCE: Prophages are phage genomes stably integrated into the host bacterium's chromosome and less frequently are maintained as extrachromosomal plasmids. Here we report that the extrachromosomal plasmid pBS32 of Bacillus subtilis encodes a prophage that, when activated, kills the host. pBS32 also encodes both the sigma factor homolog ZpdN that is necessary and sufficient for prophage induction and the protein ComI, which is a potent inhibitor of DNA uptake by natural transformation. We provide evidence that the entire pBS32 sequence may be part of the prophage and thus that competence inhibition may be linked to lysogeny.

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis-Mass Spectrometry.

In this work, we utilize capillary electrophoresis-mass spectrometry (CE-MS) in an integrated microfluidic platform to analyze an intact, lysine-linked antibody drug conjugate (ADC) in order to assess post translational modifications and drug load variants. The initial charge heterogeneity of the unconjugated IgG-2 monoclonal antibody (mAb) was assessed by separating intact charge variants. Three main charge variants were resolved in the CE dimension. These variants were attributed to pyroglutamic acid formation and decarboxylation on the primary structure of the mAb through characteristic mass shifts and changes in electrophoretic mobility. Additionally, glycoforms of the antibody charge variants were identified in the deconvoluted mass spectra. The observed glycoforms and their distribution compared favorably to a released N-glycan analysis performed on the mAb. After conjugation, the ADC was analyzed using the same microchip CE-MS method. The addition of a drug load resulted in a decrease in mobility and an increase in mass of 3145 Da. Five main species that differed in their respective drug-to-antibody ratios (DAR) were fully resolved in the CE separation, with each DAR displaying the same variant population observed on the unconjugated mAb. A DAR range of 0-4 was observed with an average of 1.7 drug loads. The DAR distribution generated from the microfluidic CE-MS data compared favorably to results from infusion-ESI-MS and imaging CE (iCE) analysis of the ADC, techniques commonly used for intact mAb and ADC characterization.

Analysis of Hemoglobin Glycation Using Microfluidic CE-MS: A Rapid, Mass Spectrometry Compatible Method for Assessing Diabetes Management.

Diabetes has become a significant health problem worldwide with the rate of diagnosis increasing rapidly in recent years. Measurement of glycated blood proteins, particularly glycated hemoglobin (HbA1c), is an important diagnostic tool used to detect and manage the condition in patients. Described here is a method using microfluidic capillary electrophoresis with mass spectrometry detection (CE-MS) to assess hemoglobin glycation in whole blood lysate. Using denaturing conditions, the hemoglobin (Hb) tetramer dissociates into the alpha and beta subunits (α- and ß-Hb), which are then separated via CE directly coupled to MS detection. Nearly baseline resolution is achieved between α-Hb, ß-Hb, and glycated ß-Hb. A second glycated ß-Hb isomer that is partially resolved from ß-Hb is detected in extracted ion electropherograms for glycated ß-Hb. Glycation on α-Hb is also detected in the α-Hb mass spectrum. Additional modifications to the ß-Hb are detected, including acetylation and a +57 Da species that could be the addition of a glyoxal moiety. Patient blood samples were analyzed using the microfluidic CE-MS method and a clinically used immunoassay to measure HbA1c. The percentage of glycated α-Hb and ß-Hb was calculated from the microfluidic CE-MS data using peak areas generated from extracted ion electropherograms. The values for glycated ß-Hb were found to correlate well with the HbA1c levels derived in the clinic, giving a slope of 1.20 and an R(2) value of 0.99 on a correlation plot. Glycation of human serum albumin (HSA) can also be measured using this technique. It was observed that patients with elevated glycated Hb levels also had higher levels of HSA glycation. Interestingly, the sample with the highest HbA1c levels did not have the highest levels of glycated HSA. Because the lifetime of HSA is shorter than Hb, this could indicate a recent lapse in glycemic control for that patient. The ability to assess both Hb and HSA glycation has the potential to provide a more complete picture of a patient's glycemic control in the months leading up to blood collection. The results presented here demonstrate that the microfluidic CE-MS method is capable of rapidly assessing Hb and HSA glycation from low volumes of whole blood with minimal sample preparation and has the potential to provide more information in a single analysis step than current technologies.